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Lianhong Sun and Wenying Shou (eds.), Engineering and Analyzing Multicellular Systems: Methods and Protocols,
Methods in Molecular Biology, vol. 1151, DOI 10.1007/978-1-4939-0554-6_1, © Springer Science+Business Media New York 2014
Chapter 1
Recent Progress in Engineering Human-Associated
Microbiomes
Stephanie J. Yaung, George M. Church, and Harris H. Wang
Abstract
Recent progress in molecular biology and genetics opens up the possibility of engineering a variety of
biological systems, from single-cellular to multicellular organisms. The consortia of microbes that reside
on the human body, the human-associated microbiota, are particularly interesting as targets for forward
engineering and manipulation due to their relevance in health and disease. New technologies in analysis
and perturbation of the human microbiota will lead to better diagnostic and therapeutic strategies against
diseases of microbial origin or pathogenesis. Here, we discuss recent advances that are bringing us closer
to realizing the true potential of an engineered human-associated microbial community.
Key words Microbiome, Microbiota, Synthetic biology, Systems biology, Microbial engineering,
Functional metagenomics, Host–microbe interactions
1 Introduction
Of the 100 trillion cells in the human body, 90 % are microbes that
naturally inhabit various body sites, including the gastrointestinal
tract, nasal and oral cavities, urogenital area, and skin [1]. An indi-
vidual’s colon is home to 1011
–1012
microbial cells/mL, the greatest
density compared to any other microbial habitat characterized to
date [2]. Many studies, such as the Human Microbiome Project
and MetaHIT, have probed the vast effects of microbiota on
human health and disease [1, 3–5]. In addition to metagenomic
sequencing [6], traditional methods of studying cells in isolation
are important for elucidating molecular bases of microbial activity.
However, cells do not exist in single-species cultures in nature. In
fact, some species are only culturable in the presence of other
microorganisms [7]. This interdependence for survival amongst
microbial species in a community attests to the importance of
intercellular interactions, both microbe–microbe and host–
microbe. Despite the fact that the human microbiota is composed
of many individual microbes, these individuals work in concert to](https://image.slidesharecdn.com/2341781-250524103414-227d46be/85/Engineering-And-Analyzing-Multicellular-Systems-Methods-And-Protocols-1st-Edition-Lianhong-Sun-20-320.jpg)
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perform tasks that rival in complexity to those of more sophisticated
multicellular systems. Thus, the human-associated microbiome
presents a ripe opportunity for forward engineering to potentially
improve human health (Fig. 1). Here, we review recent advances in
this area and outline potential avenues for future endeavors.
2 Microbiota, Host, and Disease
Contrary to traditional views, microbes are social organisms that
engage with the environment and other organisms in specific ways.
Microbes participate in intercellular communication through
contact-dependent signaling [8], quorum sensing [9], metabolic
cooperation or competition [5], spatiotemporal organization [10],
and horizontal gene transfer (HGT) [11]. Human-associated
microbes produce by-products that serve as substrates utilized by
other resident bacteria [12–14]. For instance, accumulated hydro-
gen gas from bacterial sugar fermentation is removed by acetogenic,
methanogenic, and sulfate-reducing gut bacteria [15]. In contrast to
cross-feeding relationships, microbes under stress can release bacte-
riocins to suppress the growth of competitors [16–18]. If microbes
are members of a biofilm community, they benefit from physical
protection from the environment, access to nutrients trapped and
distributed through channels in the biofilm, development of syn-
trophic relationships with other members, and the ability to share
and acquire genetic traits [19, 20]. Microbial populations also
Fig. 1 Engineering human-associated microbiota requires detailed understanding
of processes that govern the natural propagation and retention of microbes in the
host as well as environmental and adaptive pressures that drive the evolution of
cells and communities
Stephanie J. Yaung et al.](https://image.slidesharecdn.com/2341781-250524103414-227d46be/85/Engineering-And-Analyzing-Multicellular-Systems-Methods-And-Protocols-1st-Edition-Lianhong-Sun-21-320.jpg)
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genetically diversify to insure against possible unstable environmen-
tal conditions [21, 22]. Moreover, multispecies communities harbor
a dynamic gene pool consisting of mobile genetic elements, such as
transposons, plasmids, and bacteriophages, which serve as a source
of HGT to share beneficial functions with neighbors to preserve
community stability [23–26]. Densely populated communities such
as the human gut are active sites for gene transfer and reservoirs for
antibiotic resistance genes [11, 27–29].
Beyond microbe–microbe interactions, the microbiota
coevolves with the host as it develops, driving microbial adaptation
[30–33]. Core functions of microbiota benefit the host, such as
extraction of otherwise inaccessible nutrients, immune system devel-
opment, and protection against pathogen colonization [2, 34–37].
Gut microbes are critical in intestinal angiogenesis, epithelial cell
maturation, and immunological homeostasis [37–40]. For exam-
ple, the commensal Bacteroides fragilis produces polysaccharide A,
which converts host CD4+
T cells into Foxp3+
Treg cells, producing
interleukin-10 (IL-10) and inducing mucosal tolerance [41]. Host
diet, inflammatory responses, and aging also affect microbial com-
munity composition and function [42–45] (Fig. 2). Indeed, aber-
rations in host genetics, immunology, and diet can lead to
Fig. 2 Composition of the human gut microbiome during development with
respect to microbial diversity and population stability. Data compiled from recent
studies from the literature: (a) Hong 2010 [169]; (b) Saulnier 2011 [170]; (c)
Claesson 2011 [171]; (d) Yatsunenko 2012 [172]; (e) Spor 2011 [173]
Engineering Human-Associated Microbiomes](https://image.slidesharecdn.com/2341781-250524103414-227d46be/85/Engineering-And-Analyzing-Multicellular-Systems-Methods-And-Protocols-1st-Edition-Lianhong-Sun-22-320.jpg)
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microbiota-associated human diseases. Diet-induced obesity in mice
from a high-fat diet is characterized by enhanced energy harvest and
an increased Firmicutes-to-Bacteroidetes ratio [46, 47]. Furthermore,
disruptions in the homeostasis between gut microbial antigens and
host immunity can invoke allergy and autoimmunity, as in type 1
diabetes and multiple sclerosis [48–50]. It is thought that inflamma-
tory bowel disease (IBD) results from inappropriate immune
responses to intestinal bacteria; genes identified in genome-wide
association studies highlight the role of a host imbalance between
pro-inflammatory and regulatory states [48, 51].
While the host selects for microbial communities that harvest
nutrients and prime the immune system, irregular microbiota
composition may cause disease (Fig. 3), including IBD [52–54],
Host disease states
Inflammatory
bowel disease
in adults
Metabolic
syndrome
in adult
Healthy
Burkina Faso
childrena
Healthy
European
childrena
Host genetics and
environment (diet, lifestyle)
Actinobacteria
Bacteroidetes
Firmicutes
Proteobacteria
Spirochaetes
Other
Fusobacteria
Healthy Dental caries
Young adult salivag
Saliva Dental plaque
Oral microbiomef
Gut microbiome
Pediatric atopic dermatitisd
Baseline
Healthy childd
Flare Postflare
Healthy adulte
Skin microbiome
Fig. 3 Changes in the composition of human microbiota during disease states
compared to healthy states. Data compiled from recent studies from the literature:
(a) De Filippo 2010 [174]; (b) Peterson 2008 [175]; (c) Larsen 2010 [176]; (d) Kong
2012 [177]; (e) Gao 2012 [178]; (f ) Keijser 2008 [179]; (g) Yang 2012 [180]
Stephanie J. Yaung et al.](https://image.slidesharecdn.com/2341781-250524103414-227d46be/85/Engineering-And-Analyzing-Multicellular-Systems-Methods-And-Protocols-1st-Edition-Lianhong-Sun-23-320.jpg)
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lactose intolerance [55, 56], obesity [57, 58], type I diabetes
[59], arthritis [60], myocardial infarction severity [61], and
opportunistic infections by pathogens such as Clostridium difficile
and HIV [62–65]. Microbial gut metabolism links host diet not
only to body composition and obesity [66] but also to chronic
inflammatory states, such as IBD, type 2 diabetes, and cardiovas-
cular disease [67–69]. Intestinal microbes are also important in
off-target drug metabolism, rendering digoxin, acetaminophen,
and irinotecan less effective or even toxic [70–72]. In the case of
irinotecan, a chemotherapeutic used mainly for colon cancer, the
drug is metabolized by β-glucuronidases of commensal gut bacte-
ria into a toxic form that damages the intestinal lining and causes
severe diarrhea. In the oral cavity, ecological shifts in dental plaque
microbiota lead to caries (cavities), gingivitis, and periodontitis
[73]. Dental caries arise from acidic environments generated by
acidogenic (acid-forming) and aciduric (acid-tolerant) bacteria,
which metabolize sugar from the host diet. Translocation of oral
bacteria into other tissues results in infections, and cytokines from
inflamed gums released into the bloodstream stimulate systemic
inflammation. Oral bacteria have been implicated in respiratory
[74, 75] and cardiovascular diseases [76–78], though mechanisms
remain unclear.
3 Enabling Tools for Engineering the Microbiota
The human-associated microbial community presents a vast reservoir
of nonmammalian genetic information that encodes for a variety of
functions essential to the mammalian host [79]. Next-generation
sequencing technologies have enabled us for the first time to sys-
tematically probe the genetic composition of these trillions of
microbes that reside on the human body [1]. The ongoing effort
by the Human Microbiome Project and MetaHIT to catalog dom-
inant microbial strains from different body sites has generated useful
reference genomes for many of the representative species [80].
Metagenomic shot-gun sequencing approaches of whole microbial
communities, such as those found in the gut, have yielded near-
complete gene catalogs that describe abundance and diversity of
genes that contribute to maintenance and metabolism of the
microbiota [6].
In order to determine functional relationships between
human-associated microbes and their concerted effect in the mam-
malian host, we rely on functional perturbation of the microbial
community. These investigative avenues include genome-scale per-
turbation assays, specified community reconstitutions, and directed
engineering through synthetic biology (Fig. 4). Each approach
provides us with a unique angle to attack an otherwise daunting
Engineering Human-Associated Microbiomes](https://image.slidesharecdn.com/2341781-250524103414-227d46be/85/Engineering-And-Analyzing-Multicellular-Systems-Methods-And-Protocols-1st-Edition-Lianhong-Sun-24-320.jpg)
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Approaches to study the function of human-associated microbes
by genetic manipulation rely on several fundamental capabilities,
which are often the largest practical barriers to manipulate microbes
genetically. First, individual microbes need to be isolated and cul-
tured in the laboratory. Because microbes have a myriad of physi-
ologies and require different nutritional supplement for growth,
different media compositions and growth conditions need to be
laboriously tested by trial and error to isolate and culture each
microbe. These microbial culturing techniques date back to the
times of Louis Pasteur and are still the dominant approach today.
More recent microbial cultivation techniques use microfluidics and
droplet technologies to enable the discovery of synergistic interac-
tions between natural microbes that allow otherwise “unculturable”
organisms to be grown in laboratory conditions [7, 81, 82].
Upon successful microbial cultivation, the next limiting step of
microbial genetic manipulation is the transformation of foreign DNA
into cells. The passage of foreign DNA (e.g., plasmids, recombinant
fragments) into the cell requires overcoming the physical barriers
presented by the cell wall or membrane. This task is accomplished in
nature through processes such as transduction by phage, conjugation
and mating, or natural competency and DNA uptake [83, 84].
Numerous laboratory techniques have been developed for microbial
transformation including electroporation [85], biolistics [86], soni-
cation [87], and chemical or heat disruption [88]. Electroporation,
the most common of the laboratory transformation techniques,
relies on high-voltage electrocution of the bacterial sample that is
thought to transiently induce pores on the cell membrane (hence
“electroporation”) that then enable extracellular DNA to diffuse
into the cell. Various protocols for electroporation of human-associ-
ated microbes have been described and are good starting points for
developing genetic systems in these microbes [89, 90].
Upon transformation of DNA into the cell, the DNA needs to
either stably propagate intracellularly or integrate into the micro-
bial host genome through recombination or other integration
strategies. Inside the cell, stable propagation of episomal DNA
such as plasmids requires DNA replication machinery that is com-
patible with the foreign DNA [83]. Additionally, cells often use
methylation and DNA modification and restriction systems to dis-
cern foreign versus host DNA through a primitive defensive mech-
anism that fights against viruses or other invading genetic elements.
Nonetheless, these promiscuous genetic elements can often be
used as a way to integrate foreign DNA into the chromosome and
are often used for large-scale functional genomics [91].
Taking all these parameters into consideration, we have
summarized (Fig. 5) the current genetic tractability of human-
associated microbes with respect to culturability, availability of full
genome sequences, transfection methods, and expression and
manipulation systems. Expansion of these basic genetic tools is
crucial for future functional studies of human microbiota.
3.1 Challenges
of Building New
Genetic System
Engineering Human-Associated Microbiomes](https://image.slidesharecdn.com/2341781-250524103414-227d46be/85/Engineering-And-Analyzing-Multicellular-Systems-Methods-And-Protocols-1st-Edition-Lianhong-Sun-26-320.jpg)
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Fig. 5 Genetic tractability of abundant or relevant human-associated microbial genera, evaluated by the
availability of means to introduce genetic material (e.g., transformation, conjugation, or transduction), vectors,
expression systems, completed genomic sequences, and culturing methods. Circles of increasing sizes
indicate greater genetic tractability. Protocols and demonstrated methods for genetic manipulation
are listed as follows: (a) Clostridium: Phillips-Jones 1995, Jennert 2000, Young 1999, Bouillaut 2011
[181–184]; (b) Ruminococcus: Cocconcelli 1992 [185]; (c) Lactobacillus: van Pijkeren 2012, Ljungh 2009,
Damelin 2010, Sorvig 2005, Thompson 1996, Lizier 2010[107, 186–190]; (d) Enterococcus: Shepard 1995
[191]; (e) Lactococcus: Holo 1995, van Pijkeren 2012 [107, 192]; (f) Streptococcus: McLaughlin 1995, Biswas
2008 [193, 194]; (g) Staphlyococcus: Lee 1995 [195]; (h) Listeria: Alexander 1990 [196]; (i) Treponema:
Kuramitsu 2005 [197]; (j) Borrelia: Hyde 2011, Rosa 1999 [198, 199]; (k) Bifidobacterium: Mayo 2010 [200];
Stephanie J. Yaung et al.](https://image.slidesharecdn.com/2341781-250524103414-227d46be/85/Engineering-And-Analyzing-Multicellular-Systems-Methods-And-Protocols-1st-Edition-Lianhong-Sun-27-320.jpg)
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Genome-scale perturbations are a class of genetic approaches that
disrupt or perturb the expression of functional genes that contribute
to relevant phenotypes by individual microbes. To dissect the func-
tion of different genes in the cell, we have relied heavily on the use
of transposons, which are selfish genetic elements that can splice
into and out of different locations of chromosomal DNA, thereby
disrupting the coding sequence [92]. This classical approach,
known as transposon mutagenesis, has allowed us to isolate many
genetic mutants whose disrupted genes give rise to interesting
phenotypes that reflect the importance of those genes to its physiol-
ogy. Next-generation DNA sequencing has now enabled multi-
plexed genotyping of pools of transposon mutants by using
molecular barcodes that then can be applied to measure the effect
of genome-scale perturbations in different environmental condi-
tions. For example, techniques such as insertion sequencing (INSeq)
[93] utilize the inverted repeat recognition of the Himar trans-
posase, which is one nucleotide change away from the restriction
site for type II restriction enzyme MmeI, to generate paired
16–17 bp flanking genomic sequences around the transposon that
can be sequenced in pools. Thus, the defined insertion location of
every transposon in the library can be determined. By sequencing
this pooled mutant library pre- and posttreatment with any number
of environmental perturbations, one can probe the effects of differ-
ent gene disruptions on the physiology of the cell in a multiplexed
fashion. Similar techniques using other transposon systems such as
transposon sequencing (Tn-seq) [94], high-throughput insertion
tracking by deep sequencing (HITS) [95], and transposon-directed
insertion-site sequencing (TraDIS) [96] have also been developed.
In addition to transposon-based systems, shotgun expression
libraries have been useful in discovering functional DNA elements
in genomic or metagenomic DNA. Shotgun expression libraries
rely on physical shearing or restriction digestion of a donor DNA
source into smaller DNA fragments that are then cloned into a gene
expression vector and transformed into a host strain for functional
analysis. A library of metagenomic DNA samples can for example be
extracted from an environment and cloned into plasmids that are
then expressed in E. coli. Selection and sequencing of the E. coli
population for heterologous DNA that enable new function lead
to discovery of novel gene elements that perform a particular
3.2 Genome-Scale
Perturbations
Fig. 5 (continued) (l) Actinomyces: Yeung 1994 [201]; (m) Mycobacterium: Parish 2009, Sassetti 2001 [202,
203]; (n) Proprionibacterium: Luijk 2002 [204]; (o) Chlamydia: Binet 2009 [205]; (p) Porphyromonas: Belanger
2007 [206]; (q) Prevotella: Flint 2000, Salyers 1992 [207, 208]; (r) Bacteroides: Salyers 1999, Smith 1995,
Bacic 2008 [209–211]; (s) Fusobacterium: Haake 2006 [212]; (t) Helicobacter: Taylor 1992, Segal 1995 [213,
214]; (u) Camplyobacter: Taylor 1992 [214]; (v) Rickettsia: Rachek 2000 [215]; (w) Brucella: McQuiston 1995
[216]; (x) Bordetella: Scarlato 1996 [217]; (y) Neisseria: O’Dwyer 2005, Bogdon 2002, Genco 1984 [218–220];
(z) Pseudomonas: Dennis 1995 [221]
Engineering Human-Associated Microbiomes](https://image.slidesharecdn.com/2341781-250524103414-227d46be/85/Engineering-And-Analyzing-Multicellular-Systems-Methods-And-Protocols-1st-Edition-Lianhong-Sun-28-320.jpg)
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activity. This approach can easily identify activities such as antibiotic
resistance [97] but have yielded less success with other functions.
Towards forward engineering of human-associated microbes,
new genome engineering tools such as trackable multiplex recom-
bineering (TRMR) [98, 99] and multiplex automated genome
engineering (MAGE) enable efficient, site-specific modification of
the genome [100–103]. TRMR combines double-stranded
homologous recombination [104] and molecular barcodes synthe-
sized from DNA microarrays to generate populations of mutants
that are trackable by microarray or sequencing. MAGE relies on
introduction of pools of single-stranded oligonucleotides that tar-
get defined locations of the genome to introduce regulatory muta-
tions [102] or coding modifications [105]. These and other
recombineering technologies are now being developed for a variety
of other organisms including gram-negative bacteria [106], lactic
acidbacteria[107],Pseudomonassyringae[108],andMycobacterium
tuberculosis [109], and are likely to be very useful for engineering
human-associated microbes.
The community of microbes that make up the human microbiome
can be considered a “pseudo-organ” of its own. These microbes
interact with one another and the mammalian host in potentially
highly complex ways that may be difficult to decipher even with
tractable genetic systems [110]. A direct approach to study these
interactions is to build reconstituted communities of microbes
derived from monoculture isolates in defined combinations. This
de novo reconstitution approach to build synthetic communities
has significant advantages over attempts to deconvolute natural
communities. Reconstituted synthetic consortium presents a trac-
table level of complexity in terms of number of interacting micro-
bial species that can be tracked by sequencing and predicted with
quantitative models. In one such study, researchers inoculated
germ-free mice with ten representative strains of the human micro-
biota [111]. The mice were then fed with defined diets of macro-
nutrients consisting of proteins, fats, polysaccharides, and sugars.
By tracking the abundance of the ten-member microbial consor-
tium using high-throughput sequencing, the researchers could
predict over 60 % of the variation in species abundance as a result
of diet perturbations. This avenue of investigation presents a viable
approach to study the human microbiome and ways to analyze
synthetically engineered microbiota.
Engineered microbes have been utilized to reconstitute syn-
thetic communities to investigate the role of metabolic exchange.
One such important metabolic exchange is that of amino acids, as
they are the essential constituents of proteins. Various syntrophic
cross-feeding communities have been described using auxotrophic
E. coli and yeast strains that require different amino acid
supplementation for growth [112–114]. In these syntrophic
3.3 Reconstituted
Communities
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systems, metabolites that are exchanged across different biosynthetic
pathways promote more syntrophic growth than those that are
exchanged along the same pathway, which also relates to the cost
of biosynthesis of the amino acid metabolites. Amino acid
exchange is likely a large player in driving metabolism of microbial
communities as a substantial fraction of all microbes are missing
biosynthesis of various metabolites and thus require growth on
more rich and complex substrates that are found in the gut [115].
New approaches are now utilizing synthetic biology to engineer
human-associated microbiota to improve health and metabolism as
well as to monitor and fight diseases. These efforts focus on devel-
oping genetic circuits that actuate in an engineered host cell such
as E. coli that can sense and respond to changes to its environment
and in the presence of particular pathogens. For example, to detect
the human opportunistic pathogen Pseudomonas aeruginosa, which
often causes chronic cystic fibrosis infections and colonizes the gas-
trointestinal tract, E. coli was engineered to detect the small diffus-
ible molecule that is excreted by P. aeruginosa through the quorum
sensing pathway [116]. An engineered synthetic circuit was placed
in nonpathogenic E. coli, which when placed in the presence of
high-density P. aeruginosa triggered a self-lysis program that
released a narrow-spectrum bacteriocin that specifically killed the
P. aeruginosa strain. Similar strategies have also been demonstrated
to detect and respond to Vibrio cholera infection using engineered
E. coli that sense autoinducer-1 (AI1) molecules from V. cholera
quorum sensing pathway [117]. These strategies appear to yield
improved survival rates against microbial pathogenesis in murine
models [117]. Quorum sensing systems, which normally help
microbes detect local cell density, have been further enhanced to
improve robustness and performance to enable coupled short-range
and long-range feedback circuits that enable microbial communica-
tion across large distances in an engineered community.
Other microbes have been successfully engineered to perform
specific functions on human-associated surfaces such as the muco-
sal layer of the gut epithelium. Numerous diseases that occur along
the intestinal tract are targets of such engineered approaches. For
example, the probiotic strain Lactococcus lactis has been engineered
to secrete recombinant human IL-10 in the gastrointestinal tract
to reduce colitis [118, 119]. Other future applications of engi-
neered probiotics include enhancing catabolism of nutrients (e.g.,
lactose and gluten), modulation of the immune system, and
removal of pathogens by selective toxin release [116].
To probe and engineer the human-associated microbial commu-
nity, various in vitro models have been developed, ranging from
traditional batch culturing in chemostats to microfluidic systems
that incorporate host cells. Single-vessel chemostats inoculated
3.4 Microbial
Engineering Through
Synthetic Biology
3.5 In Vitro
Host Models
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with fecal samples from healthy individuals have helped identify
HGT [120] and selective bacterial colonization on different carbo-
hydrate substrates [121, 122]. A multichamber continuous culture
system mimicking spatial, nutritional, and pH properties of differ-
ent GI tract regions can be used to investigate stabilization dynam-
ics [123–125]. Similarly, the constant-depth film fermenter
resembles oral biofilm [126] and has enabled studies on biofilm for-
mation, antibiotic resistance [126], and HGT in a multispecies oral
community [127, 128]. To incorporate mammalian cells in study-
ing host–microbial interactions, organ-on-a-chip microfluidic
devices have been recently used. In one version of such a system, a
gut-on-a-chip device, the microfluidic channel is coated with extra-
cellular matrix and lined by human intestinal epithelial (Caco-2)
cells. This system mimics intestinal flow and peristaltic motion,
recapitulates columnar epithelium polarization and intestinal villi
formation, and supports the growth of commensal Lactobacillus
rhamnosus GG [129]. These microdevices offer an opportunity to
investigate host–microbiota interactions in a well-controlled man-
ner and in physiologically relevant conditions.
Inoculating with native microbiota samples provides a method
to overcome the un-cultivability of many microbes as well as to
study collective activity and discover novel functions without a
priori knowledge of community composition. However, starting
with a predefined microbial community allows a controlled setting
better suited for testing engineered systems. In one study analyz-
ing the dynamics of a community representing the four main gut
phyla in a chemostat, the authors propose that intrinsic microbial
interactions, rather than host selective pressure, play a role in the
observed colonization pattern, which was similar to what has been
documented in the human gut [130]. Similar models have been
developed for oral microbiota studies. The use of predefined oral
microbial inocula has helped elucidate metabolic cooperation in
batch culture [12] and community development in saliva-conditioned
flow cells [131].
In order to move into in vivo animal models that more closely
represent the physiology of the human host environment, researchers
have extensively utilized murine models including germ-free, gno-
tobiotic, and conventionally raised mice. Gnotobiotic animals are
born in aseptic conditions and reared in a sterile environment
where they are exposed only to known microbial species; techni-
cally, germ-free mice are a type of gnotobiotic mice that have not
been exposed to any microbes. Similar to in vitro systems, mice can
be inoculated with either a natural microbiota sample or a pre-
defined microbial community. Fecal samples, as well as oral swab
and saliva samples, can then be collected from gnotobiotic mice for
biochemical analysis and species quantification of gut and oral
cavity microbiota. In vivo models have been used to study the
3.6 In Vivo
Host Models
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transmission of antibiotic resistance in the mouse gut [132, 133]
and colonization resistance in the oral cavity [134]. Furthermore,
the choice of the inoculum donor offers opportunities to compare
different host selection pressures and microbial community
responses. Microbiota can be transplanted from conventionally
raised to germ-free animals of not only the same species but also
interspecies, as in human microbiota into mouse, called humanized
gnotobiotic mice [134]. In one study, transplants from zebrafish
gut microbiota into germ-free mice and mouse gut microbiota
into germ-free zebrafish revealed that the resulting community
conformed to the native host composition, demonstrating host
selection [135].
Altering host diet, environment, or genetic background can
also enable studies in host–microbiota interactions. One method
to gain insight into the role of microbial communities in disease is
to utilize mice with recapitulated pathologies. For example,
IL-10−/−
, ob−/−
, apoE−/−
, and TLR2−/−
or TLR5−/−
mice are models
for colitis, obesity, hypercholesterolemia, and metabolic syndrome,
respectively [46, 136–139]. To generate antigen- or pathogen-
specific phenotypes, mice can be infected with Salmonella
typhimurium to study colitis [140] or Citrobacter rodentium as a
model for attaching and effacing pathogens, such as enterohemor-
rhagic E. coli [141, 142]. Furthermore, murine models with chem-
ically induced inflammation can be tools to study chronic mucosal
inflammation; dextran sodium sulfate (DSS) can induce ulcerative
colitis, and trinitrobenzene sulfonic acid (TNBS) can stimulate
Crohn’s disease [143]. To investigate oral microbiota, there are
periodontal disease [144] and oral infection models [145, 146];
gnotobiotic rodents can also be fed a high-sucrose cariogenic diet
to promote plaque formation.
Germ-free mice inoculated with defined microbes are informa-
tive models for analyzing microbial colonization and metabolic
adaptation [147]. For example, resident bacteria and probiotic
strains adapt their substrate utilization: in the presence of
Bifidobacterium longum, Bifidobacterium animalis, or Lactobacillus
casei, Bacteroides thetaiotaomicron diversified its carbohydrate uti-
lization by shifting metabolism from mucosal glycans to dietary
plant polysaccharides [148]. Furthermore, the effect of different
diets on microbial community composition can be studied, as in
gnotobiotic mice inoculated with ten sequenced gut bacterial
species and fed with various levels of casein, cornstarch, sucrose,
and corn oil to represent protein, polysaccharide, sugar, and fat
content in the diet, respectively [111].
Over the past several decades, a large number of theoretical and
quantitative models have been developed to describe the cell and
its behavior. Constrain-based models are used to describe
metabolism of individual cells using stoichiometric representation
3.7 Computational
Frameworks for
Human Microbiomics
Engineering Human-Associated Microbiomes](https://image.slidesharecdn.com/2341781-250524103414-227d46be/85/Engineering-And-Analyzing-Multicellular-Systems-Methods-And-Protocols-1st-Edition-Lianhong-Sun-32-320.jpg)
![16
of metabolic reactions and optimization constraints [149].
Approaches such as flux balance analysis (FBA) enable the analysis
of metabolism under steady-state assumptions by linear optimiza-
tion solution methods. These methods are now being scaled to
ecosystems of cells. Recent developments using multi-level objec-
tive optimization [150] and dynamic systems [151] enable the
modeling of synthetic ecosystems of three or more members.
Using metagenomic data of the gut microbiome, Greenblum
et al. generated a community-level metabolic reconstruction net-
work of the microbiota and discovered topological variations that
are associated with obesity and IBD, giving rise to low diversity
and differences in community composition [152]. For models
that account for systems dynamics, population abundance and
metabolite concentrations can be solved independently through
different FBA models that are iterated at each time step. This
approach called dynamic multi-species metabolic modeling
(DMMM) can capture scenarios of resource competition, leading
to the identification of limiting metabolites [153]. Other comple-
mentary models include elementary mode analysis (EMA) [154]
that enables quantitative analysis of microbial ecosystems in a mul-
ticellular fashion.
4 Perspectives
Reframing the microbiota community as a core set of genes, not a
core set of species, opens a new front to the microbiome engineering
design space. In a metagenomic study of 154 individuals, no single-
gut bacterial phylotype was detected at an abundant frequency
amongst all the samples, a finding that is consistent with the idea that
the core human gut microbiome may not be best defined by promi-
nent species but by abundantly shared genes and functions [155]. We
propose that manipulation at the gene, genome, and ultimately
metagenome level offers the ability for precise multicellular engineer-
ing of desirable traits in human-associated microbiota. Besides con-
trolledperturbationsofthemicrobiometoadvanceourunderstanding
of host–microbiota interactions, metagenome-scale tools enable
novel developments in diagnostics and therapeutics.
From biosensors on the skin to reporters in the gut, there are
several opportunities in monitoring the health and disease status of
the human host, such as sensing nutritional deficiencies, immune
imbalances,environmentaltoxins,orinvadingpathogens.Prophylactic
and therapeutic avenues for human microbiome engineering include
modifying community composition, tuning metabolic activity,
mediating microbe–microbe relationships, and modulating host–
microbe interactions. Two current microbiota-associated treatments
Stephanie J. Yaung et al.](https://image.slidesharecdn.com/2341781-250524103414-227d46be/85/Engineering-And-Analyzing-Multicellular-Systems-Methods-And-Protocols-1st-Edition-Lianhong-Sun-33-320.jpg)
![17
have shown clinical efficacy: (1) fecal transplants for recurrent
Clostridium difficile infection [156] and (2) probiotics for pouchitis,
which is inflammation of the ileal pouch that is created after surgical
removal of the colon in ulcerative colitis patients [157–159]. The
main challenge is transmission of undesirable agents from donor feces
to the recipient gut in fecal transplants and native colonization resis-
tance that would impair infiltration and growth of new species in pro-
biotics [160, 161]. Nevertheless, these successful approaches
demonstrate the potential benefits of leveraging natural microorgan-
isms and entire microbial communities.
In fact, coupling organismal and functional gene-level
approaches would be a powerful way to engineer the native micro-
biota. Microbiome engineering enables multiscale system design
for the synthesis of nutrients and vitamins, enhanced digestion of
gluten and lactose, decreased acidity of the oral cavity, targeted
elimination of multidrug-resistant pathogens, and microbial mod-
ulation of the host immune system. As vehicles for drug delivery,
commensal bacteria designed to secrete heterologous genes have
been explored for treating cancer [162–164], diabetes [165],
HIV [166], and IBD [118]. For example, IL-10 has immuno-
modulatory effects in IBD but requires localized delivery at the
intestinal lining to avoid the toxic side effects and low efficacy of
systemic IL-10 injection. Ingestion of modified Lactococcus lactis
that secrete recombinant IL-10 is safe and effective in animal
models and has been promising in human clinical trials for IBD
[119, 167].
Finally, besides addressing clinical safety and efficacy criteria
for FDA regulatory approval [168], overall safety precautions
are critical considerations to minimize unintentional risks in
releasing genetically modified material into the natural environ-
ment. Rational design, such as creating auxotrophic microbes
[119], for robust stability, non-pathogenicity, and containment
of recombinant genetic systems will be essential in microbiome
engineering.
Acknowledgements
H.H.W. acknowledges the generous support from the National
Institutes of Health Director’s Early Independence Award (grant
1DP5OD009172-01). S.J.Y. acknowledges support from the
National Science Foundation Graduate Research Fellowship and
the MIT Neurometrix Presidential Graduate Fellowship. G.M.C.
acknowledges support from the Department of Energy Genomes
to Life Center (Grant DE-FG02-02ER63445).
Engineering Human-Associated Microbiomes](https://image.slidesharecdn.com/2341781-250524103414-227d46be/85/Engineering-And-Analyzing-Multicellular-Systems-Methods-And-Protocols-1st-Edition-Lianhong-Sun-34-320.jpg)
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Lianhong Sun and Wenying Shou (eds.), Engineering and Analyzing Multicellular Systems: Methods and Protocols,
Methods in Molecular Biology, vol. 1151, DOI 10.1007/978-1-4939-0554-6_2, © Springer Science+Business Media New York 2014
Chapter 2
Constructing Synthetic Microbial Communities
to Explore the Ecology and Evolution of Symbiosis
Adam James Waite and Wenying Shou
Abstract
Synthetically engineered microbial communities based on model organisms provide a simplified model of
their naturally occurring counterparts while still retaining essential features of living organisms. The degree
of control afforded by this approach has been critical in understanding how similar types of natural com-
munities might have persisted and evolved. Here, we first discuss important considerations when designing
a synthetically engineered system. Then, we describe the steps required to create a two-partner cooperative
system based on the yeast Saccharomyces cerevisiae.
Key words Evolution, Ecology, Mutualism, Cooperation, Synthetic biology, S. cerevisiae
1 Introduction
From mediating biogeochemical cycles [1] to influencing human
health [2] and disease [3], microbial communities impact all
aspects of life on earth. However, the complexity of microbial com-
munities and the difficulty in isolating and culturing microbes [4]
pose serious challenges for decoding cell–cell and cell–environment
interactions. Moreover, the evolutionary histories of microbial
communities are difficult to retrace. Alternatively, communities of
model organisms engineered to engage in defined interactions can
be deployed to address fundamental questions in ecology and evolu-
tion, such as how species coexist and coevolve [5]. In this chapter,
we discuss several considerations when designing a synthetic
community, using the construction of a two-partner cooperative
yeast system as an example. Then, we describe the methodology
in detail.
The initial consideration is the choice of organisms. Genetic
tractability and short generation times facilitate strain construction
and experimentation, as well as the discovery and interpretation of
mutations during experimental evolution. Thus, well-studied model
organisms with reference genome sequences such as Escherichia coli](https://image.slidesharecdn.com/2341781-250524103414-227d46be/85/Engineering-And-Analyzing-Multicellular-Systems-Methods-And-Protocols-1st-Edition-Lianhong-Sun-44-320.jpg)
![28
or Saccharomyces cerevisiae are ideal, although other species have
also been used [5]. While each model organism has its advantages
and disadvantages, we will use S. cerevisiae to highlight principles
that are applicable to any synthetically engineered community.
Sexual recombination is both a help and a hindrance to syn-
thetically engineered communities. On the one hand, sexual
recombination can radically simplify strain construction by allow-
ing genetic shuffling. For example, when a strain with genotype
AB is crossed with a strain of genotype ab, recombinant strains
Ab and aB can be generated. On the other hand, distinct popula-
tions in an engineered community should remain genetically
insulated from one another and therefore should not be allowed
to mate. An advantage of using S. cerevisiae is its ability to
undergo both sexual and asexual reproduction. Haploid yeast is
particularly suitable for evolution experiments since phenotypes
arising from recessive mutations are immediately apparent.
Haploid yeast can reproduce asexually as either of the two mating
types, “a” or “α.” Two cells of opposite mating type can mate to
produce an a/α diploid, which can reproduce either asexually as
diploids or sexually to form haploids. The final strains for an engi-
neered community should always be the same mating type to pre-
vent sexual recombination. However, haploid yeast switch mating
types spontaneously at very low frequency even when the gene
required for mating-type switching (HO) is defective (which is the
case for all commonly used laboratory strains). Thus, we have used
MATa cells in which the STE3 gene encoding the receptor for
MATa mating pheromone [6] is deleted. Thus, if a MATa ste3 cell
switches mating type to MATα ste3, it will fail to initiate the
mating process.
Ideally, all strains should be derived from an isogenic back-
ground so that the only mutations are the ones defined by the
researcher. We have used the strain S288C, which is one of the
common laboratory strains, and the wild vineyard isolate RM11-1a
(hereafter referred to as “RM11”). While S288C has its genome
fully annotated and easily accessible [7], it has a tendency to pro-
duce mitochondrially deficient “petite” cells [8], which are prone
to nuclear genome instability [9] and could potentially interfere
with evolution experiments. RM11 (genome sequence available at
http:/
/www.broadinstitute.org/annotation/genome/saccharomyces_
cerevisiae/) grows faster than S288C and produces very few
petites, but haploid daughter cells do not separate well from their
mothers unless the RM-11 AMN1 allele is replaced by the AMN1
allele from S288C [10]. Genetic manipulation in RM-11 is more
difficult due to its lower transformation efficiency compared to
S288C.
A major advantage of using model organisms is that they are
genetically modifiable. Foreign DNA can be transformed into yeast
as autonomously replicating and segregating circular plasmids or as
Adam James Waite and Wenying Shou](https://image.slidesharecdn.com/2341781-250524103414-227d46be/85/Engineering-And-Analyzing-Multicellular-Systems-Methods-And-Protocols-1st-Edition-Lianhong-Sun-45-320.jpg)
![29
linear DNA if genomic integration is desired. Integration is more
stable than using a plasmid. Different populations can be marked
with, for example, different antibiotic resistances or fluorescent
proteins. Currently, at least six dominant antibiotic resistance genes
are in wide use with S. cerevisiae: kan, which confers resistance to
geneticin a.k.a. G418; hph, which confers resistance to hygromycin
B; nat, which confers resistance to nourseothricin (sold as clon-
NAT by Werner BioAgents); pat, which confers resistance to
phosphinothricin; ble, which confers resistance to phleomycin;
and AUR1-C, which confers resistance to Aureobasidin A (AbA).
All of these genes are available on plasmids [11–13]. Our lab has
used G418, hygromycin B, clonNAT, and AbA resistance markers.
Using antibiotic resistance to select a subpopulation from a
co-culture is especially useful if the subpopulation is very rare, as
tens of millions of cells can be assayed on a single plate. However,
plating on media supplemented with different drugs to determine
subpopulation abundance is time-delayed since it takes at least 1 day
for colonies to grow up. It is also of low throughput due to the
small number (hundreds) of individual colonies that can be counted
on a plate. In contrast, fluorescently tagged strains can be distin-
guished using flow cytometry, which allows tens of thousands of
cells to be counted in less than a minute. While fluorescence-
activated cell sorting (FACS) can also be used to isolate subpopula-
tions, it requires an expensive instrument and is less efficient when
isolating very rare subpopulations. A large number of fluorescent
proteins are available [14], although not all of them are bright
and/or resolvable from one another using standard filter sets.
In addition, the correct folding of fluorescent proteins requires
oxygen [15] and is therefore incompatible with strict anaerobes.
We have C-terminally tagged (Fig. 1) the highly abundant proteins
FBA1 or MET6 with different fluorescent proteins in yeast. Using
the appropriate combination of lasers and filter sets (Table 1),
we can resolve mixtures of cells expressing five different fluores-
cent proteins: CFP, GFP, YFP, mOrange, and mCherry as well as
the far-red nucleic acid dye TO-PRO-3 (Invitrogen) which can be
used to stain dead cells. Plasmids containing different combina-
tions of fluorescent proteins and selectable markers (i.e., genes
for nutrient biosynthesis or antibiotic resistance) [16] are readily
available from EUROSCARF (http:/
/web.uni-frankfurt.de/
fb15/mikro/euroscarf/).
Since deleting or inserting genes will usually be accomplished
by transformations (Fig. 1), which require selectable markers, it is
convenient to be able to reuse these markers. Plasmids containing
kanMX [17] and ble [18] flanked by loxP sites are available for this
purpose (for a comprehensive list of plasmids with removable
markers, see ref. 19). In the presence of Cre recombinase, the two
loxP sites recombine, removing the intervening marker and allow-
ing for its reuse in another round of manipulation. Our lab has
Constructing Synthetic Microbial Communities](https://image.slidesharecdn.com/2341781-250524103414-227d46be/85/Engineering-And-Analyzing-Multicellular-Systems-Methods-And-Protocols-1st-Edition-Lianhong-Sun-46-320.jpg)
![31
integration of the loxP–drug–loxP cassette could be carried out in
a ura3 strain so that transformation of a URA3-marked plasmid
containing the Cre recombinase can be selected for. After induction
of Cre expression and removal of the drug marker, the URA3
plasmid can then be counterselected using 5-FOA, which only
allows survival of cells without the plasmid [20]. The uracil auxot-
rophy may then need to be removed via genetic crosses.
Alternatively, Cre expression plasmids containing antibiotic mark-
ers are also available [19].
Once the populations have been marked, interactions between
populations can be defined. Obviously, the possibilities are practi-
cally limitless, and the specifics must be left to the researcher.
We chose to base our two-partner cooperative system on comple-
mentary nutrient exchange [21].
In yeast, all of the manipulations described above can be achieved
through a small set of well-known methods [19]. These include
(1) transformation to insert or remove genetic material; (2) colony
PCR, a simple and rapid way to check whether transformation was
successful; and (3) mating, sporulation, tetrad dissection, and geno-
typing, which allows genetic features present in two different strains
to be recombined into one strain. Below we describe our current
protocols for each of these methods.
2 Materials
1. Plasmids containing genes encoding fluorescent proteins and/
or selectable markers.
2. Antibiotics: 1,000× stock G418 (200 mg/ml), 500x stock
hygromycin B (100 mg/ml), 1,000× stock clonNAT (100 mg/
ml), 1,000× stock AbA (0.5 mg/ml) (see Note 1).
3. YPD: 10 g/l Bacto-yeast extract, 20 g/l Bacto-peptone, and
bring to volume with diH2O to final 950 ml/l for later glucose
supplement. Add 20 g/l Bacto-agar (see Note 2) if making
plates. Add a magnetic stir bar, and autoclave. Using a flame to
ensure sterility, add 50 ml 40 % glucose per liter of medium and
the appropriate antibiotic, if necessary. Stir to mix. If multiple
liters of agar medium are prepared, they may be kept at 50 °C
water bath to prevent solidification of the agar.
4. SD: For liquid media, add 6.7 g/l Difco™ yeast nitrogen base
(YNB) with ammonium sulfate and without amino acids and
20 g/l glucose, and bring to volume with diH2O. Sterilize
using 0.22 μm filter. For plates, add 6.7 g/l YNB, 20 g/l
Bacto-agar, and a stir bar, and add diH2O to 950 ml/l.
Autoclave. Using sterile technique, add supplements as neces-
sary. Amino acid and nucleobase supplements can be mixed in
appropriate proportions [19] in their powder forms in a
2.1 Components for
Genetic Manipulation
Constructing Synthetic Microbial Communities](https://image.slidesharecdn.com/2341781-250524103414-227d46be/85/Engineering-And-Analyzing-Multicellular-Systems-Methods-And-Protocols-1st-Edition-Lianhong-Sun-48-320.jpg)
![32
sterilized blender and stored at room temperature. Powdered
supplements can be weighed and directly added to the media.
Finally, add 50 ml 40 % glucose. Use the glucose to wash
down any residual supplement powder adhering to the vessel
wall and stir.
5. 50 % PEG 2000 (see Note 3): Dissolve 100 g PEG in 100 ml
diH2O. Bring to 200 ml with diH2O. Sterile filter.
6. 1 M LiAc: 102 g/l Lithium acetate dihydrate (102.02 g/mol).
Sterile filter.
7. 5 mg/ml Sheared salmon sperm DNA (SS-DNA) [22].
8. Autoclaved water, tubes, and tips.
1. Sporulation media: 3 g/l potassium acetate, 0.2 g/l raffinose,
bring to final volume with diH2O. Autoclave.
2. SCE buffer: 1 M D-sorbitol, 0.1 M sodium citrate (see Note 4),
60 mM EDTA. Adjust pH to 7.0 with 38 % HCl (see Note 5).
Autoclave using a 20′ sterilization cycle, and remove promptly
(see Note 6).
3. Zymolyase 20T: 30 mg zymolyase 20T dissolved in 10 ml SCE
(see Note 7).
3 Methods
1. Design primers: For gene replacement (Fig. 1), the forward
primer should contain 45 base pairs (bp) of homology to the
genomic region immediately upstream of the “ATG” codon of
the open reading frame (ORF) of the gene to be knocked out,
followed by the sequence for the universal forward adapter
appropriate for the particular set of plasmids being used.
The reverse primer should contain the reverse complement to
the 45 bp including and immediately downstream of the stop
codon of the target ORF, followed by the reverse complement
to the universal reverse adapter appropriate for the plasmid set.
For C-terminal tagging (Fig. 1), the reverse primer is designed
as in gene replacement, and the forward primer should contain
45 bp homology to the sequence just 5′ of the stop codon of
the gene of interest.
2. PCR amplify cassette (antibiotic resistance for gene replacement
or fluorescent protein plus selectable marker for C-terminal
tagging) off a plasmid (see Note 8). Check the length of the
PCR product using gel electrophoresis.
1. Inoculate 5 ml YPD culture per transformation and shake at
30 °C until the cell density is between 3×106
and 2×107
cells/ml
(see Note 10).
2.2 Components for
Mating, Sporulation,
and Tetrad Dissection
3.1 Primer Design
and Amplification
for Gene Tagging
or Replacement
3.2 Transformation
(See Note 9)
Adam James Waite and Wenying Shou](https://image.slidesharecdn.com/2341781-250524103414-227d46be/85/Engineering-And-Analyzing-Multicellular-Systems-Methods-And-Protocols-1st-Edition-Lianhong-Sun-49-320.jpg)
![34
1. Design primers: One primer should be specific to the cassette
used for transformation (for example, the universal primer
sequence). The other primer should be outside the 45 bp
homology region used for integration (Fig. 1).
2. Using a sterile pipette tip, pick about half of a normal-sized
(~1.2 mm diameter) colony without touching the agar beneath
it. For S288C, place directly into 15 μl sterile water, vortex, and
use 1 μl in a 20 μl PCR (see Note 20). For RM11, transfer the
cells to 15 μl 0.25 % SDS (see Note 21). Vortex for 30 s, spin at
top speed (~17,949×g in a small centrifuge) for 1 min, and use
1 μl of the supernatant for a 20 μl PCR. This PCR mix must
contain a final concentration of 5 % Triton X-100 to neutralize
the protein-denaturing SDS [23]. An alternative, and more
reliable, method uses LiAc and SDS to lyse cells and requires
ethanol precipitation prior to PCR (see Note 19, ref. 22).
Specifically, cells are suspended in 100 μl 200 mM LiAc and
1 % SDS solution and incubated at 70 °C for 15 min. 300 μl
96 % ethanol is added to precipitate DNA. After brief vortexing,
DNA is collected by centrifugation at 15,000×g for 3 min.
Precipitated DNA is dissolved in 100 μl TE (10 mM Tris–HCl,
1 mM disodium EDTA, pH 8.0). After spinning cell debris down
at 15,000×g for 1 min, 1 μl supernatant is used for PCR.
1. Grow up a small patch of each haploid to be crossed towards
the top of a YPD plate (see Note 22).
2. Use a sterile toothpick to transfer a tiny amount of one strain
to an empty region of a YPD plate (see Note 23), making a
small spot (a few millimeters in diameter). With a fresh tooth-
pick, transfer a similarly tiny amount of the other strain to the
same spot and mix with the toothpick.
3. Incubate for 3.5 h (S288C) or 2.5 h (RM11) at 30 °C
(see Note 24).
4. Using a toothpick, touch the mixed spot and streak down
about a centimeter. Without re-touching the spot, make similar
streaks to the left and right of the original streak. This gives
three different dilutions of cells on the plate.
5. Use a yeast dissection microscope [19] to isolate diploids.
The cytoplasmic “bridge” between mated cells and a small bud
at its center is indicative of recently mated diploids.
1. Pre-sporulate by patching onto YPD and incubating at 30 °C
for ~10 h until a thin film of cells is formed (see Note 25).
2. Inoculate 2 ml sporulation media with about a match head’s
worth of cells. Incubate at room temperature on a rotator for
2–5 days. Check for tetrads using a light microscope using a
20–40× magnification objective [19].
3.3 Colony PCR
(See Note 19)
3.4 Mating and
Diploid Isolation
3.5 Sporulation,
Tetrad Dissection,
and Genotyping
Adam James Waite and Wenying Shou](https://image.slidesharecdn.com/2341781-250524103414-227d46be/85/Engineering-And-Analyzing-Multicellular-Systems-Methods-And-Protocols-1st-Edition-Lianhong-Sun-51-320.jpg)
![35
3. Wash cells with 1 ml sterile water and resuspend in 1 ml sterile
water. Store at 4 °C.
4. When ready to dissect, spin down 20 μl of sporulated cells and
carefully remove supernatant with a pipette. Add 20 μl SCE,
and vortex to resuspend cells. If using RM11, sonicate for
1 min.
5. Add 4 μl zymolyase 20T, and mix by pipetting up and down
(see Note 26). Incubate at room temperature for 20 min
(S288C) or 2 h (RM11) (see Note 27).
6. Gently (to avoid breaking up tetrads) pipette up digested cell
suspension and spot onto the top portion of the center strip of
a YPD plate. Tilt plate down so that the liquid rolls down the
central strip, stopping before the liquid touches the plate wall.
Let dry.
7. Use a yeast dissection microscope to separate tetrads into indi-
vidual spores. Create a grid with a maximum of two tetrads per
row [19], one to the left and one to the right of the central
strip.
8. Incubate overnight at 30 °C. Use a flame-sterilized scalpel to
remove the strip of cells from the middle of the plate (see Note 28)
and return to incubator.
9. Once the spores have germinated and grown into colonies,
replica plate onto the appropriate selective media(s). Note that
a single genetic locus should, under most circumstances, seg-
regate 2:2 [19]. For instance, two spores will be MATa and
two will be MATα. Exceptions can be caused by, for exam-
ple, gene conversion or traits that are mediated by heritable
materials transmissible through the cytoplasm, such as
mitochondria.
10. Mating types can be tested using a pair of mating-type testers,
one of each mating type. If the entire collection of spores are
auxotrophic because of mutations in a set of genes, then the
testers should be auxotrophic due to mutation in a different
gene to ensure that the resulting diploids are prototrophic. In
this case, spread ~200 μl of a saturated culture of each tester
strain on its own YPD plate and let dry. Then, replica plate the
target strains onto each plate using a sterile velvet. After half a
day, replica each plate onto an SD plate. Growth will only
occur if the strains are able to mate. If spores are prototrophic,
they need to be separately mated to each of the two tester
strains, as described above for diploid isolation. Many crosses
can be set up on one YPD plate. The mating type of a strain is
revealed by the presence or the absence of fused diploids. If this
method is necessary, it is better to narrow down the number of
strains to be tested based on other markers first.
Constructing Synthetic Microbial Communities](https://image.slidesharecdn.com/2341781-250524103414-227d46be/85/Engineering-And-Analyzing-Multicellular-Systems-Methods-And-Protocols-1st-Edition-Lianhong-Sun-52-320.jpg)
![36
4 Notes
1. Prepare stocks in deionized (di) H2O and sterile filter (except
for AbA, which should be made in ethanol and needs no sterile
filtration) and keep at −20 °C.
2. Not all sources of YNB and agar are appropriate for use with yeast.
For example, we have found that YNB and agar obtained from BD
biosciences work better than those obtained from USA Scientific.
3. The size of PEG is important. Transformation efficiency for
RM11 is very low if PEG 3500 is used. We use PEG3500 to
transform S288C, although PEG2000 should work as well.
4. NaH2PO4 can be used instead.
5. About 0.5 ml when making a final volume of 500 ml.
6. Ensure that the color of solution did not change during the
autoclave process.
7. Freeze down 1 ml aliquots at −20 °C. To use, thaw one tube and
make smaller aliquots. Store one at 4 °C for up to 2 months,
and freeze the rest for later use.
8. Always use a high-fidelity polymerase for PCR to minimize the
possibility of introducing mutations into the amplified fragment.
Here is a specific recipe for C-terminally tagging FBA1 using the
pKT plasmids [16] and KOD polymerase (EMD Millipore):
0.5 μl miniprep DNA (~100 ng), 5 μl 10x buffer, 4 μl 25 mM
MgSO4, 5 μl 8 mM dNTPs, 0.5 μl primer WSO178 (50 μM),
0.5 μl primer WSO179 (50 μM), 0.5 μl KOD
polymerase, 34 μl water (molecular biology grade). WSO178
sequence: 5′-AAGATCACCAAGTCTTTGGAAACTTTCC
G T A C C A C T A A C A C T T T A g g t g a c g g t g c t g g t t
ta-3′. WSO179 sequence: 5′-GATTCAATACTCATTAAAAA
ACTATATCAATTAATTTGAATTAACtcgatgaattcgagctcg-3′.
The 45 bp homology sequence is uppercase; the universal primer
sequence is lowercase. PCR settings: 94 °C for 2 min, 30 cycles
of {94 °C for 30 s, 55 °C for 30 s, and 70 °C for 3 min}, and
then 70 °C for 10 min. When nat is the template, it is essential
to add DMSO to a final concentration of 5 % [11].
9. Transformation is mutagenic. It is therefore best to transform
diploids and then sporulate, since one round of meiotic segrega-
tion reduces the probability of obtaining an undesired mutation
by 50 %. If multiple haploids of the desired genotype derived
from the same diploid behave similarly, then background muta-
tions are unlikely to be important. Alternatively, our lab has
found that Illumina deep sequencing can reveal the presence of
mutations caused by transformation.
10. To ensure that cultures are in exponential phase when it is
time to transform, pilot growth experiments may be helpful.
Wild-type RM11 grows very rapidly. If the density is >5×107
Adam James Waite and Wenying Shou](https://image.slidesharecdn.com/2341781-250524103414-227d46be/85/Engineering-And-Analyzing-Multicellular-Systems-Methods-And-Protocols-1st-Edition-Lianhong-Sun-53-320.jpg)
![37
cells/ml, dilute to allow the cells to complete at least two
divisions in unsaturated conditions. Transformation efficiency
remains constant for 3–4 cell divisions.
11. Keep small aliquots of SS-DNA to limit the number of freeze–
thaw cycles. Keep on ice when out of the freezer.
12. Keeping the TRAFO master mix on ice is crucial for high
efficiency.
13. Use >100 ng DNA. In general, more DNA yields more transfor-
mants, but this relationship will likely saturate at some point.
14. This can be visually confirmed by ensuring that no visible
“strands” are present in the mixture.
15. TRAFO is very viscous, so pipette slowly to ensure that the
correct volume is transferred.
16. Mixing well ensures that the SS-DNA effectively blocks non-
specific DNA binding.
17. Adjusting the amount of time for heat shock may be necessary
to achieve maximum efficiency. However, 40 min works well
for S288C and RM11.
18. Be as gentle as possible at this step, as the cells are very fragile.
19. Colony PCR is quick but can be unreliable. When it fails to
work, we have found that a quick DNA extraction before PCR
gives reliable results [23].
20. The cell suspension should be turbid.
21. Unlike S288C, boiling of RM11 cells does not provide good tem-
plate for PCR, although we do not know why. Using detergent
effectively lyses the cells and releases their DNA into solution.
22. This can be done for as little as 3 h.
23. The amount should be small enough to not leave a visible film
on the plate after transfer.
24. Different strains may require more or less time. Cells should
show visible film of growth by this time.
25. Overgrowth will lead to a reduction in sporulation efficiency.
However, a suitable number of tetrads should be present even
after 12–14 h of pre-sporulation.
26. Vortexing can oxidize the zymolyase.
27. The four spores form a three-dimensional, tetrahedral shape if
the ascus wall is undigested. After sufficient digestion, the four
spores will have a flat, diamond shape. Underdigestion of the
ascus wall will make it difficult to separate the individual spores.
Overdigestion will result in tetrads that break apart easily,
increasing the chances that four spores in the correct diamond
shape are not products of the same meiosis. Overdigestion can
also reduce spore viability.
28. Otherwise, growth of this strip will slow down the growth of
the haploids nearest to it.
Constructing Synthetic Microbial Communities](https://image.slidesharecdn.com/2341781-250524103414-227d46be/85/Engineering-And-Analyzing-Multicellular-Systems-Methods-And-Protocols-1st-Edition-Lianhong-Sun-54-320.jpg)
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- 5. Engineering andAnalyzing Multicellular Systems Lianhong Sun Wenying Shou Editors Methods and Protocols Methods in Molecular Biology 1151
- 6. ME T H O D S I N MO L E C U L A R BI O LO G Y Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK For further volumes: http://www.springer.com/series/7651
- 8. Engineering and Analyzing Multicellular Systems Methods and Protocols Edited by Lianhong Sun School of Life Sciences, University of Science & Technology of China, Hefei,Anhui,People’sRepublicofChina Wenying Shou Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA, USA
- 9. ISSN 1064-3745 ISSN 1940-6029 (electronic) ISBN 978-1-4939-0553-9 ISBN 978-1-4939-0554-6 (eBook) DOI 10.1007/978-1-4939-0554-6 Springer New York Heidelberg Dordrecht London Library of Congress Control Number: 2014934331 © Springer Science+Business Media New York 2014 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. Exempted from this legal reservation are brief excerpts in connection with reviews or scholarly analysis or material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Duplication of this publication or parts thereof is permitted only under the provisions of the Copyright Law of the Publisher’s location, in its current version, and permission for use must always be obtained from Springer. Permissions for use may be obtained through RightsLink at the Copyright Clearance Center. Violations are liable to prosecution under the respective Copyright Law. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. While the advice and information in this book are believed to be true and accurate at the date of publication, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper Humana Press is a brand of Springer Springer is part of Springer Science+Business Media (www.springer.com) Editors Lianhong Sun School of Life Sciences University of Science & Technology of China Hefei, Anhui, People’s Republic of China Wenying Shou Division of Basic Sciences Fred Hutchinson Cancer Research Center Seattle, WA, USA
- 10. v Microbial ecosystems consist of many interacting microbial species. With synthetic biology rapidly evolving from engineering genetic circuits within cells to manipulating cell–cell interactions, several synthetic microbial communities have been constructed. Such synthetic communities have been used in basic research to explore questions such as how interactions within a community shape the stability, function, patterning, and evolution of the community. In addition, synthetic communities have been constructed to solve chal- lenging engineering problems in various fields. As a consequence, a framework of engineer- ing synthetic microbial ecosystems/consortia is of importance to many users. Equally important are the transcriptomic, genomic, cell biological, and chemical methods to char- acterize communities. Ultimately, to quantitatively understand complex microbial commu- nities, predictive mathematical models can be extremely useful. This volume of Methods in Molecular Biology includes recent developments and a variety of examples on how to con- struct, analyze, and mathematically model multicellular systems. Hefei, People’s Republic of China Lianhong Sun Seattle, WA, USA Wenying Shou Preface
- 12. vii Contents Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix PART I CONSTRUCTING MULTICELLULAR SYSTEMS 1 Recent Progress in Engineering Human-Associated Microbiomes . . . . . . . . . . 3 Stephanie J. Yaung, George M. Church, and Harris H. Wang 2 Constructing Synthetic Microbial Communities to Explore the Ecology and Evolution of Symbiosis. . . . . . . . . . . . . . . . . . . . . 27 Adam James Waite and Wenying Shou 3 Combining Engineering and Evolution to Create Novel Metabolic Mutualisms Between Species . . . . . . . . . . . . . . . . . 39 Lon Chubiz, Sarah Douglas, and William Harcombe 4 Design, Construction, and Characterization Methodologies for Synthetic Microbial Consortia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 Hans C. Bernstein and Ross P. Carlson 5 An Observation Method for Autonomous Signaling-Mediated Synthetic Diversification in Escherichia coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 Ryoji Sekine, Shotaro Ayukawa, and Daisuke Kiga 6 Integration-Free Reprogramming of Human Somatic Cells to Induced Pluripotent Stem Cells (iPSCs) Without Viral Vectors, Recombinant DNA, and Genetic Modification . . . . . . . . . . . . . . . . . . . . . . . . 75 Boon Chin Heng and Martin Fussenegger 7 Transformation of Bacillus subtilis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 Xiao-Zhou Zhang, Chun You, and Yi-Heng Percival Zhang 8 Culturing Anaerobes to Use as a Model System for Studying the Evolution of Syntrophic Mutualism. . . . . . . . . . . . . . . . . . . . 103 Sujung Lim, Sergey Stolyar, and Kristina Hillesland 9 Therapeutic Microbes for Infectious Disease . . . . . . . . . . . . . . . . . . . . . . . . . . 117 Choon Kit Wong, Mui Hua Tan, Bahareh Haji Rasouliha, In Young Hwang, Hua Ling, Chueh Loo Poh, and Matthew Wook Chang PART II ANALYZING AND MODELING MULTICELLULAR SYSTEMS 10 Quantitative Measurement and Analysis in a Synthetic Pattern Formation Multicellular System. . . . . . . . . . . . . . . . . . . 137 Xiongfei Fu and Wei Huang 11 Transcriptome Analysis of a Microbial Coculture in which the Cell Populations Are Separated by a Membrane. . . . . . . . . . . . . . 151 Kazufumi Hosoda, Naoaki Ono, Shingo Suzuki, and Tetsuya Yomo
- 13. viii 12 Identification of Mutations in Laboratory-Evolved Microbes from Next-Generation Sequencing Data Using breseq . . . . . . . . . . . . . . . . . . . . . 165 Daniel E. Deatherage and Jeffrey E. Barrick 13 3D-Fluorescence In Situ Hybridization of Intact, Anaerobic Biofilm . . . . . . . . 189 Kristen A. Brileya, Laura B. Camilleri, and Matthew W. Fields 14 The Characterization of Living Bacterial Colonies Using Nanospray Desorption Electrospray Ionization Mass Spectrometry. . . . . . . . . . . . . . . . . . 199 Brandi S. Heath, Matthew J. Marshall, and Julia Laskin 15 Modeling Community Population Dynamics with the Open-Source Language R . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209 Robin Green and Wenying Shou 16 Simulating Microbial Community Patterning Using Biocellion. . . . . . . . . . . . . 233 Seunghwa Kang, Simon Kahan, and Babak Momeni Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255 Contents
- 14. ix SHOTARO AYUKAWA • Academy of Computational Life Sciences, Tokyo Institute of Technology, Kanagawa, Japan JEFFREY E. BARRICK • Department of Molecular Biosciences, Center for Systems and Synthetic Biology, Center for Computational Biology and Bioinformatics, Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, USA HANS C. BERNSTEIN • Department of Chemical and Biological Engineering, Center for Biofilm Engineering, Montana State University, Bozeman, MT, USA KRISTEN A. BRILEYA • Department of Microbiology, Center for Biofilm Engineering, Montana State University, Bozeman, MT, USA LAURA B. CAMILLERI • Department of Microbiology, Center for Biofilm Engineering, Montana State University, Bozeman, MT, USA ROSS P. CARLSON • Department of Chemical and Biological Engineering, Center for Biofilm Engineering, Montana State University, Bozeman, MT, USA MATTHEW WOOK CHANG • Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore LON CHUBIZ • Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA, USA GEORGE M. CHURCH • Department of Genetics, Harvard Medical School, Boston, MA, USA; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA DANIEL E. DEATHERAGE • Department of Molecular Biosciences, Center for Systems and Synthetic Biology, Center for Computational Biology and Bioinformatics, Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, USA SARAH DOUGLAS • Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA, USA MATTHEW W. FIELDS • Department of Microbiology, Center for Biofilm Engineering, Montana State University, Bozeman, MT, USA XIONGFEI FU • Department of Physics, The University of Hong Kong, Pokfulam, Hong Kong, China MARTIN FUSSENEGGER • Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland ROBIN GREEN • Molecular and Cellular Biology Program, University of Washington, Seattle, WA, USA; Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA, USA WILLIAM HARCOMBE • Department of Ecology, Evolution, and Behavior, University of Minnesota, St. Paul, MN, USA BRANDI S. HEATH • Physical Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA BOON CHIN HENG • Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland Contributors
- 15. x KRISTINA HILLESLAND • Biological Sciences Division, School of STEM, UW Bothell, Bothell, WA, USA KAZUFUMI HOSODA • Graduate School of Information Science and Technology, Osaka University, Suita, Osaka, Japan WEI HUANG • Department of Biology, South University of Science and Technology of China Nanshan, Shenzhen, China; Department of Biochemistry, The University of Hong Kong, Pokfulam, Hong Kong, China IN YOUNG HWANG • Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore SIMON KAHAN • Northwest Institute for Advanced Computing, University of Washington, Seattle, WA, USA SEUNGHWA KANG • Pacific Northwest National Laboratory, Seattle, WA, USA DAISUKE KIGA • Department of Computational Intelligence and Systems Science, Tokyo Institute of Technology, Kanagawa, Japan JULIA LASKIN • Physical Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA SUJUNG LIM • Biological Sciences Division, School of STEM, UW Bothell, Bothell, WA, USA HUA LING • Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore CHUEH LOO POH • School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore, Singapore MATTHEW J. MARSHALL • Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA BABAK MOMENI • Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA, USA NAOAKI ONO • Graduate School of Information Science, Nara Institute of Science and Technology, Ikoma, Nara, Japan BAHAREH HAJI RASOULIHA • School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore, Singapore RYOJI SEKINE • Department of Computational Intelligence and Systems Science, Tokyo Institute of Technology, Kanagawa, Japan WENYING SHOU • Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA, USA SERGEY STOLYAR • Institute for Systems Biology, Seattle, WA, USA SHINGO SUZUKI • RIKEN Quantitative Biology Center, Furuedai, Osaka, Japan MUI HUA TAN • School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore, Singapore ADAM JAMES WAITE • Molecular and Cellular Biology Program, University of Washington, Seattle, WA, USA; Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA, USA HARRIS H. WANG • Department of Systems Biology, Columbia University Medical Center, New York, NY, USA; Department of Pathology and Cell Biology, Columbia University Medical Center, New York, NY, USA CHOON KIT WONG • School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore, Singapore Contributors
- 16. xi STEPHANIE J. YAUNG • Program in Medical Engineering Medical Physics, Harvard-MIT Health Sciences and Technology, Cambridge, MA, USA; Department of Genetics, Harvard Medical School, Boston, MA, USA; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA TETSUYA YOMO • Graduate School of Information Science and Technology, Osaka University, Suita, Osaka, Japan; Graduate School of Frontier Bioscience, Osaka University, Suita, Osaka, Japan; Exploratory Research for Advanced Technology, Suita, Osaka, Japan CHUN YOU • Biological Systems Engineering Department, Virginia Tech, Blacksburg, VA, USA XIAO-ZHOU ZHANG • Biological Systems Engineering Department, Virginia Tech, Blacksburg, VA, USA; Gate Fuels Inc., Blacksburg, VA, USA YI-HENG PERCIVAL ZHANG • Biological Systems Engineering Department, Virginia Tech, Blacksburg, VA, USA; Gate Fuels Inc., Blacksburg, VA, USA Contributors
- 18. Part I Constructing Multicellular Systems
- 20. 3 Lianhong Sun and Wenying Shou (eds.), Engineering and Analyzing Multicellular Systems: Methods and Protocols, Methods in Molecular Biology, vol. 1151, DOI 10.1007/978-1-4939-0554-6_1, © Springer Science+Business Media New York 2014 Chapter 1 Recent Progress in Engineering Human-Associated Microbiomes Stephanie J. Yaung, George M. Church, and Harris H. Wang Abstract Recent progress in molecular biology and genetics opens up the possibility of engineering a variety of biological systems, from single-cellular to multicellular organisms. The consortia of microbes that reside on the human body, the human-associated microbiota, are particularly interesting as targets for forward engineering and manipulation due to their relevance in health and disease. New technologies in analysis and perturbation of the human microbiota will lead to better diagnostic and therapeutic strategies against diseases of microbial origin or pathogenesis. Here, we discuss recent advances that are bringing us closer to realizing the true potential of an engineered human-associated microbial community. Key words Microbiome, Microbiota, Synthetic biology, Systems biology, Microbial engineering, Functional metagenomics, Host–microbe interactions 1 Introduction Of the 100 trillion cells in the human body, 90 % are microbes that naturally inhabit various body sites, including the gastrointestinal tract, nasal and oral cavities, urogenital area, and skin [1]. An indi- vidual’s colon is home to 1011 –1012 microbial cells/mL, the greatest density compared to any other microbial habitat characterized to date [2]. Many studies, such as the Human Microbiome Project and MetaHIT, have probed the vast effects of microbiota on human health and disease [1, 3–5]. In addition to metagenomic sequencing [6], traditional methods of studying cells in isolation are important for elucidating molecular bases of microbial activity. However, cells do not exist in single-species cultures in nature. In fact, some species are only culturable in the presence of other microorganisms [7]. This interdependence for survival amongst microbial species in a community attests to the importance of intercellular interactions, both microbe–microbe and host– microbe. Despite the fact that the human microbiota is composed of many individual microbes, these individuals work in concert to
- 21. 4 perform tasks that rival in complexity to those of more sophisticated multicellular systems. Thus, the human-associated microbiome presents a ripe opportunity for forward engineering to potentially improve human health (Fig. 1). Here, we review recent advances in this area and outline potential avenues for future endeavors. 2 Microbiota, Host, and Disease Contrary to traditional views, microbes are social organisms that engage with the environment and other organisms in specific ways. Microbes participate in intercellular communication through contact-dependent signaling [8], quorum sensing [9], metabolic cooperation or competition [5], spatiotemporal organization [10], and horizontal gene transfer (HGT) [11]. Human-associated microbes produce by-products that serve as substrates utilized by other resident bacteria [12–14]. For instance, accumulated hydro- gen gas from bacterial sugar fermentation is removed by acetogenic, methanogenic, and sulfate-reducing gut bacteria [15]. In contrast to cross-feeding relationships, microbes under stress can release bacte- riocins to suppress the growth of competitors [16–18]. If microbes are members of a biofilm community, they benefit from physical protection from the environment, access to nutrients trapped and distributed through channels in the biofilm, development of syn- trophic relationships with other members, and the ability to share and acquire genetic traits [19, 20]. Microbial populations also Fig. 1 Engineering human-associated microbiota requires detailed understanding of processes that govern the natural propagation and retention of microbes in the host as well as environmental and adaptive pressures that drive the evolution of cells and communities Stephanie J. Yaung et al.
- 22. 5 genetically diversify to insure against possible unstable environmen- tal conditions [21, 22]. Moreover, multispecies communities harbor a dynamic gene pool consisting of mobile genetic elements, such as transposons, plasmids, and bacteriophages, which serve as a source of HGT to share beneficial functions with neighbors to preserve community stability [23–26]. Densely populated communities such as the human gut are active sites for gene transfer and reservoirs for antibiotic resistance genes [11, 27–29]. Beyond microbe–microbe interactions, the microbiota coevolves with the host as it develops, driving microbial adaptation [30–33]. Core functions of microbiota benefit the host, such as extraction of otherwise inaccessible nutrients, immune system devel- opment, and protection against pathogen colonization [2, 34–37]. Gut microbes are critical in intestinal angiogenesis, epithelial cell maturation, and immunological homeostasis [37–40]. For exam- ple, the commensal Bacteroides fragilis produces polysaccharide A, which converts host CD4+ T cells into Foxp3+ Treg cells, producing interleukin-10 (IL-10) and inducing mucosal tolerance [41]. Host diet, inflammatory responses, and aging also affect microbial com- munity composition and function [42–45] (Fig. 2). Indeed, aber- rations in host genetics, immunology, and diet can lead to Fig. 2 Composition of the human gut microbiome during development with respect to microbial diversity and population stability. Data compiled from recent studies from the literature: (a) Hong 2010 [169]; (b) Saulnier 2011 [170]; (c) Claesson 2011 [171]; (d) Yatsunenko 2012 [172]; (e) Spor 2011 [173] Engineering Human-Associated Microbiomes
- 23. 6 microbiota-associated human diseases. Diet-induced obesity in mice from a high-fat diet is characterized by enhanced energy harvest and an increased Firmicutes-to-Bacteroidetes ratio [46, 47]. Furthermore, disruptions in the homeostasis between gut microbial antigens and host immunity can invoke allergy and autoimmunity, as in type 1 diabetes and multiple sclerosis [48–50]. It is thought that inflamma- tory bowel disease (IBD) results from inappropriate immune responses to intestinal bacteria; genes identified in genome-wide association studies highlight the role of a host imbalance between pro-inflammatory and regulatory states [48, 51]. While the host selects for microbial communities that harvest nutrients and prime the immune system, irregular microbiota composition may cause disease (Fig. 3), including IBD [52–54], Host disease states Inflammatory bowel disease in adults Metabolic syndrome in adult Healthy Burkina Faso childrena Healthy European childrena Host genetics and environment (diet, lifestyle) Actinobacteria Bacteroidetes Firmicutes Proteobacteria Spirochaetes Other Fusobacteria Healthy Dental caries Young adult salivag Saliva Dental plaque Oral microbiomef Gut microbiome Pediatric atopic dermatitisd Baseline Healthy childd Flare Postflare Healthy adulte Skin microbiome Fig. 3 Changes in the composition of human microbiota during disease states compared to healthy states. Data compiled from recent studies from the literature: (a) De Filippo 2010 [174]; (b) Peterson 2008 [175]; (c) Larsen 2010 [176]; (d) Kong 2012 [177]; (e) Gao 2012 [178]; (f ) Keijser 2008 [179]; (g) Yang 2012 [180] Stephanie J. Yaung et al.
- 24. 7 lactose intolerance [55, 56], obesity [57, 58], type I diabetes [59], arthritis [60], myocardial infarction severity [61], and opportunistic infections by pathogens such as Clostridium difficile and HIV [62–65]. Microbial gut metabolism links host diet not only to body composition and obesity [66] but also to chronic inflammatory states, such as IBD, type 2 diabetes, and cardiovas- cular disease [67–69]. Intestinal microbes are also important in off-target drug metabolism, rendering digoxin, acetaminophen, and irinotecan less effective or even toxic [70–72]. In the case of irinotecan, a chemotherapeutic used mainly for colon cancer, the drug is metabolized by β-glucuronidases of commensal gut bacte- ria into a toxic form that damages the intestinal lining and causes severe diarrhea. In the oral cavity, ecological shifts in dental plaque microbiota lead to caries (cavities), gingivitis, and periodontitis [73]. Dental caries arise from acidic environments generated by acidogenic (acid-forming) and aciduric (acid-tolerant) bacteria, which metabolize sugar from the host diet. Translocation of oral bacteria into other tissues results in infections, and cytokines from inflamed gums released into the bloodstream stimulate systemic inflammation. Oral bacteria have been implicated in respiratory [74, 75] and cardiovascular diseases [76–78], though mechanisms remain unclear. 3 Enabling Tools for Engineering the Microbiota The human-associated microbial community presents a vast reservoir of nonmammalian genetic information that encodes for a variety of functions essential to the mammalian host [79]. Next-generation sequencing technologies have enabled us for the first time to sys- tematically probe the genetic composition of these trillions of microbes that reside on the human body [1]. The ongoing effort by the Human Microbiome Project and MetaHIT to catalog dom- inant microbial strains from different body sites has generated useful reference genomes for many of the representative species [80]. Metagenomic shot-gun sequencing approaches of whole microbial communities, such as those found in the gut, have yielded near- complete gene catalogs that describe abundance and diversity of genes that contribute to maintenance and metabolism of the microbiota [6]. In order to determine functional relationships between human-associated microbes and their concerted effect in the mam- malian host, we rely on functional perturbation of the microbial community. These investigative avenues include genome-scale per- turbation assays, specified community reconstitutions, and directed engineering through synthetic biology (Fig. 4). Each approach provides us with a unique angle to attack an otherwise daunting Engineering Human-Associated Microbiomes
- 25. 8 challenge of de-convolving a highly intertwined set of microbial interactions in a very heterogeneous environment and a difficult- to-manipulate human host. Advances in both in vitro and in vivo host models have thus also facilitated research endeavors in this area, which we discuss in the following sections. Fig. 4 General approaches to engineer the human microbiome through design, quantitative modeling, genome- scale perturbation, and analysis in in vitro and in vivo models, with the ultimate goal of producing demand- meeting applications to improve sensing, prevention, and treatment of diseases Stephanie J. Yaung et al.
- 26. 9 Approaches to study the function of human-associated microbes by genetic manipulation rely on several fundamental capabilities, which are often the largest practical barriers to manipulate microbes genetically. First, individual microbes need to be isolated and cul- tured in the laboratory. Because microbes have a myriad of physi- ologies and require different nutritional supplement for growth, different media compositions and growth conditions need to be laboriously tested by trial and error to isolate and culture each microbe. These microbial culturing techniques date back to the times of Louis Pasteur and are still the dominant approach today. More recent microbial cultivation techniques use microfluidics and droplet technologies to enable the discovery of synergistic interac- tions between natural microbes that allow otherwise “unculturable” organisms to be grown in laboratory conditions [7, 81, 82]. Upon successful microbial cultivation, the next limiting step of microbial genetic manipulation is the transformation of foreign DNA into cells. The passage of foreign DNA (e.g., plasmids, recombinant fragments) into the cell requires overcoming the physical barriers presented by the cell wall or membrane. This task is accomplished in nature through processes such as transduction by phage, conjugation and mating, or natural competency and DNA uptake [83, 84]. Numerous laboratory techniques have been developed for microbial transformation including electroporation [85], biolistics [86], soni- cation [87], and chemical or heat disruption [88]. Electroporation, the most common of the laboratory transformation techniques, relies on high-voltage electrocution of the bacterial sample that is thought to transiently induce pores on the cell membrane (hence “electroporation”) that then enable extracellular DNA to diffuse into the cell. Various protocols for electroporation of human-associ- ated microbes have been described and are good starting points for developing genetic systems in these microbes [89, 90]. Upon transformation of DNA into the cell, the DNA needs to either stably propagate intracellularly or integrate into the micro- bial host genome through recombination or other integration strategies. Inside the cell, stable propagation of episomal DNA such as plasmids requires DNA replication machinery that is com- patible with the foreign DNA [83]. Additionally, cells often use methylation and DNA modification and restriction systems to dis- cern foreign versus host DNA through a primitive defensive mech- anism that fights against viruses or other invading genetic elements. Nonetheless, these promiscuous genetic elements can often be used as a way to integrate foreign DNA into the chromosome and are often used for large-scale functional genomics [91]. Taking all these parameters into consideration, we have summarized (Fig. 5) the current genetic tractability of human- associated microbes with respect to culturability, availability of full genome sequences, transfection methods, and expression and manipulation systems. Expansion of these basic genetic tools is crucial for future functional studies of human microbiota. 3.1 Challenges of Building New Genetic System Engineering Human-Associated Microbiomes
- 27. 10 Fig. 5 Genetic tractability of abundant or relevant human-associated microbial genera, evaluated by the availability of means to introduce genetic material (e.g., transformation, conjugation, or transduction), vectors, expression systems, completed genomic sequences, and culturing methods. Circles of increasing sizes indicate greater genetic tractability. Protocols and demonstrated methods for genetic manipulation are listed as follows: (a) Clostridium: Phillips-Jones 1995, Jennert 2000, Young 1999, Bouillaut 2011 [181–184]; (b) Ruminococcus: Cocconcelli 1992 [185]; (c) Lactobacillus: van Pijkeren 2012, Ljungh 2009, Damelin 2010, Sorvig 2005, Thompson 1996, Lizier 2010[107, 186–190]; (d) Enterococcus: Shepard 1995 [191]; (e) Lactococcus: Holo 1995, van Pijkeren 2012 [107, 192]; (f) Streptococcus: McLaughlin 1995, Biswas 2008 [193, 194]; (g) Staphlyococcus: Lee 1995 [195]; (h) Listeria: Alexander 1990 [196]; (i) Treponema: Kuramitsu 2005 [197]; (j) Borrelia: Hyde 2011, Rosa 1999 [198, 199]; (k) Bifidobacterium: Mayo 2010 [200]; Stephanie J. Yaung et al.
- 28. 11 Genome-scale perturbations are a class of genetic approaches that disrupt or perturb the expression of functional genes that contribute to relevant phenotypes by individual microbes. To dissect the func- tion of different genes in the cell, we have relied heavily on the use of transposons, which are selfish genetic elements that can splice into and out of different locations of chromosomal DNA, thereby disrupting the coding sequence [92]. This classical approach, known as transposon mutagenesis, has allowed us to isolate many genetic mutants whose disrupted genes give rise to interesting phenotypes that reflect the importance of those genes to its physiol- ogy. Next-generation DNA sequencing has now enabled multi- plexed genotyping of pools of transposon mutants by using molecular barcodes that then can be applied to measure the effect of genome-scale perturbations in different environmental condi- tions. For example, techniques such as insertion sequencing (INSeq) [93] utilize the inverted repeat recognition of the Himar trans- posase, which is one nucleotide change away from the restriction site for type II restriction enzyme MmeI, to generate paired 16–17 bp flanking genomic sequences around the transposon that can be sequenced in pools. Thus, the defined insertion location of every transposon in the library can be determined. By sequencing this pooled mutant library pre- and posttreatment with any number of environmental perturbations, one can probe the effects of differ- ent gene disruptions on the physiology of the cell in a multiplexed fashion. Similar techniques using other transposon systems such as transposon sequencing (Tn-seq) [94], high-throughput insertion tracking by deep sequencing (HITS) [95], and transposon-directed insertion-site sequencing (TraDIS) [96] have also been developed. In addition to transposon-based systems, shotgun expression libraries have been useful in discovering functional DNA elements in genomic or metagenomic DNA. Shotgun expression libraries rely on physical shearing or restriction digestion of a donor DNA source into smaller DNA fragments that are then cloned into a gene expression vector and transformed into a host strain for functional analysis. A library of metagenomic DNA samples can for example be extracted from an environment and cloned into plasmids that are then expressed in E. coli. Selection and sequencing of the E. coli population for heterologous DNA that enable new function lead to discovery of novel gene elements that perform a particular 3.2 Genome-Scale Perturbations Fig. 5 (continued) (l) Actinomyces: Yeung 1994 [201]; (m) Mycobacterium: Parish 2009, Sassetti 2001 [202, 203]; (n) Proprionibacterium: Luijk 2002 [204]; (o) Chlamydia: Binet 2009 [205]; (p) Porphyromonas: Belanger 2007 [206]; (q) Prevotella: Flint 2000, Salyers 1992 [207, 208]; (r) Bacteroides: Salyers 1999, Smith 1995, Bacic 2008 [209–211]; (s) Fusobacterium: Haake 2006 [212]; (t) Helicobacter: Taylor 1992, Segal 1995 [213, 214]; (u) Camplyobacter: Taylor 1992 [214]; (v) Rickettsia: Rachek 2000 [215]; (w) Brucella: McQuiston 1995 [216]; (x) Bordetella: Scarlato 1996 [217]; (y) Neisseria: O’Dwyer 2005, Bogdon 2002, Genco 1984 [218–220]; (z) Pseudomonas: Dennis 1995 [221] Engineering Human-Associated Microbiomes
- 29. 12 activity. This approach can easily identify activities such as antibiotic resistance [97] but have yielded less success with other functions. Towards forward engineering of human-associated microbes, new genome engineering tools such as trackable multiplex recom- bineering (TRMR) [98, 99] and multiplex automated genome engineering (MAGE) enable efficient, site-specific modification of the genome [100–103]. TRMR combines double-stranded homologous recombination [104] and molecular barcodes synthe- sized from DNA microarrays to generate populations of mutants that are trackable by microarray or sequencing. MAGE relies on introduction of pools of single-stranded oligonucleotides that tar- get defined locations of the genome to introduce regulatory muta- tions [102] or coding modifications [105]. These and other recombineering technologies are now being developed for a variety of other organisms including gram-negative bacteria [106], lactic acidbacteria[107],Pseudomonassyringae[108],andMycobacterium tuberculosis [109], and are likely to be very useful for engineering human-associated microbes. The community of microbes that make up the human microbiome can be considered a “pseudo-organ” of its own. These microbes interact with one another and the mammalian host in potentially highly complex ways that may be difficult to decipher even with tractable genetic systems [110]. A direct approach to study these interactions is to build reconstituted communities of microbes derived from monoculture isolates in defined combinations. This de novo reconstitution approach to build synthetic communities has significant advantages over attempts to deconvolute natural communities. Reconstituted synthetic consortium presents a trac- table level of complexity in terms of number of interacting micro- bial species that can be tracked by sequencing and predicted with quantitative models. In one such study, researchers inoculated germ-free mice with ten representative strains of the human micro- biota [111]. The mice were then fed with defined diets of macro- nutrients consisting of proteins, fats, polysaccharides, and sugars. By tracking the abundance of the ten-member microbial consor- tium using high-throughput sequencing, the researchers could predict over 60 % of the variation in species abundance as a result of diet perturbations. This avenue of investigation presents a viable approach to study the human microbiome and ways to analyze synthetically engineered microbiota. Engineered microbes have been utilized to reconstitute syn- thetic communities to investigate the role of metabolic exchange. One such important metabolic exchange is that of amino acids, as they are the essential constituents of proteins. Various syntrophic cross-feeding communities have been described using auxotrophic E. coli and yeast strains that require different amino acid supplementation for growth [112–114]. In these syntrophic 3.3 Reconstituted Communities Stephanie J. Yaung et al.
- 30. 13 systems, metabolites that are exchanged across different biosynthetic pathways promote more syntrophic growth than those that are exchanged along the same pathway, which also relates to the cost of biosynthesis of the amino acid metabolites. Amino acid exchange is likely a large player in driving metabolism of microbial communities as a substantial fraction of all microbes are missing biosynthesis of various metabolites and thus require growth on more rich and complex substrates that are found in the gut [115]. New approaches are now utilizing synthetic biology to engineer human-associated microbiota to improve health and metabolism as well as to monitor and fight diseases. These efforts focus on devel- oping genetic circuits that actuate in an engineered host cell such as E. coli that can sense and respond to changes to its environment and in the presence of particular pathogens. For example, to detect the human opportunistic pathogen Pseudomonas aeruginosa, which often causes chronic cystic fibrosis infections and colonizes the gas- trointestinal tract, E. coli was engineered to detect the small diffus- ible molecule that is excreted by P. aeruginosa through the quorum sensing pathway [116]. An engineered synthetic circuit was placed in nonpathogenic E. coli, which when placed in the presence of high-density P. aeruginosa triggered a self-lysis program that released a narrow-spectrum bacteriocin that specifically killed the P. aeruginosa strain. Similar strategies have also been demonstrated to detect and respond to Vibrio cholera infection using engineered E. coli that sense autoinducer-1 (AI1) molecules from V. cholera quorum sensing pathway [117]. These strategies appear to yield improved survival rates against microbial pathogenesis in murine models [117]. Quorum sensing systems, which normally help microbes detect local cell density, have been further enhanced to improve robustness and performance to enable coupled short-range and long-range feedback circuits that enable microbial communica- tion across large distances in an engineered community. Other microbes have been successfully engineered to perform specific functions on human-associated surfaces such as the muco- sal layer of the gut epithelium. Numerous diseases that occur along the intestinal tract are targets of such engineered approaches. For example, the probiotic strain Lactococcus lactis has been engineered to secrete recombinant human IL-10 in the gastrointestinal tract to reduce colitis [118, 119]. Other future applications of engi- neered probiotics include enhancing catabolism of nutrients (e.g., lactose and gluten), modulation of the immune system, and removal of pathogens by selective toxin release [116]. To probe and engineer the human-associated microbial commu- nity, various in vitro models have been developed, ranging from traditional batch culturing in chemostats to microfluidic systems that incorporate host cells. Single-vessel chemostats inoculated 3.4 Microbial Engineering Through Synthetic Biology 3.5 In Vitro Host Models Engineering Human-Associated Microbiomes
- 31. 14 with fecal samples from healthy individuals have helped identify HGT [120] and selective bacterial colonization on different carbo- hydrate substrates [121, 122]. A multichamber continuous culture system mimicking spatial, nutritional, and pH properties of differ- ent GI tract regions can be used to investigate stabilization dynam- ics [123–125]. Similarly, the constant-depth film fermenter resembles oral biofilm [126] and has enabled studies on biofilm for- mation, antibiotic resistance [126], and HGT in a multispecies oral community [127, 128]. To incorporate mammalian cells in study- ing host–microbial interactions, organ-on-a-chip microfluidic devices have been recently used. In one version of such a system, a gut-on-a-chip device, the microfluidic channel is coated with extra- cellular matrix and lined by human intestinal epithelial (Caco-2) cells. This system mimics intestinal flow and peristaltic motion, recapitulates columnar epithelium polarization and intestinal villi formation, and supports the growth of commensal Lactobacillus rhamnosus GG [129]. These microdevices offer an opportunity to investigate host–microbiota interactions in a well-controlled man- ner and in physiologically relevant conditions. Inoculating with native microbiota samples provides a method to overcome the un-cultivability of many microbes as well as to study collective activity and discover novel functions without a priori knowledge of community composition. However, starting with a predefined microbial community allows a controlled setting better suited for testing engineered systems. In one study analyz- ing the dynamics of a community representing the four main gut phyla in a chemostat, the authors propose that intrinsic microbial interactions, rather than host selective pressure, play a role in the observed colonization pattern, which was similar to what has been documented in the human gut [130]. Similar models have been developed for oral microbiota studies. The use of predefined oral microbial inocula has helped elucidate metabolic cooperation in batch culture [12] and community development in saliva-conditioned flow cells [131]. In order to move into in vivo animal models that more closely represent the physiology of the human host environment, researchers have extensively utilized murine models including germ-free, gno- tobiotic, and conventionally raised mice. Gnotobiotic animals are born in aseptic conditions and reared in a sterile environment where they are exposed only to known microbial species; techni- cally, germ-free mice are a type of gnotobiotic mice that have not been exposed to any microbes. Similar to in vitro systems, mice can be inoculated with either a natural microbiota sample or a pre- defined microbial community. Fecal samples, as well as oral swab and saliva samples, can then be collected from gnotobiotic mice for biochemical analysis and species quantification of gut and oral cavity microbiota. In vivo models have been used to study the 3.6 In Vivo Host Models Stephanie J. Yaung et al.
- 32. 15 transmission of antibiotic resistance in the mouse gut [132, 133] and colonization resistance in the oral cavity [134]. Furthermore, the choice of the inoculum donor offers opportunities to compare different host selection pressures and microbial community responses. Microbiota can be transplanted from conventionally raised to germ-free animals of not only the same species but also interspecies, as in human microbiota into mouse, called humanized gnotobiotic mice [134]. In one study, transplants from zebrafish gut microbiota into germ-free mice and mouse gut microbiota into germ-free zebrafish revealed that the resulting community conformed to the native host composition, demonstrating host selection [135]. Altering host diet, environment, or genetic background can also enable studies in host–microbiota interactions. One method to gain insight into the role of microbial communities in disease is to utilize mice with recapitulated pathologies. For example, IL-10−/− , ob−/− , apoE−/− , and TLR2−/− or TLR5−/− mice are models for colitis, obesity, hypercholesterolemia, and metabolic syndrome, respectively [46, 136–139]. To generate antigen- or pathogen- specific phenotypes, mice can be infected with Salmonella typhimurium to study colitis [140] or Citrobacter rodentium as a model for attaching and effacing pathogens, such as enterohemor- rhagic E. coli [141, 142]. Furthermore, murine models with chem- ically induced inflammation can be tools to study chronic mucosal inflammation; dextran sodium sulfate (DSS) can induce ulcerative colitis, and trinitrobenzene sulfonic acid (TNBS) can stimulate Crohn’s disease [143]. To investigate oral microbiota, there are periodontal disease [144] and oral infection models [145, 146]; gnotobiotic rodents can also be fed a high-sucrose cariogenic diet to promote plaque formation. Germ-free mice inoculated with defined microbes are informa- tive models for analyzing microbial colonization and metabolic adaptation [147]. For example, resident bacteria and probiotic strains adapt their substrate utilization: in the presence of Bifidobacterium longum, Bifidobacterium animalis, or Lactobacillus casei, Bacteroides thetaiotaomicron diversified its carbohydrate uti- lization by shifting metabolism from mucosal glycans to dietary plant polysaccharides [148]. Furthermore, the effect of different diets on microbial community composition can be studied, as in gnotobiotic mice inoculated with ten sequenced gut bacterial species and fed with various levels of casein, cornstarch, sucrose, and corn oil to represent protein, polysaccharide, sugar, and fat content in the diet, respectively [111]. Over the past several decades, a large number of theoretical and quantitative models have been developed to describe the cell and its behavior. Constrain-based models are used to describe metabolism of individual cells using stoichiometric representation 3.7 Computational Frameworks for Human Microbiomics Engineering Human-Associated Microbiomes
- 33. 16 of metabolic reactions and optimization constraints [149]. Approaches such as flux balance analysis (FBA) enable the analysis of metabolism under steady-state assumptions by linear optimiza- tion solution methods. These methods are now being scaled to ecosystems of cells. Recent developments using multi-level objec- tive optimization [150] and dynamic systems [151] enable the modeling of synthetic ecosystems of three or more members. Using metagenomic data of the gut microbiome, Greenblum et al. generated a community-level metabolic reconstruction net- work of the microbiota and discovered topological variations that are associated with obesity and IBD, giving rise to low diversity and differences in community composition [152]. For models that account for systems dynamics, population abundance and metabolite concentrations can be solved independently through different FBA models that are iterated at each time step. This approach called dynamic multi-species metabolic modeling (DMMM) can capture scenarios of resource competition, leading to the identification of limiting metabolites [153]. Other comple- mentary models include elementary mode analysis (EMA) [154] that enables quantitative analysis of microbial ecosystems in a mul- ticellular fashion. 4 Perspectives Reframing the microbiota community as a core set of genes, not a core set of species, opens a new front to the microbiome engineering design space. In a metagenomic study of 154 individuals, no single- gut bacterial phylotype was detected at an abundant frequency amongst all the samples, a finding that is consistent with the idea that the core human gut microbiome may not be best defined by promi- nent species but by abundantly shared genes and functions [155]. We propose that manipulation at the gene, genome, and ultimately metagenome level offers the ability for precise multicellular engineer- ing of desirable traits in human-associated microbiota. Besides con- trolledperturbationsofthemicrobiometoadvanceourunderstanding of host–microbiota interactions, metagenome-scale tools enable novel developments in diagnostics and therapeutics. From biosensors on the skin to reporters in the gut, there are several opportunities in monitoring the health and disease status of the human host, such as sensing nutritional deficiencies, immune imbalances,environmentaltoxins,orinvadingpathogens.Prophylactic and therapeutic avenues for human microbiome engineering include modifying community composition, tuning metabolic activity, mediating microbe–microbe relationships, and modulating host– microbe interactions. Two current microbiota-associated treatments Stephanie J. Yaung et al.
- 34. 17 have shown clinical efficacy: (1) fecal transplants for recurrent Clostridium difficile infection [156] and (2) probiotics for pouchitis, which is inflammation of the ileal pouch that is created after surgical removal of the colon in ulcerative colitis patients [157–159]. The main challenge is transmission of undesirable agents from donor feces to the recipient gut in fecal transplants and native colonization resis- tance that would impair infiltration and growth of new species in pro- biotics [160, 161]. Nevertheless, these successful approaches demonstrate the potential benefits of leveraging natural microorgan- isms and entire microbial communities. In fact, coupling organismal and functional gene-level approaches would be a powerful way to engineer the native micro- biota. Microbiome engineering enables multiscale system design for the synthesis of nutrients and vitamins, enhanced digestion of gluten and lactose, decreased acidity of the oral cavity, targeted elimination of multidrug-resistant pathogens, and microbial mod- ulation of the host immune system. As vehicles for drug delivery, commensal bacteria designed to secrete heterologous genes have been explored for treating cancer [162–164], diabetes [165], HIV [166], and IBD [118]. For example, IL-10 has immuno- modulatory effects in IBD but requires localized delivery at the intestinal lining to avoid the toxic side effects and low efficacy of systemic IL-10 injection. Ingestion of modified Lactococcus lactis that secrete recombinant IL-10 is safe and effective in animal models and has been promising in human clinical trials for IBD [119, 167]. Finally, besides addressing clinical safety and efficacy criteria for FDA regulatory approval [168], overall safety precautions are critical considerations to minimize unintentional risks in releasing genetically modified material into the natural environ- ment. Rational design, such as creating auxotrophic microbes [119], for robust stability, non-pathogenicity, and containment of recombinant genetic systems will be essential in microbiome engineering. Acknowledgements H.H.W. acknowledges the generous support from the National Institutes of Health Director’s Early Independence Award (grant 1DP5OD009172-01). S.J.Y. acknowledges support from the National Science Foundation Graduate Research Fellowship and the MIT Neurometrix Presidential Graduate Fellowship. G.M.C. acknowledges support from the Department of Energy Genomes to Life Center (Grant DE-FG02-02ER63445). Engineering Human-Associated Microbiomes
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- 44. 27 Lianhong Sun and Wenying Shou (eds.), Engineering and Analyzing Multicellular Systems: Methods and Protocols, Methods in Molecular Biology, vol. 1151, DOI 10.1007/978-1-4939-0554-6_2, © Springer Science+Business Media New York 2014 Chapter 2 Constructing Synthetic Microbial Communities to Explore the Ecology and Evolution of Symbiosis Adam James Waite and Wenying Shou Abstract Synthetically engineered microbial communities based on model organisms provide a simplified model of their naturally occurring counterparts while still retaining essential features of living organisms. The degree of control afforded by this approach has been critical in understanding how similar types of natural com- munities might have persisted and evolved. Here, we first discuss important considerations when designing a synthetically engineered system. Then, we describe the steps required to create a two-partner cooperative system based on the yeast Saccharomyces cerevisiae. Key words Evolution, Ecology, Mutualism, Cooperation, Synthetic biology, S. cerevisiae 1 Introduction From mediating biogeochemical cycles [1] to influencing human health [2] and disease [3], microbial communities impact all aspects of life on earth. However, the complexity of microbial com- munities and the difficulty in isolating and culturing microbes [4] pose serious challenges for decoding cell–cell and cell–environment interactions. Moreover, the evolutionary histories of microbial communities are difficult to retrace. Alternatively, communities of model organisms engineered to engage in defined interactions can be deployed to address fundamental questions in ecology and evolu- tion, such as how species coexist and coevolve [5]. In this chapter, we discuss several considerations when designing a synthetic community, using the construction of a two-partner cooperative yeast system as an example. Then, we describe the methodology in detail. The initial consideration is the choice of organisms. Genetic tractability and short generation times facilitate strain construction and experimentation, as well as the discovery and interpretation of mutations during experimental evolution. Thus, well-studied model organisms with reference genome sequences such as Escherichia coli
- 45. 28 or Saccharomyces cerevisiae are ideal, although other species have also been used [5]. While each model organism has its advantages and disadvantages, we will use S. cerevisiae to highlight principles that are applicable to any synthetically engineered community. Sexual recombination is both a help and a hindrance to syn- thetically engineered communities. On the one hand, sexual recombination can radically simplify strain construction by allow- ing genetic shuffling. For example, when a strain with genotype AB is crossed with a strain of genotype ab, recombinant strains Ab and aB can be generated. On the other hand, distinct popula- tions in an engineered community should remain genetically insulated from one another and therefore should not be allowed to mate. An advantage of using S. cerevisiae is its ability to undergo both sexual and asexual reproduction. Haploid yeast is particularly suitable for evolution experiments since phenotypes arising from recessive mutations are immediately apparent. Haploid yeast can reproduce asexually as either of the two mating types, “a” or “α.” Two cells of opposite mating type can mate to produce an a/α diploid, which can reproduce either asexually as diploids or sexually to form haploids. The final strains for an engi- neered community should always be the same mating type to pre- vent sexual recombination. However, haploid yeast switch mating types spontaneously at very low frequency even when the gene required for mating-type switching (HO) is defective (which is the case for all commonly used laboratory strains). Thus, we have used MATa cells in which the STE3 gene encoding the receptor for MATa mating pheromone [6] is deleted. Thus, if a MATa ste3 cell switches mating type to MATα ste3, it will fail to initiate the mating process. Ideally, all strains should be derived from an isogenic back- ground so that the only mutations are the ones defined by the researcher. We have used the strain S288C, which is one of the common laboratory strains, and the wild vineyard isolate RM11-1a (hereafter referred to as “RM11”). While S288C has its genome fully annotated and easily accessible [7], it has a tendency to pro- duce mitochondrially deficient “petite” cells [8], which are prone to nuclear genome instability [9] and could potentially interfere with evolution experiments. RM11 (genome sequence available at http:/ /www.broadinstitute.org/annotation/genome/saccharomyces_ cerevisiae/) grows faster than S288C and produces very few petites, but haploid daughter cells do not separate well from their mothers unless the RM-11 AMN1 allele is replaced by the AMN1 allele from S288C [10]. Genetic manipulation in RM-11 is more difficult due to its lower transformation efficiency compared to S288C. A major advantage of using model organisms is that they are genetically modifiable. Foreign DNA can be transformed into yeast as autonomously replicating and segregating circular plasmids or as Adam James Waite and Wenying Shou
- 46. 29 linear DNA if genomic integration is desired. Integration is more stable than using a plasmid. Different populations can be marked with, for example, different antibiotic resistances or fluorescent proteins. Currently, at least six dominant antibiotic resistance genes are in wide use with S. cerevisiae: kan, which confers resistance to geneticin a.k.a. G418; hph, which confers resistance to hygromycin B; nat, which confers resistance to nourseothricin (sold as clon- NAT by Werner BioAgents); pat, which confers resistance to phosphinothricin; ble, which confers resistance to phleomycin; and AUR1-C, which confers resistance to Aureobasidin A (AbA). All of these genes are available on plasmids [11–13]. Our lab has used G418, hygromycin B, clonNAT, and AbA resistance markers. Using antibiotic resistance to select a subpopulation from a co-culture is especially useful if the subpopulation is very rare, as tens of millions of cells can be assayed on a single plate. However, plating on media supplemented with different drugs to determine subpopulation abundance is time-delayed since it takes at least 1 day for colonies to grow up. It is also of low throughput due to the small number (hundreds) of individual colonies that can be counted on a plate. In contrast, fluorescently tagged strains can be distin- guished using flow cytometry, which allows tens of thousands of cells to be counted in less than a minute. While fluorescence- activated cell sorting (FACS) can also be used to isolate subpopula- tions, it requires an expensive instrument and is less efficient when isolating very rare subpopulations. A large number of fluorescent proteins are available [14], although not all of them are bright and/or resolvable from one another using standard filter sets. In addition, the correct folding of fluorescent proteins requires oxygen [15] and is therefore incompatible with strict anaerobes. We have C-terminally tagged (Fig. 1) the highly abundant proteins FBA1 or MET6 with different fluorescent proteins in yeast. Using the appropriate combination of lasers and filter sets (Table 1), we can resolve mixtures of cells expressing five different fluores- cent proteins: CFP, GFP, YFP, mOrange, and mCherry as well as the far-red nucleic acid dye TO-PRO-3 (Invitrogen) which can be used to stain dead cells. Plasmids containing different combina- tions of fluorescent proteins and selectable markers (i.e., genes for nutrient biosynthesis or antibiotic resistance) [16] are readily available from EUROSCARF (http:/ /web.uni-frankfurt.de/ fb15/mikro/euroscarf/). Since deleting or inserting genes will usually be accomplished by transformations (Fig. 1), which require selectable markers, it is convenient to be able to reuse these markers. Plasmids containing kanMX [17] and ble [18] flanked by loxP sites are available for this purpose (for a comprehensive list of plasmids with removable markers, see ref. 19). In the presence of Cre recombinase, the two loxP sites recombine, removing the intervening marker and allow- ing for its reuse in another round of manipulation. Our lab has Constructing Synthetic Microbial Communities
- 47. 30 modified these plasmids to contain nat (WSB116) and hph (WSB117). We generally do not use auxotrophy (the inability to synthesize an essential metabolite) to mark strains except when both selection and counterselection are required. For example, PCR Marker Transformation + Marker Marker Gene X Gene X Gene X Gene replacement Marker Checking primer 45 bp homology sequence Universal primer sequence Genomic DNA C-terminal tagging Fig. 1 Schematic of gene replacement and C-terminal tagging. In both cases, the selectable “marker” is PCR amplified off a plasmid using a pair of hybrid primers. The 3′ end of the primers contains sequences specific to the plasmid (thin green lines) in a region ideally identical among a family of marker plasmids to achieve flexibility. The 5′ end of the primers is 45 bp homologous to yeast genomic DNA (thick blue lines). For gene replacement, the forward homology is to the region immediately 5′ of the start codon of the open reading frame (ORF) of the gene of interest (“Gene X”), while the reverse homology is the reverse complement of the region immediately 3′ of the stop codon of the ORF. For C-terminal tagging, the reverse homology is the same as for gene replacement, while the forward homology is the 45 bp leading up to but not including the stop codon of the ORF.After transfor- mation, checking primers (thin black arrow lines) are used in colony PCR to verify proper integration of the DNA fragment.One primer has homology to the plasmid sequence,while the other has homology specific to the region outside of the 45 bp homology used for integration. For C-terminal tagging, the depicted location of checking primers is preferred, as it results in a shorter (and therefore easier to amplify) PCR product Table 1 Lasers and filter sets used to simultaneously resolve five fluorescent proteins and one fluorescent dye Laser (nm) Filtera Fluorophore 405 450/50 CFP 488 505/10 GFP 530/30 YFP/citrine 561 590/20 mOrange 615/25 mCherry 639 660/16 TO-PRO-3 a The number before the slash indicates the center wavelength in nanometers; the number after the slash is the total bandwidth passed by the filter. For example, “450/50” indicates a filter that passes wavelengths from 425 to 475 nm Adam James Waite and Wenying Shou
- 48. 31 integration of the loxP–drug–loxP cassette could be carried out in a ura3 strain so that transformation of a URA3-marked plasmid containing the Cre recombinase can be selected for. After induction of Cre expression and removal of the drug marker, the URA3 plasmid can then be counterselected using 5-FOA, which only allows survival of cells without the plasmid [20]. The uracil auxot- rophy may then need to be removed via genetic crosses. Alternatively, Cre expression plasmids containing antibiotic mark- ers are also available [19]. Once the populations have been marked, interactions between populations can be defined. Obviously, the possibilities are practi- cally limitless, and the specifics must be left to the researcher. We chose to base our two-partner cooperative system on comple- mentary nutrient exchange [21]. In yeast, all of the manipulations described above can be achieved through a small set of well-known methods [19]. These include (1) transformation to insert or remove genetic material; (2) colony PCR, a simple and rapid way to check whether transformation was successful; and (3) mating, sporulation, tetrad dissection, and geno- typing, which allows genetic features present in two different strains to be recombined into one strain. Below we describe our current protocols for each of these methods. 2 Materials 1. Plasmids containing genes encoding fluorescent proteins and/ or selectable markers. 2. Antibiotics: 1,000× stock G418 (200 mg/ml), 500x stock hygromycin B (100 mg/ml), 1,000× stock clonNAT (100 mg/ ml), 1,000× stock AbA (0.5 mg/ml) (see Note 1). 3. YPD: 10 g/l Bacto-yeast extract, 20 g/l Bacto-peptone, and bring to volume with diH2O to final 950 ml/l for later glucose supplement. Add 20 g/l Bacto-agar (see Note 2) if making plates. Add a magnetic stir bar, and autoclave. Using a flame to ensure sterility, add 50 ml 40 % glucose per liter of medium and the appropriate antibiotic, if necessary. Stir to mix. If multiple liters of agar medium are prepared, they may be kept at 50 °C water bath to prevent solidification of the agar. 4. SD: For liquid media, add 6.7 g/l Difco™ yeast nitrogen base (YNB) with ammonium sulfate and without amino acids and 20 g/l glucose, and bring to volume with diH2O. Sterilize using 0.22 μm filter. For plates, add 6.7 g/l YNB, 20 g/l Bacto-agar, and a stir bar, and add diH2O to 950 ml/l. Autoclave. Using sterile technique, add supplements as neces- sary. Amino acid and nucleobase supplements can be mixed in appropriate proportions [19] in their powder forms in a 2.1 Components for Genetic Manipulation Constructing Synthetic Microbial Communities
- 49. 32 sterilized blender and stored at room temperature. Powdered supplements can be weighed and directly added to the media. Finally, add 50 ml 40 % glucose. Use the glucose to wash down any residual supplement powder adhering to the vessel wall and stir. 5. 50 % PEG 2000 (see Note 3): Dissolve 100 g PEG in 100 ml diH2O. Bring to 200 ml with diH2O. Sterile filter. 6. 1 M LiAc: 102 g/l Lithium acetate dihydrate (102.02 g/mol). Sterile filter. 7. 5 mg/ml Sheared salmon sperm DNA (SS-DNA) [22]. 8. Autoclaved water, tubes, and tips. 1. Sporulation media: 3 g/l potassium acetate, 0.2 g/l raffinose, bring to final volume with diH2O. Autoclave. 2. SCE buffer: 1 M D-sorbitol, 0.1 M sodium citrate (see Note 4), 60 mM EDTA. Adjust pH to 7.0 with 38 % HCl (see Note 5). Autoclave using a 20′ sterilization cycle, and remove promptly (see Note 6). 3. Zymolyase 20T: 30 mg zymolyase 20T dissolved in 10 ml SCE (see Note 7). 3 Methods 1. Design primers: For gene replacement (Fig. 1), the forward primer should contain 45 base pairs (bp) of homology to the genomic region immediately upstream of the “ATG” codon of the open reading frame (ORF) of the gene to be knocked out, followed by the sequence for the universal forward adapter appropriate for the particular set of plasmids being used. The reverse primer should contain the reverse complement to the 45 bp including and immediately downstream of the stop codon of the target ORF, followed by the reverse complement to the universal reverse adapter appropriate for the plasmid set. For C-terminal tagging (Fig. 1), the reverse primer is designed as in gene replacement, and the forward primer should contain 45 bp homology to the sequence just 5′ of the stop codon of the gene of interest. 2. PCR amplify cassette (antibiotic resistance for gene replacement or fluorescent protein plus selectable marker for C-terminal tagging) off a plasmid (see Note 8). Check the length of the PCR product using gel electrophoresis. 1. Inoculate 5 ml YPD culture per transformation and shake at 30 °C until the cell density is between 3×106 and 2×107 cells/ml (see Note 10). 2.2 Components for Mating, Sporulation, and Tetrad Dissection 3.1 Primer Design and Amplification for Gene Tagging or Replacement 3.2 Transformation (See Note 9) Adam James Waite and Wenying Shou
- 50. 33 2. Prepare a boiling water bath for the SS-DNA. While the water is warming up, harvest the culture in a sterile 50 ml centrifuge tube at 425×g for 2 min. 3. Pour off the YPD medium, and resuspend the cells in 25 ml of sterile water and centrifuge again. 4. Pour off the water, resuspend the cells in 100 μL 0.1 M LiAc, and transfer the suspension to a 1.5 ml microfuge tube. 5. Pellet the cells at top speed for 15 s, and remove supernatant with a micropipette. 6. Resuspend the cells in about 43 μl of 0.1 M LiAc to a final volume of 50 μl (2×109 cells/ml) per transformation. 7. By now the water bath should be boiling. Boil SS-DNA (with cap lock on) for 5 min, and then quickly transfer it to ice (see Note 11). Vortex briefly to speed up cooling, and then keep on ice. 8. Vortex the cell suspension and pipette 50 μl into 1.5 ml tubes. Pellet the cells at top speed for 15 s, and remove supernatant with a pipette. Vortex to loosen up the pellet. 9. Prepare the transformation mixture (TRAFO) master mix (1.2× the total volume required so that pipetting errors can be accommodated) and keep on ice (see Note 12): 240 μl 50 % w/v PEG (see Note 3), 36 μl 1.0 M LiAc, 20 μl 5 mg/ml SS-DNA, and 64-x μl sterile dH2O per transformation, where x μl is the volume of DNA to be added (see Note 13). Vortex until completely homogenous (see Note 14). 10. Add 360-x μl TRAFO to each cell pellet (see Note 15), and mix well by pipetting up and down (see Note 16). Add x μl DNA, and mix well again by vortexing or pipetting. 11. Heat shock cells by placing the tubes in a 42 °C water bath for 40 min (see Note 17). Mix by inverting every ~10 min. 12. Centrifuge at 3,824×g for 15 s, and remove TRAFO with a pipette or simply by decanting. Wash cells by resuspending the pellet in 1 ml YPD. Spin again at 3,824×g for 15 s (see Note 18). Remove YPD, and pipette 1 ml fresh YPD into each tube. Resuspend the pellet by pipetting it up and down gently. 13. If selection is on complementation of nutrient auxotrophy, cells can be directly plated on selective medium. If selection is on drug resistance, cells need to be incubated for ~2–3 h at 30 °C in 1 ml YPD to express the resistance gene before being plated. 14. To plate, first centrifuge at 3,824×g for 15 s. Discard 700 μl of the supernatant. Resuspend cells in the remaining 300 μl and plate on 80 % of the surface. Use a sterile toothpick to streak from the plated area to the empty area to maximize the chance of obtaining single colonies. 15. Incubate at 30 °C. Colonies should be visible in 2–3 days. Constructing Synthetic Microbial Communities
- 51. 34 1. Design primers: One primer should be specific to the cassette used for transformation (for example, the universal primer sequence). The other primer should be outside the 45 bp homology region used for integration (Fig. 1). 2. Using a sterile pipette tip, pick about half of a normal-sized (~1.2 mm diameter) colony without touching the agar beneath it. For S288C, place directly into 15 μl sterile water, vortex, and use 1 μl in a 20 μl PCR (see Note 20). For RM11, transfer the cells to 15 μl 0.25 % SDS (see Note 21). Vortex for 30 s, spin at top speed (~17,949×g in a small centrifuge) for 1 min, and use 1 μl of the supernatant for a 20 μl PCR. This PCR mix must contain a final concentration of 5 % Triton X-100 to neutralize the protein-denaturing SDS [23]. An alternative, and more reliable, method uses LiAc and SDS to lyse cells and requires ethanol precipitation prior to PCR (see Note 19, ref. 22). Specifically, cells are suspended in 100 μl 200 mM LiAc and 1 % SDS solution and incubated at 70 °C for 15 min. 300 μl 96 % ethanol is added to precipitate DNA. After brief vortexing, DNA is collected by centrifugation at 15,000×g for 3 min. Precipitated DNA is dissolved in 100 μl TE (10 mM Tris–HCl, 1 mM disodium EDTA, pH 8.0). After spinning cell debris down at 15,000×g for 1 min, 1 μl supernatant is used for PCR. 1. Grow up a small patch of each haploid to be crossed towards the top of a YPD plate (see Note 22). 2. Use a sterile toothpick to transfer a tiny amount of one strain to an empty region of a YPD plate (see Note 23), making a small spot (a few millimeters in diameter). With a fresh tooth- pick, transfer a similarly tiny amount of the other strain to the same spot and mix with the toothpick. 3. Incubate for 3.5 h (S288C) or 2.5 h (RM11) at 30 °C (see Note 24). 4. Using a toothpick, touch the mixed spot and streak down about a centimeter. Without re-touching the spot, make similar streaks to the left and right of the original streak. This gives three different dilutions of cells on the plate. 5. Use a yeast dissection microscope [19] to isolate diploids. The cytoplasmic “bridge” between mated cells and a small bud at its center is indicative of recently mated diploids. 1. Pre-sporulate by patching onto YPD and incubating at 30 °C for ~10 h until a thin film of cells is formed (see Note 25). 2. Inoculate 2 ml sporulation media with about a match head’s worth of cells. Incubate at room temperature on a rotator for 2–5 days. Check for tetrads using a light microscope using a 20–40× magnification objective [19]. 3.3 Colony PCR (See Note 19) 3.4 Mating and Diploid Isolation 3.5 Sporulation, Tetrad Dissection, and Genotyping Adam James Waite and Wenying Shou
- 52. 35 3. Wash cells with 1 ml sterile water and resuspend in 1 ml sterile water. Store at 4 °C. 4. When ready to dissect, spin down 20 μl of sporulated cells and carefully remove supernatant with a pipette. Add 20 μl SCE, and vortex to resuspend cells. If using RM11, sonicate for 1 min. 5. Add 4 μl zymolyase 20T, and mix by pipetting up and down (see Note 26). Incubate at room temperature for 20 min (S288C) or 2 h (RM11) (see Note 27). 6. Gently (to avoid breaking up tetrads) pipette up digested cell suspension and spot onto the top portion of the center strip of a YPD plate. Tilt plate down so that the liquid rolls down the central strip, stopping before the liquid touches the plate wall. Let dry. 7. Use a yeast dissection microscope to separate tetrads into indi- vidual spores. Create a grid with a maximum of two tetrads per row [19], one to the left and one to the right of the central strip. 8. Incubate overnight at 30 °C. Use a flame-sterilized scalpel to remove the strip of cells from the middle of the plate (see Note 28) and return to incubator. 9. Once the spores have germinated and grown into colonies, replica plate onto the appropriate selective media(s). Note that a single genetic locus should, under most circumstances, seg- regate 2:2 [19]. For instance, two spores will be MATa and two will be MATα. Exceptions can be caused by, for exam- ple, gene conversion or traits that are mediated by heritable materials transmissible through the cytoplasm, such as mitochondria. 10. Mating types can be tested using a pair of mating-type testers, one of each mating type. If the entire collection of spores are auxotrophic because of mutations in a set of genes, then the testers should be auxotrophic due to mutation in a different gene to ensure that the resulting diploids are prototrophic. In this case, spread ~200 μl of a saturated culture of each tester strain on its own YPD plate and let dry. Then, replica plate the target strains onto each plate using a sterile velvet. After half a day, replica each plate onto an SD plate. Growth will only occur if the strains are able to mate. If spores are prototrophic, they need to be separately mated to each of the two tester strains, as described above for diploid isolation. Many crosses can be set up on one YPD plate. The mating type of a strain is revealed by the presence or the absence of fused diploids. If this method is necessary, it is better to narrow down the number of strains to be tested based on other markers first. Constructing Synthetic Microbial Communities
- 53. 36 4 Notes 1. Prepare stocks in deionized (di) H2O and sterile filter (except for AbA, which should be made in ethanol and needs no sterile filtration) and keep at −20 °C. 2. Not all sources of YNB and agar are appropriate for use with yeast. For example, we have found that YNB and agar obtained from BD biosciences work better than those obtained from USA Scientific. 3. The size of PEG is important. Transformation efficiency for RM11 is very low if PEG 3500 is used. We use PEG3500 to transform S288C, although PEG2000 should work as well. 4. NaH2PO4 can be used instead. 5. About 0.5 ml when making a final volume of 500 ml. 6. Ensure that the color of solution did not change during the autoclave process. 7. Freeze down 1 ml aliquots at −20 °C. To use, thaw one tube and make smaller aliquots. Store one at 4 °C for up to 2 months, and freeze the rest for later use. 8. Always use a high-fidelity polymerase for PCR to minimize the possibility of introducing mutations into the amplified fragment. Here is a specific recipe for C-terminally tagging FBA1 using the pKT plasmids [16] and KOD polymerase (EMD Millipore): 0.5 μl miniprep DNA (~100 ng), 5 μl 10x buffer, 4 μl 25 mM MgSO4, 5 μl 8 mM dNTPs, 0.5 μl primer WSO178 (50 μM), 0.5 μl primer WSO179 (50 μM), 0.5 μl KOD polymerase, 34 μl water (molecular biology grade). WSO178 sequence: 5′-AAGATCACCAAGTCTTTGGAAACTTTCC G T A C C A C T A A C A C T T T A g g t g a c g g t g c t g g t t ta-3′. WSO179 sequence: 5′-GATTCAATACTCATTAAAAA ACTATATCAATTAATTTGAATTAACtcgatgaattcgagctcg-3′. The 45 bp homology sequence is uppercase; the universal primer sequence is lowercase. PCR settings: 94 °C for 2 min, 30 cycles of {94 °C for 30 s, 55 °C for 30 s, and 70 °C for 3 min}, and then 70 °C for 10 min. When nat is the template, it is essential to add DMSO to a final concentration of 5 % [11]. 9. Transformation is mutagenic. It is therefore best to transform diploids and then sporulate, since one round of meiotic segrega- tion reduces the probability of obtaining an undesired mutation by 50 %. If multiple haploids of the desired genotype derived from the same diploid behave similarly, then background muta- tions are unlikely to be important. Alternatively, our lab has found that Illumina deep sequencing can reveal the presence of mutations caused by transformation. 10. To ensure that cultures are in exponential phase when it is time to transform, pilot growth experiments may be helpful. Wild-type RM11 grows very rapidly. If the density is >5×107 Adam James Waite and Wenying Shou
- 54. 37 cells/ml, dilute to allow the cells to complete at least two divisions in unsaturated conditions. Transformation efficiency remains constant for 3–4 cell divisions. 11. Keep small aliquots of SS-DNA to limit the number of freeze– thaw cycles. Keep on ice when out of the freezer. 12. Keeping the TRAFO master mix on ice is crucial for high efficiency. 13. Use >100 ng DNA. In general, more DNA yields more transfor- mants, but this relationship will likely saturate at some point. 14. This can be visually confirmed by ensuring that no visible “strands” are present in the mixture. 15. TRAFO is very viscous, so pipette slowly to ensure that the correct volume is transferred. 16. Mixing well ensures that the SS-DNA effectively blocks non- specific DNA binding. 17. Adjusting the amount of time for heat shock may be necessary to achieve maximum efficiency. However, 40 min works well for S288C and RM11. 18. Be as gentle as possible at this step, as the cells are very fragile. 19. Colony PCR is quick but can be unreliable. When it fails to work, we have found that a quick DNA extraction before PCR gives reliable results [23]. 20. The cell suspension should be turbid. 21. Unlike S288C, boiling of RM11 cells does not provide good tem- plate for PCR, although we do not know why. Using detergent effectively lyses the cells and releases their DNA into solution. 22. This can be done for as little as 3 h. 23. The amount should be small enough to not leave a visible film on the plate after transfer. 24. Different strains may require more or less time. Cells should show visible film of growth by this time. 25. Overgrowth will lead to a reduction in sporulation efficiency. However, a suitable number of tetrads should be present even after 12–14 h of pre-sporulation. 26. Vortexing can oxidize the zymolyase. 27. The four spores form a three-dimensional, tetrahedral shape if the ascus wall is undigested. After sufficient digestion, the four spores will have a flat, diamond shape. Underdigestion of the ascus wall will make it difficult to separate the individual spores. Overdigestion will result in tetrads that break apart easily, increasing the chances that four spores in the correct diamond shape are not products of the same meiosis. Overdigestion can also reduce spore viability. 28. Otherwise, growth of this strip will slow down the growth of the haploids nearest to it. Constructing Synthetic Microbial Communities
- 55. Another Random Document on Scribd Without Any Related Topics
- 56. XIV. Dagene var begyndte at længes. Fra tidlig om Morgenen, indtil Daglyset forsvandt, sad han ved Staffeliet. — Han vilde. Han havde tegnet tre eller fire Kartoner til et stort Figurbillede, kasseret dem alle og saa taget fat paa den femte. Heller ikke den var han tilfreds med, men han følte sin Uformuenhed til at gjøre det bedre. Saa blot videre — videre. — Han spændte Lærredet paa Blændrammen og tog fat paa at male Billedet. Atter og atter smurte han det over igjen. Naar han havde malet flittig hele Dagen og endelig syntes, at Gnisten var i Færd med at komme, saa det ligesom lysnede op i ham, blev det kun endnu værre, naar han den næste Morgen kom ind i Atelieret og saa Resultatet af den foregaaende Dags Arbejde. Hans Billede stod tungt og indholdsløst, uden Friskhed, uden Liv. Saa kunde han sidde og falde helt sammen, med Hovedet begravet i sine Hænder og med en forfærdelig knugende Følelse af, at Evnerne svigtede ham. Og dermed var igjen mange Dages møjsommeligt Arbejde spildt. Til sine Tider forekom det ham, at han ikke havde en eneste frisk eller ny Tanke, og at han ligesaa gjærne kunde opgive det hele. Til andre Tider myldrede det med Ideer om, hvorledes han vilde male, og hvorledes det skulde være — det stod altsammen saa klart for ham — men naar han saa førte Penslen op til Lærredet, nægtede Haanden at udføre sin Part, og hvad han malede blev tørt, kjedeligt og haandværksmæssigt.
- 57. Men han vilde. Og han begyndte forfra og forfra igjen. Omsider — en af de sidste Dage før Kunstnernes Arbejder skulde være indleverede til den aarlige Foraarsudstilling — blev hans Billede færdigt. Aldrig havde noget Arbejde kostet ham saa megen Kval. Han gad ikke se det. Og han var bange for at se det. Han havde vendt det om mod Væggen i Atelieret og lod det staa saaledes, indtil Karlen kom, der skulde bringe det op paa Charlottenborg. Og da det var borte, vidste han ikke rigtig, om han skulde føle sig lettet derved eller endnu mere mismodig og tvivlende end i alle de lange Arbejdsdage, da det havde staaet paa hans Staffeli. Men han var sig bevidst, at han havde lagt alt det i Billedet, som han havde evnet. Det kunde jo ogsaa være, at det var bedre, end han selv troede. Maaske var det den trykkede Sindsstemning, hvori han befandt sig, der fik ham til at se saa modfalden og utilfreds paa det. Sikkert var det, at det vilde blive afgjørende for ham, om dette Billed gjorde Lykke eller ikke. Han syntes næsten, at han maatte staa eller falde med det. Han havde med stor Spænding ventet paa Maleriernes Ophængning for at se, hvorledes hans »Vesterhavs-Fiskere« vilde se ud ved Siden af de andre Arbejder. Han var deroppe Dagen før den sædvanlige Aabningshøjtidelighed og traf sammen med adskillige af sine Kammerater. Men han havde ikke behøvet at høre deres mere eller mindre skaansomme Ytringer for at sige sig selv, at hans Arbejde var mislykket — en øjensynlig Tilbagegang. Thi trods den svigtende Produktionsevne havde han beholdt sit kritiske Blik baade for sig selv og andre. Han saa mere tydelig, end nogen kunde fortælle ham det, at hans Arbejde var forfejlet. Den samme umandige Svaghed, denne Halvhed mellem Energi og Uformuenhed, som han ofte var sig bevidst i sin Karakter som Menneske, syntes at træde frem ogsaa i hans Værk. I hans Billede var der en vis pedantisk Stivhed, blandet med et mislykket Forsøg
- 58. paa en kjækkere og friere Malemaade, og noget lignende gjorde sig gjældende i selve Indholdet. Der var Tilløb til en sund, frisk Friluftsstemning, men den knækkede over paa Halvvejen og blev til sygelig Følsomhed. Billedet var og blev mislykket. Der var adskillige dygtige Arbejder af hans Kammerater — Billeder, der vidnede om Udvikling og om voxende Kræfter. Han skammede sig ved at mærke hos sig selv, at han misundte dem. De andre gik frem. Skulde han være nødt til at opgive Kampen som frugtesløs. — Var Gnisten virkelig haabløst slukket? Eller havde den ikke været stærk nok til at blive andet for ham end en skuffende Lygtemand? Nej, det var umuligt. Han følte det saa sandt og vist hos sig selv, at han havde baade Kunstnerens Øje og Sind. Livet og Naturen aabenbarede sig villig for ham. Han var sig bevidst, at han forstod at tyde dem, og selv i dette mismodige Øjeblik mærkede han, hvorledes det gjærede i hans Sind med spirende Tanker og Syner. — Det var umuligt, at han havde kunnet tage fejl af sit Kald — at han kun skulde have Evne til at se og opfatte som Kunstneren, men ikke den skabende Kraft. Men Paavirkningen — baade den indre og ydre — havde manglet ham i altfor lang Tid. Efter Mødet med Margrethe Aaby var der fulgt en Tomhed og Sløvhed, som han ikke kunde faa Bugt med. Hun havde henvist ham og sig selv til Pligtens lige Vej. Han gjorde sig Umage for at følge den — og bildte sig ind, at det nærmest var, fordi han var for fejg til at gjøre andet — men det skete med en underlig sløv Viljeløshed, thi han havde vænnet sig til at se al Ting graat i graat. — Og dog var Redning maaske mulig endnu. Kunde han komme bort, rejse et Aars Tid, glemme en hel Del, som han gjærne vilde glemme, og samle friske Tanker — saa vilde Gnisten muligvis blusse op igjen.
- 59. Men skulde han blive hjemme, tynget af sin huslige Misère, smaalige Sorger for at leve — og de truede med at blive meget følelige, saafremt han ikke fik sit Billede solgt — men først og fremmest med den knugende Bevidsthed, at hans Evner ubarmhjærtig svigtede ham, saa syntes han, at det maatte føre til haabløs Undergang. Samme Dag, han havde indsendt sit Billede, havde han givet en Ansøgning ind om et af Akademiets store Stipendier. Det var bedrøveligt nok at tænke paa, at hans Fremtid paa en Maade syntes at afhænge af, om han fik den lumpne Sum Penge eller ikke. Men de »lumpne« Penge vilde sætte ham i Stand til at rejse ud og vinde friske Kræfter, skabe et nyt Væld i den udtørrede Kilde, og derfor blev det alligevel — hvor prosaisk det end var — et Livsspørgsmaal for ham. Men det havde de ærværdige Professorer næppe nogen Anelse om. Naar hans Billede blev fundet utilfredsstillende, var det ikke rimeligt, at han vilde faa Stipendiet. Man vilde vistnok i alt Fald komme til det Resultat, at han havde bedst af at vente et Par Aar paa en saadan Opmuntring. Og imidlertid — men det var jo alligevel muligt, at det gik bedre, end han troede. Det var en kold, sludfuld Dag i Slutningen af April. En af de gjækkende Dage, hvor Solen pludselig bryder frem mellem regntunge Skyer og varsler om Foraar og Sommer for strax efter at skjule sig igjen og lade det falske Smil efterfølge af stride Regnstrømme og raslende Haglbyger. Gader og Torve laa kølig forfriskede i et Regnbad, der havde efterladt brede Vandpytter paa Stenbroen, og de letsindige Indvaanere af begge Kjøn, der havde ladet sig friste af en Smule blegnæbet Middagssol til altfor tidlig at stikke i Foraarstøjet, hastede nu med blaalig anløbne Kinder og røde Næsetipper langs med
- 60. Husrækkerne, under ihærdige, men ofte frugtesløse Anstrængelser for at holde Paraplyen stik mod Vinden. — Henning havde taget en Beslutning. Han vilde tale med en af de Professorer, der var beskikkede til at uddele Stipendierne. Han vilde ærlig gjøre rede for, hvor magtpaaliggende det var ham at komme ud for at modtage en befrugtende Paavirkning, som han følte, at han trængte haardt til. Det havde kostet ham en Del Overvindelse at holde fast ved dette Forsæt og give sig i Færd med at udføre det, men der stod saa meget paa Spil, at han ikke vilde lade noget Middel uforsøgt til at rejse sig igjen efter det sidste Nederlag. — Saa begav han sig hin sludfulde Aprildag paa Vejen til den velkjendte Bygning, hvor Professoren havde Embedsbolig. Det var henimod den Tid af Dagen, da Udstillingen lukkes, og i Porten mødte han en Sværm af besøgende: kritisk udseende Herrer og Damer, der stak deres Katalog og Blyantspen i Lommen, idet de nærmede sig Udgangen; Gjæster fra Provinserne, der saa' saa ivrige og travle ud, som om de endnu havde et Par Maleriudstillinger og diverse Museer at støve igjennem, før de med en rolig Samvittighed kunde lægge deres mødige Lemmer til Hvile i Hotellernes rummelige Senge. Nogle blev staaende i Porten med de allerede opslaaede Paraplyer for at vente, til Bygen var trukken over, og i Sværmen saa Henning et Par bekjendte Ansigter, som han skyndte sig forbi for ikke at blive nødt til at indlade sig i en ligegyldig Samtale, som han ikke følte sig videre oplagt til. — Han skraaede over Gaarden, gik ind ad Porten til højre, hvor der stod en Stabel Kasser, der havde gjemt eller endnu indeholdt Billeder, bestemte til »anden Ophængning«, og stod i Begreb med at gaa op ad den brede, snirklede Trappe, da han blev standset af et af Akademiets Bude, gamle Blæsenberg, hvis ulastelige, sorte Frakke, stive Halsbind og omhyggelig børstede Filthat havde et saa officielt
- 61. Præg, at ingen kongelig Kontorchef havde behøvet at skamme sig ved at bytte Klæder med ham. Blæsenberg havde derfor ogsaa i mange Aar gaaet under Hædersnavnet »Chefen« blandt Akademiets Elever, der kjendte hans knirkende Støvler i halvtresindstyve Skridts Afstand. Denne indflydelsesrige Personlighed standsede Henning paa Trappen med et fortroligt: »Goddag Hr. Bentsen! Det var grumme rart, jeg traf Dem, for jeg gaar her med et Brev, som jeg skulde ha'e brungen ud til Dem. Det var saa grumme heldigt. Værsgo', Hr. Bentsen!« Med disse Ord leverede han Henning et stort, embedsmæssig udseende Brev i blaa Konvolut, og da Henning modtog det uden at synes tilbøjelig til at indlade sig i nærmere Samtale, gik Blæsenberg videre efter at have gjort en kort Bemærkning om det grumme triste Vejr. — Henning blev staaende med Brevet i Haanden. Han havde paa Fornemmelsen, hvad det indeholdt, men tøvede med at aabne det. Saa rev han endelig Konvolutten op. — Skrivelsen var affattet i den sædvanlige Kancellistil. Stipendiet var ikke tilstaaet ham. — Han gik tilbage over Gaarden, ud gjennem Hovedporten, der imidlertid var bleven rømmet af det udstillingsbesøgende Publikum. Den smækkede i bag ved ham med et lydeligt Drøn, som om den for evig Tid vilde lukke ham udenfor de indviede Mure. Han blev staaende og saa ud over Torvet, der laa som en blinkende, mørk Flade foran ham. Regnen piskede ned mod Brostenene og paa de endnu bladløse Trær rundt om »Hesten«. Sporvogne og drivvaade Drosker rullede forbi; Folk gik med Paraplyen helt nede over Hovedet; Vandet sivede ud af Tagrenderne med sit ensformige Prik-Prik. De røde Lygter blev satte udenfor Theatret, og de første Theatergængere — Folk fra Provinsen, der holdt sig til Plakaternes Bemærkning om, at »Indgangen aabnes Kl. 6½« — begyndte saa smaat at indfinde sig. —
- 62. Han blev staaende, uvis om, hvor han skulde gaa hen. Hjem gad han ikke gaa. Dér ventede ham ingen Opmuntring. Han tænkte paa, hvor helt anderledes det vilde have været, dersom han hjemme havde fundet den Kjærlighed og Forstaaelse, som kan hjælpe en Mand til at overvinde selv de største Hindringer og forsone ham med de bitreste Skuffelser, han møder ude i Verden. Men det nyttede ikke at gruble derover. Det var nu en Gang saa og kunde ikke blive anderledes. Han følte ikke længere nogen Bitterhed mod Minna, snarere en bedrøvelig Medynk baade med hende og sig selv. Men Midlerne til at redde sig fra Skibbruddet — hvor skulde han finde dem? Saa faldt det ham pludselig ind at henvende sig til en af Stadens Mæcenater, en Mand, der havde Ord for at have hjulpet mange unge Kunstnere frem. Han vilde forklare ham, hvorledes Sagerne stod — naturligvis med Undtagelse af sine huslige Forhold — fortælle ham, at hans Fremtid som Kunstner beroede paa, at han kunde komme bort et Aars Tid for at faa friske Kræfter, og bede ham om de nødvendige Penge som et Laan. Hvad var et Par tusende Kroner for en Mand, der ejede flere Millioner, og hvis Formue voxede hvert Aar med adskillige Hundredetusender. Men for ham, Henning, var den forholdsvis ubetydelige Sum ensbetydende med nyt Liv og en ny Tilværelse. Desuden mente han, at han kunde stille nogen Sikkerhed for et saadant Beløb. Jo mere han tænkte derover, des mere sandsynligt forekom det ham, at det kunde lykkes, og han fik omsider Mod til at vove Forsøget. Da han først var kommen saa vidt, syntes han, at det var bedst lige strax at sætte Beslutningen i Værk. — Klokken var hen ved syv, altsaa ikke den Tid, man plejer at gjøre Visit hos fornemme Folk. Men lige meget — han vilde ikke opsætte
- 63. det. Han begav sig paa Vej til det Kvarter af Byen, hvor den store Mand boede. Tjeneren, der modtog hans Kort, saa spørgende op og ned ad ham og bemærkede, at Excellencen ikke modtog paa den Tid, men han skulde gjærne aflevere Kortet. Dermed gik han og vendte kort efter tilbage for at bede Henning træde ind. Excellencen skulde strax være til Tjeneste. Henning befandt sig i en rummelig Hjørnestue, halvt Bibliothek, halvt Arbejdsværelse, udstyret i gammeldags Stil, med al den Luxus, som en betydelig Formue i Forening med en fint udviklet Smag kan tilvejebringe. Over Skrivebordet hang en ægte Rembrandt, paa den modsatte Væg nogle Stykker af gamle danske Mestere. — Overalt bløde, dæmpede Farver, tunge, mørkerøde Silkegardiner, en mat bronceret Lysekrone, Bøger, indbundne i brunt Maroquin; bredbladede, mørkegrønne Planter i smagfulde Stativer, Stole med lave, magelige Sæder, et Tigerskind med udstoppet Hoved inde under Skrivebordet. — Der gik ti og atter ti Minuter. Henning hørte nu og da en fjærn Klirren af Glas og andet Bordservice. Excellencen havde aabenbart endnu ikke rejst sig fra Middagsbordet. — Folk plejer at være i en blid og medgjørlig Stemning, naar de har spist godt. Han vilde haabe, at Excellencen maatte være tilfreds med sin Middag. — Saa ventede han endnu en halv Snes Minuter. Haglene piskede mod Ruderne, og Lyset, der selv midt paa Dagen havde Møje med at trænge ind mellem de folderige Gardiner, blev mere og mere dæmpet. Han havde sat sig paa en af de smaa Hjørnesofaer og lænede Hovedet mod Rygstødet. Hans Blik strejfede ørkesløst de forskjellige Gjenstande i Værelset, medens han forestillede sig, om Excellencen, hvem han aldrig havde set personlig, var en høj, imponerende Skikkelse eller en lille, tør, mager Mand; om hans Stemme var barsk og bydende eller muligvis fin og sleben som en ægte Hofmands. —
- 64. Men lidt efter lidt forlod hans Tanker disse Enemærker og vandrede langt bort fra den store Mand og hans Arbejdskabinet. Værelset var opfyldt af en let, behagelig Vellugt, der blandede sig med den stærke Varme fra en massiv Fajanceovn, og uden at han selv vidste af det, faldt hans Hoved tilbage mod Sofaens Rygstød; de Billeder, der drog forbi ham, antog mere og mere taageagtige Skikkelser. — Et pludseligt Lysskær vækkede ham af den Døs, hvori Trætheden og den lange Ventetid havde bragt ham. Han sprang op fra sin magelige Stilling og gjorde et forvirret Buk for Excellencen, der var traadt ind i Stuen, fulgt af en Tjener, som satte en Lampe hen paa Skrivebordet og derpaa forsvandt. »Jeg beder Dem — bliv kun siddende,« sagde Hs. Exc. med et fint, maaske en lille Smule ironisk Smil. »Tør jeg spørge, hvormed jeg kan være til Tjeneste? — Jeg mindes ikke før at have havt den Fornøjelse ....« »Jeg tør maaske antage, at mit Navn ikke er Deres Excellence helt ubekjendt,« sagde Henning. »Nej —« Excellencen raadførte sig med Visitkortet, som han endnu havde i Haanden. — »De har vist flere Gange havt udstillet — ikke sandt? Om jeg husker rigtig, har De ogsaa et Billede deroppe i Aar — nogle Fiskere fra Vestkysten — tror jeg?« »Ja desværre,« svarede Henning. »Det er et mislykket Arbejde, som jeg ikke burde have udstillet. Jeg ...« »Aa hvad — De er en ung Mand. Har De været mindre heldig i Aar, kan De jo tage Revanche en anden Gang. De har jo Tiden for Dem. — Men hvormed ...« Excellencen kastede et Blik paa Uret ligesom for uden al Fortrydelse at minde den unge Mand om, at hans Tid var mere kostbar, og at han ikke havde den for sig i en tilsvarende Grad, hvad der kunde være en hel Del i, naar man tog Hensyn til, at han var en meget gammel Mand.
- 65. »Hvormed ...?« Henning søgte med saa faa Ord som muligt at komme frem med, hvad han havde paa Hjærte, og imidlertid sad den lille sirlige, hvidhaarede Mand og hørte paa ham, stadig med samme uforanderlige, halvt ironiske, halvt opmuntrende Smil. »Det maa vistnok synes Dem underligt, at jeg kommer med en saadan Anmodning uden at være personlig kjendt af Dem,« sluttede Henning, »og jeg skulde sikkert heller ikke have gjort det, hvis jeg ikke havde en saa bestemt Følelse af, at hele min Fremtid ...« »Ja vist, ja vist,« afbrød den lille Excellence. »Saaledes er det med dem alle — med dem alle. Det gjælder altid hele deres Fremtid. Men det er umuligt at hjælpe enhver. Der stilles saa mange Krav. Man vil gjærne gjøre, hvad man kan. — Men det er umuligt — ganske umuligt. — Jeg forsikrer Dem, kjære unge Ven, at selv den største Formue vilde spille Bankerot, hvis man skulde træde hjælpende til i alle Tilfælde. Nej, det er umuligt — ganske umuligt.« »Jeg havde ikke tænkt mig at modtage denne Sum som en Gave,« sagde Henning, hvis Kinder begyndte at farves røde, ikke saa meget af Fortrydelse over Afslaget, thi det kunde altid være lige saa vel motiveret som hans Anmodning, og der var jo desuden en hel Del Sandhed i, hvad den gamle Mand sagde — men fordi han mere og mere følte det ydmygende i Situationen. »— — Det var ikke min Mening at modtage det som Gave, men som et Laan. Jeg havde tænkt at kunne give f. Ex. en Livspolice i Sikkerhed, og ...« Han stammede lidt i det ... »naar jeg skaffede behørig Sikkerhed for Præmiens Betaling, vilde Gjælden jo i værste Tilfælde blive betalt ved min Død, og Deres Excellence eller Deres eventuelle Arvinger vilde saaledes under ingen Omstændigheder kunne gaa tabt af Kapitalen ...« »Meget rigtigt — meget rigtigt. Men jeg beklager ikke at kunne indlade mig paa den Slags Sager. — Det er jo muligt, at De kan faa det ordnet saaledes i et eller andet Pengeinstitut ...«
- 66. Excellencen, der i de sidste Minuter utaalmodig havde ladet en Papirkniv glide frem og tilbage mellem sine spidse, hvide Fingre, hostede ganske sagte, og det opmuntrende Smil blev mere køligt. »Saa maa jeg endnu blot bede Dem undskylde, at jeg har henvendt mig til Dem,« sagde Henning og rejste sig. »Trods Deres Excellences Bemærkning kan jeg ikke andet end gjentage, at jeg ikke vilde have gjort dette Skridt, hvis jeg ikke havde været meget haardt tvungen dertil.« Den lille Mand saa op med et hastigt Glimt i sine skarpe, graa Øjne. »Ja — det gjør mig meget ondt. Hermed kan jeg ikke være Dem til Tjeneste. — Kan derimod et mindre Beløb muligvis hjælpe Dem i en øjeblikkelig Forlegenhed, saa ...« Han gjorde en Bevægelse med Haanden hen til Skrivebordsskuffen, men Udtrykket i Hennings Ansigt overbeviste ham om, at han havde misforstaaet hans sidste Ytring. »Saa beklager jeg, at De maa gaa fra mig med uforrettet Sag. Altid meget kjedeligt — meget kjedeligt for begge Parter.« Henning bukkede tavs, og den lille Excellence fulgte ham med sin aldrig svigtende, tilknappede Høflighed et Par Skridt henimod Døren. — Regnen og Haglbygerne var holdt op. Der viste sig en klar Lysning mellem Skyerne, og Blæsten, der jog fejende omkring Gadehjørnerne, havde tørret Fortovene, saa kun de større og mere udholdende Vandpytter var blevne tilbage i Stenbroens ujævne Fordybninger. Henning gik langsomt hjemad, lige saa aandelig træt, som han var det legemlig. Den ydmygende Situation, han havde lagt bag ved sig, vedblev at forfølge ham. Det var, ligesom den paanødte ham en Følelse af, hvor lidet vidt han havde bragt det. I gamle Dage havde han fundet en
- 67. Slags Tilfredsstillelse i at anstille Beregninger over, at naar han blev saa og saa gammel, skulde han være naaet saa eller saa langt frem. Det var en Art Milepæle, han opstillede paa sin Fremtidsvej. Men det opgav han nu. Der laa saa mange svigtede Forsætter bag ved, at han blev led ved dem og ligesom undsaa sig ved at føje ny til — for ogsaa at bryde dem. Der havde været en Tid, hvor den Ydmygelse, han nylig havde lidt, vilde have opflammet hans Energi og ægget ham til at trodse sine Uheld. — Han kom til at tænke paa den Dag, da han som ganske ungt Menneske — ikke stort mere end en Dreng — var kommen op med sit Maleri til Fabriksinspektøren, der med saarende Ord havde raadet ham til at slaa de Nykker af Hovedet. Den Gang var der blevet vakt en saadan Trods i hans Sind, og han vidste af Erfaring, at den kan hærde baade Vilje og Evner. Men ogsaa denne Følelse lod nu til at være død for ham. Han kæmpede med sig selv for at kalde den til Live igjen, men det vilde ikke lykkes. Men han kunde saa tydelig se, hvorledes det altsammen var kommet og maatte komme saaledes. Han var sig sine egne Fejl fuldkommen bevidst, men alligevel formaaede han ikke at mande sig op til at begynde Kampen forfra. Paavirkningen maatte komme ude fra. Skæbnen maatte sende ham den, thi inde i ham selv var Visen sungen til Ende. — Han naaede omsider hjem, stadig forfulgt af de samme Tanker, der atter og atter vendte tilbage i det samme ensformige Kredsløb. — Da han havde spist til Aften med Minna, gik han ind i Atelieret og kastede sig paa Sofaen uden at bryde sig om at tænde Lys. Han laa og stirrede paa det tomme Staffeli, grundende over, naar han vel vilde faa begyndt paa et nyt Arbejde. Deres Sovekammer stødte op til den bageste Væg i Atelieret, og en Tid lang kunde han høre Minna pusle derinde. Men lidt efter gik hun ud af Stuen, og al Ting blev ganske stille.
- 68. Han havde vel ligget et Par Timers Tid paa Sofaen uden at sove, men i en halvt drømmende Tilstand, under hvilken han maatte gjøre Vold paa sig, naar han vilde knytte Tankerne sammenhængende til hinanden, da han pludselig blev skræmmet op ved en kort, rallende Hoste, der lød inde fra Sovekammeret. Den varede kun et Øjeblik, men havde noget uhyggeligt ved sig, lignede slet ikke den Maade, hvorpaa Børn undertiden hoster i Søvne, og Henning, der bestandig, endog uden den mindste Grund, plagedes af en overdreven, sygelig Ængstelse for Barnet, sprang forskrækket op og ilede ind i Dagligstuen. Men Minna, der havde været nærmere ved Sovekammeret, var allerede kommen ham i Forkjøbet, havde været inde og taget Barnet op og kom nu løbende ind, bærende det paa Armen. »Men Gud — han kvæles — se — hvad fejler dog lille Aage?« Hun stod og rystede over hele Kroppen, medens hun holdt Barnet, hvis Bryst arbejdede med en krampagtig Stønnen. Dets Ansigt var blegt, med et let blaaligt Anstrøg, og Øjnene havde et forpint Udtryk, som om de ytrede en stum Bøn om Hjælp mod den usynlige Fjende, der plagede det lille Legeme. »Se — Henning — han kan ikke faa Luft!« skreg Minna. Han var lige saa bleg og forskrækket som hun og famlede efter sin Hat for at løbe efter Lægen. Minna havde vendt Barnet om paa sin Arm og slog det i Ryggen for at fremskynde en Opkastning, dersom det skulde have sunket noget. Men derpaa fulgte den samme uhyggelige, rallende Hoste som første Gang, kun mere besværlig og langvarig. Henning vendte sig om i Døren og saa med et fortvivlet Blik hen paa Moderen og Barnet. Han hørte endnu Minna skrige: »Aa Gud — det kan da ikke være Strubehoste!« — Men derpaa fór han ned ad Trappen, gjennem den lille Forhave og hen ad Vejen, som om han havde havt Vinger. —
- 69. Han løb hele Vejen ind til Byen, saa hurtig som en ung kraftig Mand kan løbe, naar det gjælder om at frelse et Liv, der er ham mere dyrebart end hans eget. Han løb, saa det peb og gispede i hans Bryst; sagtnede ikke et eneste Sekund sin voldsomme Fart, pressede kun Haanden mod det smertende Sting, han følte i Siden, og løb videre og videre uden at ænse, hvorledes Blodet strømmede til hans Hoved og pressede sammen om hans Hjærte. Der var en dødelig Angst i hans Sind. Musklerne i hans Ansigt fortrak sig i den stumme Smerte. Hans Øjne brændte, og han havde en tør, kvælende Fornemmelse i Halsen. Hans Søn — hans prægtige Dreng dø! Hans hidsede Fantasi forestillede ham, at det allerede var sket. Han saa den smukke, raske Dreng ligge Lig, med Ligets voxagtige Bleghed over de elskede Træk. Han saa dem komme med Dødens uhyggelige Attributer for at skrinlægge det kjæreste, han ejede. Han hørte dem hugge Sømmene til — saa tydelig, at Nerverne i hans Hoved dirrede derved. Hans Bryst snøredes sammen, og han løb — løb som en afsindig. — Lægen var heldigvis hjemme. Han forklarede ham, saa godt han kunde, hvad der var sket, og et Par Minuter efter sad de i en Droske og rullede afsted til hans Hjem. De fandt Minna gaaende op og ned ad Gulvet med det syge Barn i sine Arme. Lægen forlangte en Ske, som han holdt paa den lilles Tunge for at se ham ned i Halsen. Henning maatte holde Lyset imens. Doktoren rystede paa Hovedet, spurgte, om Barnet havde været hæs i Løbet af Dagen. Nej, lille Aage havde været fuldkommen rask og lige saa kvik og munter, som han plejede. Kunde det være muligt, at det var den skrækkelige Strubehoste?
- 70. Doktoren rystede atter paa Hovedet. Det var vanskeligt at afgjøre i Øjeblikket. Det var muligt, at det kun var en Halskatarrh, et Tilfælde, som man kaldte for falsk Strubehoste, og som tit plejede at angribe Smaabørn ganske pludselig. Han bad dem berolige sig og haabe det bedste. Barnet skulde have koghede Omslag om Halsen og hver halve Time en Theskefuld af nogle Brystdraaber, paa hvilke de nu skulde faa Recepten. Henning løb hen paa Apotheket. Han ringede gjentagne Gange paa Natklokken, før den vagthavende indfandt sig. Saa blev han lukket ind i det kvalme Lokale med dets underlige krydrede Luft. Han maatte vente, indtil den søvndrukne Apothekerlærling fik Medikamentet tillavet, og de fem eller ti Minuter, der gik, forekom ham som lige saa mange Aarhundreder. Hans Søn — hans lille, prægtige Dreng! Det var de samme Ord, formende det samme Billede, der atter og atter gjentog sig for ham. Og paa samme Tid følte han en heftig Trang til at anraabe Gud om at have Medlidenhed med hans Hjærteangst. Men han kunde ikke føje Ord og Tanker sammen til en Bøn. Det var enkelte, forpinte Skrig, der undslap hans Bryst og banede sig Vej did op, hvor der — han mindedes det fra sin Børnelærdom — skal være idel Lys og Retfærdighed. Det blev ved at kæmpe og arbejde i ham, medens han skyndte sig tilbage til sit Hjem, stadig med den samme kvælende hede Fornemmelse i Brystet; med det samme Billede af hans lille Yndling stillende sig frem for hans opskræmmede Tanker og med den samme heftige, men afmægtige Trang til at give sin Fortvivlelse Luft i Graad og Bøn. — Derpaa fulgte Natten, den ubeskrivelig lange, kvalfulde Nat, medens de begge sad oppe og vaagede hos det syge Barn, lyttende med ængstelig Spænding til dets Aandedrag, stirrende ufravendt paa det, saa længe det laa hensunket i et kort Blund, og begge to følende det samme isnende Stik i Hjærtet, hver Gang Hosten kom igjen.
- 71. Var det en Straf for deres Vildfarelser, for deres Mangel paa Kjærlighed og Overbærenhed med hinanden — for alt det stygge, der havde været i deres Samliv? Kunde Skæbnen ville ramme dem saa forfærdelig haardt? Overfor den store Sorg, der truede Henning, forekom det ham, at alt andet, hvad der havde brudt ham ned i de sidste Aar, blev saa uendelig smaat og betydningsløst. Dette var den første store Hjærtesorg, og den var kommen saa pludselig og overvældende. Han foragtede sig selv, fordi han før var falden sammen og havde bildt sig ind, at hans Liv var tomt og indholdsløst, hans Kræfter opbrugte. Nu saa han først, at han kunde lide et Tab, der var helt anderledes tungt. Maatte han dog blot blive sparet for det. Alt andet kunde gjenoprettes. Kun ikke det — kun ikke det. — De laa paa Knæ, hver ved sin Side af Sofaen inde i Dagligstuen, hvor der var blevet redt til det syge Barn. Lampen brændte paa Bordet og kastede sit Skær hen paa det lille Ansigt, der fortrak sig urolig i Søvne. Hver Erindring, der knyttede sig til Barnet, kaldtes atter frem — Erindringer, der vilde synes latterlig smaa for alle andre, men som fik deres Hjærter til at bæve og snørede deres Bryst sammen i kvalt Graad. De første, usikre Skridt paa egen Haand, de første, stammende Ord, den første Fødselsdag og det første Stykke Legetøj. Hvert lille Fremskridt, der var forekommet dem som et mærkeligt Vidunder. Barnets pudsige Indfald, dets muntre Leg og dets ømme Kjærtegn — denne friske Barnemund, der bød sig saa lokkende til Kys, og disse runde, bløde Arme, der saa utallige Gange havde slynget sig om deres Nakke med et Overmaal af barnlig Kjærlighed. Og alt det skulde være forbi. Nej — nej. Kun ikke det — kun ikke det! Dagslyset begyndte at kæmpe med Skæret fra Lampen og fik mere og mere Indpas gjennem de nedrullede Gardiner. Skyggerne, der
- 72. havde ligget og forstukket sig under Møblerne, trak sig længere og længere tilbage og forsvandt tilsidst helt for den frembrydende Morgendæmring. — Det havde allerede en god Stund været saa lyst udenfor, at man kunde have slukket Lampen og rullet Gardinerne op, men ingen af dem havde Tanke derfor. Den fjedrede Befolkning i Træerne ude i Alléen begyndte at istemme sin Morgenkoncert, først en enkelt Sanger og derpaa hele Skaren. Men de hørte det ikke. Solen sendte sine første smaa Lysglimt ind mellem Rullegardinet og Vindueskarmen. De lagde ikke Mærke dertil. Barnet var faldet i en fast Søvn; det syntes at trække Vejret lettere, og den uhyggelige, rallende Lyd i Brystet og Struben kom svagere og med længere Mellemrum; Farven lod ogsaa saa smaat til at vende tilbage paa Kinderne. De fulgte disse Symptomer, og Haabet begyndte at vaagne hos dem, men de turde ikke stole paa det. De saa' spørgende paa hinanden, vexlede nogle Ord med en Stemme, der dirrede af Bevægelse, og bøjede sig hvert Minut over det slumrende Barn for at lytte til dets Aandedrag, snart grebne af Uro over den lange Søvn, snart givende sig mere og mere hen til Haabet om, at Faren var overstaaet. — Pigen, der var bleven oppe for at hjælpe dem med at passe den lille Patient, havde, uden at de vidste af det, lavet Kaffe ude i Køkkenet og kom nu ind med en Præsenterbakke, som hun satte paa Bordet i Dagligstuen. I det samme vaagnede den lille Dreng, smilede til dem og rakte sine Arme frem. Nattens frygtelige Angst og Spænding gjorde paa én Gang Plads for en Følelse, saa glad og befriende, som de næppe før havde kjendt den. De lagde sig paa Knæ ved Barnets Seng, bedækkede hans Pande, Mund og Hænder med lidenskabelige Kys, saa at Drengen saa' halvt forundret, halvt ængstelig paa dem. »Lille Aage bliver snart rask — du skal ikke græde, søde Moder,« stammede den lille Fyr.
- 73. Deres Blikke mødtes; stiltiende rakte de hinanden Haanden, kæmpende hver paa sin Side for at skjule, hvorledes Graaden stansede Ordene i deres Mund. Saa kastede Minna sig pludselig om hans Hals med en lidenskabelig Hulken. »Aa, Henning — Henning,« mumlede hun — »Vorherre har været meget bedre mod mig, end jeg har fortjent — Gid jeg aldrig maa glemme det ...« Hun trykkede sit Hoved op til hans Bryst, og han bøjede sig ned og kyssede hendes Pande. Da Lægen kom igjen op ad Formiddagen, erklærede han, at der ikke var nogen som helst Grund til yderligere Ængstelse. Det havde kun været et Tilfælde af den omtalte, falske Strubehoste, hvis Begyndelsessymptomer vanskelig lod sig skjelne fra den ægte.
- 74. XV. Han var bleven revet ud af sin Sløvhed og mindet om, at han endnu havde noget at leve og arbejde for. Allerede et Par Dage efter, at lille Aage var bleven rask, stod der et Udkast til et nyt Billede paa hans Staffeli, og han mærkede, at noget af den gamle Arbejdslyst var kommet over ham igjen. — Han arbejdede flittig, gjorde sig Umage for at jage alle Graavejrstanker paa Flugt, om de end meldte sig nok saa paatrængende, og søgte paany at afvinde sit Forhold til Minna de bedste og lyseste Sider. Hun prøvede, saa godt hun kunde, paa at komme ham i Møde. De vilde aabenbart begge to saa gjærne holde fast ved deres gode Forsæt, naar det blot maatte lykkes. I den første Tid gjorde hun Vold paa sig selv for at synes blidere og mere tilfreds, end det laa i hendes Natur at være. Men hun forstod ikke at forstille sig, og hun led ved denne Tvang. Det førte ikke til andet end til en Slags kunstig tilvejebragt Vaabenstilstand, der var lige saa trykkende som den tidligere aabenbare Krig, og det tjente kun til endnu tydeligere at vise dem, hvor fjærnt de stod hinanden, og hvor haabløst det var at tro paa, at det nogensinde kunde blive anderledes. — Og der var saa meget, i hvilket de kunde have trængt til at have en Støtte i hinanden. Henning maatte prøve en hel Del af de Ydmygelser, som Pengenød fører med sig, og Minna forstod ligesaa lidt nu som tidligere at finde sig taalmodig i den Slags Ting. Hun klagede bittert over alt det, de maatte gaa igjennem, drog gjærne Sammenligninger med dem, om hvem hun mente, at de havde det meget bedre end hun selv. — Kunde Henning ikke
- 75. begynde paa noget andet end at være Maler; det førte jo dog aldrig til noget. Hun sagde det ikke i nogen ond Mening; kun fordi hun hverken forstod ham eller hans Arbejde. Men det saarede ham, og han tænkte med Bitterhed paa, hvem der vel skulde tro paa ham, naar ikke en Gang hans Hustru gjorde det. — Han havde sat al sin Arbejdsevne ind paa sit ny Billede. »Enkens Søn« skulde det hedde; det forestillede en Moder ved sit Barns, en halvvoxen Søns Dødsleje. Han arbejdede undertiden med en overdreven Anspændelse af alle sine Evner og nærede en sygelig Ængstelse for, at den Gnist, som han næsten med Vold og Magt holdt fast, skulde svigte ham, før han blev færdig. Saa var det ud paa Sommeren. De havde ikke havt Raad til nogen længere Rejse, men for at bringe lidt Afvexling i den trykkende Ensformighed havde Henning lejet en tarvelig Sommerbolig i et Fiskerleje nogle Mil fra Hovedstaden. De havde boet derude en Maaneds Tid, da Henning en Dag maatte tage til Byen af den meget tvingende Aarsag, at de ikke havde flere Penge i Huset. Det var usædvanlig varmt Vejr. Luften havde lige fra tidlig om Morgenen truet med Torden, og Brostenene inde i Byens Gader brændte en under Fødderne. Ude i Alléerne, hvor Træerne havde faaet det støvede og forjaskede Udseende, der viser, at den smukkeste Del af Sommeren er forbi, vandrede Folk i store Skarer afsted til de forskjellige Forlystelsesanstalter uden at bryde sig om Tordenskyerne, der trak sammen i stedse tættere Masser, medens nu og da en pludselig, let Susen i Trætoppene spaaede om, hvad der var i Vente. — Henning havde travet omkring i Byen fra tidlig paa Formiddagen. Og da han omsider havde faaet udrettet, hvad han skulde, var det sidste Tog og det sidste Dampskib afgaaet. Saa var der ikke andet for end at overnatte i Byen.
- 76. Han var bleven uvant med at tilbringe en Aften paa egen Haand og vidste ikke, hvorledes han bedst skulde faa Ende paa den. Det havde stadig hørt med til Gaaderne i Minnas Karakter, at skjønt hun var sær og uvenlig, naar han var hjemme, blev hun dog meget ilde til Mode, naar han en Aften vilde gaa ud alene, og Henning, der efterhaanden havde brudt fuldstændig med sin gamle Omgangskreds, havde vænnet sig til at føje hende i dette Punkt. — Han slæntrede langsomt ud ad Broen og Alléen, medens han med en ørkesløs Mine betragtede de livlige Menneskegrupper, som vandrede forbi ham med al den Travlhed, der er betegnende for Kjøbenhavneren, naar han er ude for at more sig. Før han rigtig vidste af det, var han kommen i Nærheden af »Runddelen«. Han naaede det lille Forstadstheater, paa hvilket der gives Forestillinger i Sommersæsonen, og i Mangel af bedre Sysselsættelse gav han sig til at studere en af de gloende røde Plakater, der var slaaet op paa hver sin Side af Indgangen. Spasereturen gjennem den skyggefulde Allé, hvor det dæmrende Halvmørke havde fortrængt Dagens brændende Solhede, og Synet af den brogede Menneskesværm havde stemt ham lidt livligere, og det faldt ham ind, at han lige saa gjærne kunde nyde sit Aftensmaaltid i det lille Theater som paa ethvert andet Sted. Han løste Billet og traadte ind paa Tilskuerpladsen, over hvilken der hang en graalig Taage af Øl- og Tobaksdunster, som halvvejs skjulte det talrig tilstedeværende Publikum, der havde grupperet sig omkring de smaa firkantede, under Vægten af Smørrebrødstallerkener, Iskageassietter, Ølkrus, Toddyglas og Chokoladekopper bugnende Borde. Tonerne fra Orkestret skar sig trangbrystet Vej gjennem den øredøvende Summen af snadrende Stemmer, men forhindrede ikke disse Stemmers Ejermænd i at forlange hvert eneste Musikstykke da capo et Par Gange, saa lidt som den utrættelige Dirigent i at
- 77. efterkomme de for ham og hans Orkester saa smigrende Opfordringer. Skjønt der ikke lod til at være saa megen tom Plads i Salen, at man kunde sætte sin Støvlehæl der, lykkedes det dog en Opvarter med beundringsværdig Behændighed at bugsere Henning helt op foran Scenen, hvor han med en lige saa utrolig taskenspilleragtig Dygtighed tilvejebragte baade en Stol og et Bord. Henning tog Plads for at nyde de Forfriskninger, han havde bestilt. Han sad og kiggede i Programmet og tænkte allerede saa smaat paa at gaa sin Vej, saa snart han var færdig med sit Smørrebrød og Øl, thi Luften var saa trykkende hed og kvalm, at ikke en Gang Programmets lovende Meddelelse om et nyt Stykke med en Debutantinde indeholdt den allersvageste Fristelse for ham til at blive. — Men Tæppet gik op, før han kunde slippe ud, og han besluttede da at finde sig taalmodig i sin Skæbne, til Stykket var spillet til Ende. Imidlertid morede han sig med at betragte Tilskuernes Fysiognomier. Der var smaa Sypiger og unge Kavallerer, hvis Bekjendtskab syntes at grunde sig mere paa en hastig tilvejebragt Fortrolighed end paa en langvarig Prøve. Der var ærbare Borgerfamilier med deres voxne Døtre; unge, flot klædte Herrer, der aabenbart tjente den letfodede Mercurius, og hvis støjende Bifaldsytringer dundrede gjennem Salen; der var nylig indkaldte Jenser med deres Kjærester, og gamle Pensionister, som tydelig nok hørte til de trofaste Stamgjæster, fordi de mente, at de lige saa gjærne kunde drikke deres Øl her som et hvert andet Sted. Der var solide og trivelige Madammer, der af Hjærtens Lyst nød Komedien med den troskyldigste Andagt præget i deres rødmussede Ansigter. Der var unge og gamle, blaserede Herrer med Lorgnetten kneben ind foran det venstre Øje; blegnæbede Fabrikpiger, som lo paafaldende højt og saa sig omkring med udfordrende Blikke. Der var en og anden Skuespiller fra et af Hovedstadstheatrene i Selskab med et Par Forfattere eller Journalister. —
- 78. Henning, som havde været optagen af at udskille Bestanddelene i det brogede Selskab, blev pludselig opmærksom paa, hvad der foregik paa Scenen, ved at høre Klangen af en ualmindelig frisk og klar Pigestemme, der lød paa en for Øret højst velgjørende Maade mellem de andre Skuespilleres mere eller mindre falske og forskregne Stemmer. I det samme brød en vældig Bifaldssalve løs paa Tilskuerpladsen, og nogle Buketter af den Slags, som bydes til Salg paa offentlige Forlystelsessteder, kastedes op foran Lamperækken. Det maatte vel være Debutantinden. Han saa op og blev saa paa en Gang ved at stirre paa Skuespillerinden. Det var en endnu ganske ung Pige, paa højst en Snes Aar. Det mørke, blanke Haar var strøget glat tilbage fra en ualmindelig smukt formet Pande. Øjnene havde et ejendommelig alvorligt og behersket Udtryk. Skikkelsen var rank og smækker. Den sorte Fløjls Spencer sluttede om en Figur, som vilde have været værdig til at staa Model for en Phidias' Fremstilling af klassisk Skjønhed. Hun sang sin Kuplet med ukunstlet Fordringsløshed uden paa nogen fremtrædende Maade at bejle til Publikums Gunst og tillige med et musikalsk Foredrag, som disse Brædder vistnok sjældent var Vidne til. Derpaa havde hun nogle Repliker, lige saa meningsløse som hele Rollen og Stykket, men hun sagde dem med en Kvikhed, der indbragte hende en ny, larmende Bifaldssalve fra Tilskuerne. — Og dog var det intet af disse Fortrin hos Debutantinden, der i saa høj Grad lagde Beslag paa Hennings Opmærksomhed. Men jo mere han saa paa hende, des mere forbavset blev han over den skuffende Lighed. Det var, som om Margrethe Aaby var traadt lyslevende frem for ham paa Forstadstheatrets Brædder, kun noget højere og rankere og med en mere selvbevidst Holdning. Men Ansigtet, Udtrykket i Øjnene og
- 79. det lille kløgtige Træk om Munden syntes at være ganske det samme. Han saa' saa vedholdende paa hende, at han tilsidst fangede hendes Blik, og det forekom ham, at hun i et Sekund saa' derned, hvor han sad. Saa gik hun videre i Rollen, og han vedblev at stirre paa hende, indtil hun havde sunget Slutningsverset, og Tæppet faldt. En dundrende Applaus, forstærket med Banken af Stokke og Paraplyer mod Borde og Gulv, attesterede Publikums Tilfredshed med den ny Skuespillerinde. Henning løb Programmet igjennem, og da han havde overtydet sig om, at hun ikke optraadte mere den Aften, gik han hastig ud af Salen. Udenfor begyndte der at falde enkelte tunge Regndraaber, og han indsugede begjærlig den kølige Luftning, der slog ham i Møde. Han havde en Fornemmelse, som om han var vaagnet op af en Drøm; var næsten tilbøjelig til at tro, at hans Fantasi havde spillet ham et Puds. En saa mærkværdig Lighed mellem to Mennesker, der ikke stod i den fjærneste Forbindelse med hinanden! — Han tog Theaterprogrammet op af Lommen, og ved den første Gaslygte, han kom til, blev han staaende for endnu en Gang at læse hendes Navn. Laura Schmidt stod der med ganske almindelige, prosaiske Bogstaver. — Han kunde ikke slippe Billedet af denne Dobbeltgængerske. Han følte et heftigt Ønske om at se hende igjen for at overbevise sig om, at Ligheden ikke var saa slaaende, som han fra først af havde troet. Han dannede sig en uklar Forestilling om, at det ligesom vilde øve en beroligende Virkning paa ham, hvis han kunde komme til et saadant Resultat ved at se hende endnu en Gang. Da han skulde til at rejse hjem den paafølgende Morgen, faldt det ham et Øjeblik ind at telegrafere til Minna, at han blev en Dag
- 80. længere i Byen, men han opgav strax denne Plan. Han fandt, at det dog næppe var den rette Maade til at gjenvinde sin Sindsligevægt. Der var endnu Tid til at komme afsted med det først afgaaende Skib. Saa skyndte han sig med sin Paaklædning og begav sig sporenstregs til Dampskibsstationen for at vende tilbage til Fiskerlejet. — Derude blev han med Minna og lille Aage en hel Maaned endnu. Skjønt han flere Gange havde noget at besørge, undgik han at tage til Byen. Han havde troet, at det var lykkedes ham at trænge Erindringen om Margrethe Aaby og deres sidste Møde i Baggrunden. Han havde i alt Fald kæmpet ærlig for at gjøre det, og Bevidstheden om hans Følelsers absolutte Haabløshed havde maaske tildels hjulpet ham dertil. Men nu kom det altsammen igjen med fornyet Styrke. — Omsider var de sidste Landliggere og de sidste Badegjæster flyttede ind til Byen, og Minna havde allerede en god Stund klaget over, at hun kjedede sig ihjel i de ynkelige smaa Huller, der udgjorde deres Sommerbolig. Naar man ikke havde Raad til at bo ordentlig, skulde man hellere lade være at ligge paa Landet, sagde hun. Saa flyttede de da hjem igjen. Den sidste Del af Sommeren var gaaet, uden at han havde faaet noget videre bestilt, og det var paa Tiden, at han atter tog alvorlig fat paa sit store Billede. — Han malede flittig, efter at de var komne til Ro i deres gamle Lejlighed, men hvor megen Umage han end gjorde sig for at fordybe sig helt og holdent i sit Arbejde, kunde han dog ikke forhindre, at hans Tanker med mere Haardnakkethed end nogensinde før vendte tilbage til Margrethe Aaby eller lige saa ofte til Skuespillerinden ude paa Forstadstheatret, saaledes at disse to smeltede sammen til én og samme Skikkelse. Et Par Gange prøvede han paa at nærme sig Minna, ligesom han vilde søge Beskyttelse mod de Udskejelser, hans Fantasi tillod sig,
- 81. men Minna havde glemt sin alvorlige Gaaen i Rette med sig selv og viste hans Tilnærmelser tilbage paa den gamle, saarende Maade. Han blev tilsidst ked af sin frugtesløse Kamp og overtalte sig til at tro, at han havde havt Ret i sin første Betragtning, og at han gjorde bedst i at se denne Dobbeltgængerske endnu en Gang med lysvaagne Øjne hellere end at lade hende regere uindskrænket i hans Fantasi. — Det var en sex Uger efter den første Aften, han havde spaseret ud til Forstadstheatret. Den mellemliggende Tid havde jaget den korte Sommer paa Flugt. Løvet havde faaet det første gullige Anstrøg, og Bladene hvirvlede med et melankolsk Suk ned mod Jorden, hver Gang Blæsten sendte et skarpt Pust gjennem de ærværdige gamle Lindetrær. — Men de mørke Aftener skræmmede ikke Folk fra at vandre i store Skarer ud til Sommertheatret, hvis særlige Attraction var den aarlige »Revue« med sine paa Øjeblikkets brændende Spørgsmaal møntede Brandere. Denne Sæson var Revuens Handling forlagt til Kina, og Theatrets ny Primadonna optraadte i en af Hovedrollerne — som Prinsesse af det himmelske Rige. — Endog ved at se hende i dette fantastiske Kustume følte han sig betagen af den frappante Lighed. Han lagde Mærke til — og det var med en ubevidst Tilfredsstillelse — at hun holdt sit Spil indenfor visse, af en naturlig god Smag afstukne Grændser, hvor meget Rollen end indbød til allehaande sceniske Udskejelser. Der var over hendes Person og Optræden udbredt en kvindelig Anstand, som traadte endnu stærkere frem i disse Omgivelser, og alligevel forstod hun at spille saaledes, at hun helt og holdent erobrede det stærkt blandede Publikum. Bifaldet, der lød, naar hun havde sunget en af sine Viser, var ligefrem bedøvende. Men hun tog imod det paa en fuldkommen
- 82. rolig, lidt ringeagtende Maade. Henning bestræbte sig for at forstørre ethvert Træk hos hende, som kunde udvidske Ligheden med Margrethe. Der var Øjeblikke, hvor den irriterede ham; hvor han følte Uvilje mod dette Pigebarn, der understod sig i at ligne en Person, hvem hun rimeligvis stod saa dybt under i alle andre Henseender end den tilfældige, ydre Lighed. Men det vilde ikke lykkes ham. Han gjorde sig forgjæves Umage for at aflure hende en uskjøn Bevægelse, et Blik eller et Smil, der kunde virke frastødende paa ham — altsammen til ingen Nytte. Saa bildte han sig tilsidst ind, at hele denne taabelige Illusion beroede paa Lamperækkens skuffende Lys og den Frastand, hvori han saa hende. Paa nært Hold vilde han sikkert faa et helt andet Indtryk af hende, og dermed vilde Kogleriet være forbi. Han fandt paa de mærkeligste Paaskud til at gaa hjemme fra, og den ene Aften efter den anden tilbragte han nogle Timer ude i Forstadstheatret. Det var et Par Gange forekommet ham, at hun havde set ned paa den Plads, hvor han sad, og derpaa taget Øjnene til sig med en Rødmen, der kom og forsvandt lige hastig. — En Aften, da hun havde optraadt i det sidste Stykke før Forestillingens Slutning, tog han sig for at skyde alle Betænkeligheder til Side og gaa op bag Kulisserne. Han kjendte lidt til Direktøren, der næppe vilde have noget imod at præsentere ham for Theatrets Primadonna. Gjennem en Bagdør og ad en skrøbelig Pindeværkstrappe kom han op bag Scenen, omkring hvilken der løb en snæver Gang med Døre ind til Skuespillernes Paaklædningsværelser. Luften, der slog ham i Møde, var mættet af Gasos, Cigarrøg og en stærk, gjennemtrængende Duft af Patchouli. Døren til et af de smaa Rum, der gjorde Tjeneste som Paaklædningsværelser, stod paa Klem og fremviste Reversen af en
- 83. Skuespiller i Skjorteærmer, der arbejdede ihærdig paa at fjærne Sminken af sit Ansigt ved Hjælp af Fedt. Medens Henning stod et Øjeblik i Tvivl om, hvad Vej han skulde gaa, kom Direktøren ham i Møde i egen Person, endnu iført sit Kustume som kinesisk Kejser og dampende af alle Kræfter paa en Cigaret. »For Pokker — jeg syntes jo nok, jeg kjendte Dem. Værs'god, kom indenfor. Det var pænt af Dem at se herop.« Han førte Henning ind i et af de omtalte, smaa Rum, der laa nærmest Trappen og benyttedes paa en Gang til Direktørens Paaklædnings- og Arbejdsværelse. Ved den ene Væg stod et langt Fyrretræs Bord; midt paa dette et Toiletspejl med Gasblus paa hver Side, foruden Sminkekrukker, Æsker med Pudder, Parykker, Skæg, fedtede Manuskripter, Cigarstumper, et Par Bajerflasker, snavsede Flipper og Mansketter, krøllede Slips, en sort Silkehat, Stumper af kulørt Tarlatan og Shirting — hulter til bulter mellem hinanden. Sofaen ved den modsatte Væg var skjult under en ligesaa broget Mangfoldighed af de mest heterogene Beklædningsgjenstande, ligefra kinesiske Mandarinkaaber til en moderne sort Selskabskjole. Direktøren samlede det sammen i en mægtig Bylt, som han smed hen i en Krog af Stuen, og bad derpaa Henning tage Plads, medens han selv satte sig overskrævs paa en Stol, der stod foran et gammelt Skrivebord henne ved Vinduet. »Naa — hvad siger De om Revuen i Aar? Det er saagu' den bedste, vi endnu har havt herude. Og den trækker voldsomt. Baade den og vor ny Akkvisition blandt Damepersonalet. De kan tro, det har været en brillant Sæson.« »Javist —« sagde Henning lidt nervøst. »Hvor har De for Resten faaet fat i Deres ny Primadonna?«
- 84. »Et rent Tilfælde, kjære Ven — et rent Tilfælde. Men hun er mageløs — ikke sandt? Og saa en komplet Dame!« udbrød Direktøren begejstret. »Sikke Bevægelser, sikke Manerer — kvikke og utvungne og dog fuldkommen ladylike — det er saagu' det, man aldrig kan faa dresseret de andre til, hvor meget man saa herser med dem.« »Hun har vel tidligere optraadt i Provinserne?« spurgte Henning og tændte med en ligegyldig Mine den Cigaret, Direktøren bød ham. »Nej — Fanden heller. Aldrig før sat sine Fødder paa Brædderne. Hun skulde have været til det kongelige til næste Sæson — jo, gu' er det sandt,« forsikrede Direktøren med en stærk Gestus, da han lagde Mærke til, at Henning saa' op med en vantro Mine. »Hun havde allerede aflagt Prøve — glimrende Udfald — men saa blev hendes gamle nok syg — hendes Fader er en gammel Provinsskuespiller — og jeg fik at vide, at hun søgte Engagement for Sommeren, fordi hun var nødt til at klare for dem derhjemme. — Ja, ser De, — da jeg netop var svært i Knibe for en Primadonna, og jeg havde hørt, at hun skulde være noget extra i Subrettefaget, saa tilbød jeg hende Engagement herude paa saa glimrende Vilkaar, forstaar De, at hun omsider slog til, skjønt — ærlig talt — jeg mærkede nok, at hun gjorde det forbandet nødig.« »Se se! Det lyder jo helt interessant. Naa — og hendes Rygte — det er vel grundmuret?« — Henning havde lidt ondt ved at faa Ordene frem i en let henkastet Tone. »Hendes Rygte, min Fa'er!« — Direktøren gjorde atter sin ejendommelige, store Gestus. — »Om det saa var min egen Datter, kunde jeg ikke ønske hende et bedre Rygte. Hun møder præcis til Prøver og Forestillinger, kan altid sine Roller, er høflig og tjenstvillig mod de andre, men taaler ingen Næsvisheder. Kommer man hende for nær, saa har hun saadan en egen energisk Maade at holde Folk tre Skridt fra Livet, saa de skal nok tage sig i Agt for at komme igjen. Om Aftenen efter Forestillingen kommer der et gammelt Skabilkenhoved af en Tjenestepige og henter hende, og de kjører
- 85. hjem sammen. Det hører med til Engageringsvilkaarene, at der paa Theatrets Regning holder en Droske og venter paa hende, naar Forestillingen er forbi. Nej, min Fa'er, hun er sikker nok,« sluttede Direktøren sin Lovtale, der var fremført med en Tungefærdighed, som havde gjort ham helt stakaandet. »En hel Sfinx, synes jeg,« bemærkede Henning. »Jeg skal for Resten ganske oprigtig sige Dem, hvorfor jeg har fattet en vis Interesse for Deres Vidunder. Hun ligner paafaldende en Dame, som jeg nærer stor Agtelse for. ... Ved De hvad — kan De ikke præsentere mig for hende?« tilføjede han lidt nølende. »Pokker heller — De, krrrtsch — Gavstrik!« Med disse Ord gjorde Direktøren et spøgefuldt Udfald med sin Pegefinger mod Hennings Skjortekrave, men skiftede pludselig Tone og forsikrede med et uhyre alvorligt Ansigt, at det var absolut umuligt. Han turde ligefrem ikke. »Aa Snak — det vilde jo være Snærperi,« paastod Henning, der blev mere og mere ivrig. »Det skal naturligvis foregaa ligesom ganske tilfældig. De beder hende under et eller andet Paaskud om at komme herind; saa sidder jeg her, og det er da hverken mere eller mindre end simpel Høflighed, at De præsenterer os for hinanden.« »Naa ja — paa den Maade — lad gaa.« Direktøren rejste sig, stak Hovedet ud af Døren og raabte ud over Trappegelænderet: »Aa, Truelsen — vil De sige til Frøken Schmidt, at jeg gjærne vil tale med hende, før hun gaar.« »Javel, Hr. Direktør,« lød en Stemme nedefra. Direktøren vendte tilbage til sin forrige Plads og underholdt Henning med forskjellige interessante Historier fra Theaterverdenen. Men Henning hørte kun det halve af dem, medens han i stærk Spænding ventede paa hendes Indtrædelse. Det varede en halv Snes Minuter. Saa lød der Skridt udenfor, en kort Banken paa Døren, og hun traadte ind. —
- 86. Ligheden var endnu mere paafaldende, som hun stod der i en simpel, men tækkelig, mørk Dragt, der fremhævede hendes ungdommelige Skikkelse. Og hun forekom ham langt smukkere end paa Scenen. Han lagde Mærke til, at hendes Stemme havde en frisk, behagelig Klang, da hun svarede Direktøren paa nogle Spørgsmaal om en Rolle, han havde givet hende. Hun gjengjældte Hennings Hilsen, men vendte sig strax igjen om til Direktøren, vexlede nogle Bemærkninger med ham om det ny Stykke, hun skulde spille i, og trak sig derpaa tilbage, hilsende begge Herrerne med en let Hovedbøjning. »Naa — det fik vi ikke megen Fornøjelse af,« udbrød Direktøren, da hun var gaaet. »Hun er i Grunden forbandet kort for Hovedet, men et mageløst Pigebarn alligevel — ikke sandt? Og der er Evner, min Fa'er, virkelige Evner! Stop lidt — havde jeg bare tænkt paa det, kunde jeg for Resten have skaffet Dem en bedre Lejlighed til at gjøre hendes Bekjendtskab. Og De kunde med det samme have gjort mig en stor Tjeneste ... Hvem Pokker skal jeg nu faa til det? ... Det var da ogsaa nederdrægtig dumt — ne-derdrægtig!« Direktøren kløede sig med en fortrædelig Mine bag Øret. Henning spurgte, hvad det drejede sig om. »Det skal jeg sige Dem. Vi indstuderer et Stykke, hvori der bruges et Portræt i naturlig Størrelse af den Skuespillerinde, der spiller Hovedrollen. I alt Fald for Kustumets Vedkommende er det nødvendigt, at vi faar en nogenlunde korrekt Gjengivelse, ellers kunde man jo leje et Portræt hos en eller anden Marskandiser. Naa, det skal naturligvis være rent Hurtigmaleri, forstaar De; bare Frisuren og de samme Farver, der er i Dragten. Det var rigtignok ikke noget Arbejde for Dem, men det havde ellers været en ypperlig Lejlighed til at studere Deres Sfinx — hvad? Og De havde oven i Kjøbet gjort mig en stor Tjeneste dermed. Hun havde blot behøvet at sidde for Dem en Times Tid ... Det var dog skammeligt, at jeg ikke før tænkte paa det!«
- 87. Direktøren vedblev at klø sig i Nakken, medens Henning sad og rokkede urolig frem og tilbage i Sofaen. Naar alt kom til alt, hvad ondt var der saa i, at han portræterede den unge Skuespillerinde? Skulde en Kunstner, fordi han var Ægtemand, ikke have Lov til at male et kvindeligt Ansigt, der interesserede ham? Tilsidst blev Fristelsen ham for stærk, og efter at Direktøren en rum Tid havde kradset sig bag Øret og slaaet sig med den flade Haand paa Panden for at fremkalde en Ide, der var lige saa god som den, der var røget i Lyset, vendte Henning sig om mod ham og fremkom med sit Forslag. »Det er vel ikke for sent endnu, hvis De ønsker, at jeg skal male et Portræt af Deres Primadonna,« sagde han lidt hastig. »Nej — vil De virkelig?« Direktøren saa op med et glædestraalende Blik. »De er en Perle! Saa skriver jeg endnu i Aften et Par Ord til hende og siger, at jeg har faaet en af vore virkelige, anerkjendte Kunstnere til at tage det omtalte Portræt af hende. De kan saa dejlig sidde ovre i min Dagligstue; ikke en Sjæl skal forstyrre Dem. Det var prægtigt!« »Naar skal jeg komme?« spurgte Henning, stadig lidt febrilsk. »Hvis det passer Dem, kan vi jo sætte i Morgen Formiddag Klokken tolv? — Saa skal jeg sørge for, at Frøkenen venter paa Dem ovre hos mig. Det er altsaa et Ord?« »Ja.« Direktøren trykkede varmt Hennings Haand med den Forsikring, at han gjorde ham en knusende Tjeneste, og Henning skyndte sig bort — i en noget urolig Stemning.
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