![12 D.G. Gibson
either double-stranded (ds) DNA fragments (8) or single-stranded
(ss) oligos (9). In addition, homologous recombination in yeast can
be used to build DNA fragments from overlapping constituent parts.
This was first demonstrated when a plasmid was constructed from
two dsDNA fragments containing homologous ends (10). Two
nonhomologous dsDNA fragments can also be bridged by single-
stranded oligonucleotides that join the ends of the two fragments
(11). Previously, we showed that six overlapping dsDNA fragments
could be assembled by yeast into an entire Mycoplasma genitalium
genome (6). Subsequently, this process was improved, and 25 over-
lapping fragments, between 17 kb and 35 kb in length, were assem-
bled at once into this genome (12). More recently, we reported on
the synthesis of a 1.08-Mbp Mycoplasma mycoides genome, which
was used to produce a cell controlled only by this synthetic genome
(7). Using yeast recombination, the synthetic M. mycoides genome
was assembled in three stages from 1,078 overlapping 1,080-bp
DNA fragments that were each chemically synthesized.
To exclusively use yeast in the production of whole genomes
and large constructs of any reasonable sequence, what remained
was the demonstration of the assembly of chemically synthesized
oligonucleotides into appropriate dsDNA molecules, which we
reported in 2009 (13). There we showed that yeast could take up
and assemble at least 38 overlapping single-stranded oligonucle-
otides and a linear double-stranded vector in one transformation
event to produce ~1.2-kb dsDNA fragments. These oligonucle-
otides can overlap by as few as 20 bp and can be as long as 200
nucleotides in length. A protocol for synthesizing kilobase-sized
DNA fragments in yeast from a series of overlapping oligos is
described here.
1. Yeast/E. coli shuttle vector [e.g., pRS313 (ATCC 77142),
pRS314 (ATCC 77143), pRS315 (ATCC 77144), and pRS316
(ATCC 77145)].
2. Primers for assembly vector amplification.
3. Overlapping synthetic oligonucleotides to be assembled.
4. High-fidelity polymerase chain reaction (PCR) amplification
kit (e.g., Phusion®
polymerase (New England BioLabs®
,
Inc. [NEB])).
5. Gel extraction kit (e.g., QIAquick Gel Extraction Kit, Qiagen).
6. Tris–EDTA buffer pH 8.0 (TE buffer).
7. DNA analysis software (e.g., Vector NTI®
[Invitrogen],
Clone Manager [Sci-Ed], and CLC Genomics Workbench
[CLC bio]).
2. Materials
2.1. Design
and Preparation
of the Oligonucleotides
and Assembly Vector](https://image.slidesharecdn.com/1261456-250522215141-84d2b676/85/Gene-Synthesis-Methods-And-Protocols-1st-Edition-Julie-A-Marchand-29-320.jpg)
![26 M.C. Huang et al.
The DNA sequence to be synthesized can be designed manually or
using computer software. We strongly recommend using computer
software [e.g., DNAWorks (http:/
/helixweb.nih.gov/dnaworks)
(17) or TmPrime (http:/
/prime.ibn.a-star.edu.sg/) (18)] to design
the gene sequence. These computer programs allow for the con-
struction of oligonucleotides with a uniform melting temperature
which increases the yield of the assembled full-length DNA prod-
uct of the PCR gene assembly. Additionally, these programs also
analyze the potential for mishybridization and secondary structures
among the oligonucleotides, which you may want to check before
conducting the gene assembly. For DNA with high sequence
repeats, the PCR-based gene synthesis may not be the best choice,
and the LCR-based approach is more effective for these challeng-
ing DNAs (19).
We use TmPrime to design the gene sequence. Figure 2 illustrates
all the parameters needed to generate the oligonucleotide set when
using TmPrime. Most of the parameters are self-explanatory. For
instance, the user is asked to provide gene information, gene assem-
bly buffer condition, oligonucleotide and outer primer concentra-
tions, optional parameters for long DNA assembly, and parameters
for mispriming analysis. The software will report the melting tem-
peratures, oligonucleotide sequences, potential formation of sec-
ondary structures, and statistical information for the oligo sets of
each pool in a PDF file (see Note 5). To ensure successful TopDown
gene synthesis, oligos are designed to have a melting temperature
that is ~8°C higher than that of the outer primers to minimize the
competition between PCR assembly and PCR amplification of the
assembled product in the one-step gene synthesis (see Note 6).
The sequence of the human calcium-binding protein A4 promoter
(S100A4, 752 bp; chr1:1503312036–1503311284) has been
selected here as the target DNA for demonstration. The average
melting temperature of the designed oligonucleotides is 66°C and
consists of a pool of 30 oligos ranging in length from 41 nucle-
otides (nt) to 66 nt.
The primers that anneal to the target gene can be designed using
IDT SciTools (http:/
/www.idtdna.com/SciTools/SciTools.aspx)
(20) and TmPrime with an outer primer concentration of 300–
400 nM. It is important that the primers are designed to anneal
only to the outermost 3¢ and 5¢ ends of the target gene.
1. Set up the master mix containing the inner oligos: add 2 ml
(100 mM) of each of the oligos to a 600-ml microfuge tube,
and add deionized distilled water to a final volume of 200 ml.
3. Methods
3.1. Reagent Setup:
Designing the DNA
of Interest
3.1.1. Design
of Oligonucleotides
and Outer Primers
3.1.2. Designing
Gene-Specific Primers
3.2. Real-Time
TopDown Gene
Synthesis](https://image.slidesharecdn.com/1261456-250522215141-84d2b676/85/Gene-Synthesis-Methods-And-Protocols-1st-Edition-Julie-A-Marchand-43-320.jpg)
![The little town of Ballycastle does not contain much to occupy the
traveller: behind the church stands a ruined old mansion with round turrets,
that must have been a stately tower in former days. The town is more
modern, but almost as dismal as the tower. A little street behind it slides off
into a potato-field—the peaceful barrier of the place; and hence I could see
the tall rock of Bengore, with the sea beyond it, and a pleasing landscape
stretching towards it.
Dr. Hamilton’s elegant and learned book has an awful picture of yonder
head of Bengore; and hard by it the Guide-book says is a coal-mine, where
Mr. Barrow found a globular stone hammer, which, he infers, was used in
the coal-mine before weapons of iron were invented. The former writer
insinuates that the mine must have been worked more than a thousand years
ago, ‘before the turbulent chaos of events that succeeded the eighth
century.’ Shall I go and see a coal-mine that may have been worked a
thousand years since? Why go see it? says idleness. To be able to say that I
have seen it. Sheridan’s advice to his son here came into my mind;[32] and I
shall reserve a description of the mine, and an antiquarian dissertation
regarding it, for publication elsewhere.
Ballycastle must not be left without recording the fact that one of the
snuggest inns in the country is kept by the postmaster there; who has also a
stable full of good horses for travellers who take his little inn on the way to
the Giant’s Causeway.
The road to the Causeway is bleak, wild, and hilly. The cabins along the
road are scarcely better than those of Kerry, the inmates as ragged, and
more fierce and dark-looking. I never was so pestered by juvenile beggars
as in the dismal village of Ballintoy. A crowd of them rushed after the car,
calling for money in a fierce manner, as if it was their right; dogs as fierce
as the children came yelling after the vehicle; and the faces which scowled](https://image.slidesharecdn.com/1261456-250522215141-84d2b676/85/Gene-Synthesis-Methods-And-Protocols-1st-Edition-Julie-A-Marchand-63-320.jpg)
![Witnessing the sight
Of that dire disaster,
Out began to laugh
Missis, maid, and master;
Such a merry peal,
‘Specially Miss Peg’s was
(As the glass of ale
Trickling down my legs was),
That the joyful sound
Of that ringing laughter
Echoed in my ears
Many a long day after.
When the laugh was done.
Peg, the pretty hussy,
Moved about the room
Wonderfully busy;
Now she looks to see
If the kettle keep hot,
Now she rubs the spoons,
Now she cleans the teapot:
Now she sets the cups
Trimly and secure,
Now she scours a pot,
And so it was I drew her.
Thus it was I drew her
Scouring of a kettle,[33]
(Faith! her blushing cheeks
Redden’d on the metal!)
Ah! but ‘tis in vain
That I try to sketch it;](https://image.slidesharecdn.com/1261456-250522215141-84d2b676/85/Gene-Synthesis-Methods-And-Protocols-1st-Edition-Julie-A-Marchand-83-320.jpg)
Gene Synthesis Methods And Protocols 1st Edition Julie A Marchand
- 1. Gene Synthesis Methods And Protocols 1st Edition Julie A Marchand download https://ebookbell.com/product/gene-synthesis-methods-and- protocols-1st-edition-julie-a-marchand-2522912 Explore and download more ebooks at ebookbell.com
- 2. Here are some recommended products that we believe you will be interested in. You can click the link to download. Synthetic Gene Circuits Methods And Protocols Filippo Menolascina https://ebookbell.com/product/synthetic-gene-circuits-methods-and- protocols-filippo-menolascina-22375554 Synthetic Gene Network Modeling Analysis And Robust Design Methods Borsen Chen Yuchao Wang https://ebookbell.com/product/synthetic-gene-network-modeling- analysis-and-robust-design-methods-borsen-chen-yuchao-wang-4732302 Synthetic Gene Networks Methods In Molecular Biology V813 2012th Edition Wilfried Weber https://ebookbell.com/product/synthetic-gene-networks-methods-in- molecular-biology-v813-2012th-edition-wilfried-weber-2486142 Carbon Nanotubes Synthesis Structure Properties And Applications 1st Edition Dresselhaus Mildred S Ed Dresselhaus Gene Ed Avouris Phaedon Ed https://ebookbell.com/product/carbon-nanotubes-synthesis-structure- properties-and-applications-1st-edition-dresselhaus-mildred-s-ed- dresselhaus-gene-ed-avouris-phaedon-ed-4332808
- 3. Forest Hydrology And Biogeochemistry Synthesis Of Past Research And Future Directions 1st Edition Kevin J Mcguire https://ebookbell.com/product/forest-hydrology-and-biogeochemistry- synthesis-of-past-research-and-future-directions-1st-edition-kevin-j- mcguire-2448990 Semiconducting Polymers Controlled Synthesis And Microstructure 1st Edition Geng https://ebookbell.com/product/semiconducting-polymers-controlled- synthesis-and-microstructure-1st-edition-geng-5699670 Analysis And Synthesis Of Delta Operator Systems With Actuator Saturation 1st Ed Hongjiu Yang https://ebookbell.com/product/analysis-and-synthesis-of-delta- operator-systems-with-actuator-saturation-1st-ed-hongjiu-yang-10493582 Advancing Development Of Synthetic Gene Regulators With The Power Of Highthroughput Sequencing In Chemical Biology Chandran https://ebookbell.com/product/advancing-development-of-synthetic-gene- regulators-with-the-power-of-highthroughput-sequencing-in-chemical- biology-chandran-6754558 Synthetic Nucleic Acids As Inhibitors Of Gene Expression Mechanisms Applications And Therapeutic Implications 1st Edition Levon Michael Khachigian Editor https://ebookbell.com/product/synthetic-nucleic-acids-as-inhibitors- of-gene-expression-mechanisms-applications-and-therapeutic- implications-1st-edition-levon-michael-khachigian-editor-2183908
- 6. ME T H O D S I N MO L E C U L A R BI O L O 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. Gene Synthesis Methods and Protocols Edited by Jean Peccoud VirginiaBioinformaticsInstitute,VirginiaTech,Blacksburg,VA,USA
- 9. ISSN 1064-3745 e-ISSN 1940-6029 ISBN 978-1-61779-563-3 e-ISBN 978-1-61779-564-0 DOI 10.1007 /978-1-61779-564-0 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2012930137 © Springer Science+Business Media, LLC 2012 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Humana Press is part of Springer Science+Business Media (www.springer.com) Editor Jean Peccoud, Ph.D. Virginia Bioinformatics Institute Virginia Tech Blacksburg, VA, USA jpeccoud@vbi.vt.edu
- 10. v Preface The de novo fabrication of custom DNA molecules is a transformative technology that significantly affects the biotechnology industry. Basic genetic engineering techniques for manipulating DNA in vitro opened an incredible field of opportunity in the life sciences. However, genetic engineering has now moved beyond the introduction of single genes into cells to multigene cassettes, and is rapidly progressing toward whole genome engineering. In this new context, the synthesis of DNA molecules has resurged as the time and cost- limiting step in genetic engineering. Today, most multigene engineering projects involve ad hoc methods of DNA assembly. A variety of PCR-based methods are in common use alongside more traditional restriction enzyme-based assembly methods. Their essential feature is the piecing together of existing DNAs that are cloned from natural sources. These techniques present a number of limita- tions. The use of restriction sites within natural sequences necessitates a labor intensive custom cloning strategy that is difficult to automate. As a result, molecular biologists often reach a tacit compromise between obtaining a desired sequence and the number of steps in the cloning process they are willing or able to undertake in constructing it. Theoretically, DNA fabrication methods that are rooted in chemical synthesis could transform synthesis into a generic, predictable, and scalable process allowing the generation of any user-defined DNA sequence. By liberating the process from the confines of preexisting sequences, the problem of composition design becomes orthogonal to the problem of physical construction. Therefore, as gene synthesis becomes a commodity, biologists will spend more time designing custom DNA molecules and characterizing their performance, and less time constructing them. One day, DNA may be fabricated using a purely chemical process. Today, however, DNA fabrication still involves sophisticated cloning techniques, but nevertheless a transi- tion period has already emerged. Academic and commercial operators experiment with complex processes that combine the assembly of chemically synthesized oligos with cloning steps in attempts to construct long DNA molecules. Even though a number of companies have rushed to and sometimes later walked away from the gene synthesis market, DNA fabrication is not a black box that would involve radically different techniques than those commonly used in a molecular biology laboratory, nor does it require expensive equip- ment. Depending on the context it might make sense to outsource DNA fabrication to an external vendor, but in other cases there might be value in performing part of the process in house. In fact, gene synthesis projects are approachable by undergraduate students enabled by straightforward protocols and training in a relatively small set of molecular biology skills. In any case, it is important to understand that the fabrication of small DNA fragment (less than 1 kb) is often very straightforward, but the assembly of longer DNA molecules raises a number of inherent technical difficulties that need to be understood.
- 11. vi Preface This book provides step-by-step protocols for the different stages of a DNA fabrication process. Section I focuses on protocols used for the assembly of oligonucleotides in building blocks also called synthons. The cloning of synthons into larger fragments up to the size of bacterial genomes is the focus of Section II. Bioinformatics protocols and software applica- tions necessary to design gene synthesis protocols are described in Section III. Finally, Section IV describes the educational and biosecurity impacts of gene synthesis. Any laboratory relying on recombinant DNA technology for its research is a potential user of gene synthesis. Few laboratories will develop a completely home grown gene syn- thesis process. Oligonucleotide synthesis or sequencing will most likely be outsourced to a core facility or a commercial operator. In other cases, the synthesis of longer fragments may also be outsourced. By providing step-by-step descriptions of all the different stages of a complex gene synthesis process, this book will help readers refine their understanding of gene synthesis and determine what part of the process they can or should do in their labora- tory and what parts should be contracted to a specialized service provider. Blacksburg, VA, USA Jean Peccoud, Ph.D.
- 12. vii Contents Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix PART I ASSEMBLY OF OLIGONUCLEOTIDES IN SYNTHONS 1 Building Block Synthesis Using the Polymerase Chain Assembly Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Julie A. Marchand and Jean Peccoud 2 Oligonucleotide Assembly in Yeast to Produce Synthetic DNA Fragments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Daniel G. Gibson 3 TopDown Real-Time Gene Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Mo Chao Huang, Wai Chye Cheong, Hongye Ye, and Mo-Huang Li 4 De Novo DNA Synthesis Using Single-Molecule PCR . . . . . . . . . . . . . . . . . . 35 Tuval Ben Yehezkel, Gregory Linshiz, and Ehud Shapiro PART II SYNTHON ASSEMBLY 5 SLIC: A Method for Sequence- and Ligation-Independent Cloning . . . . . . . . 51 Mamie Z. Li and Stephen J. Elledge 6 Assembly of Standardized DNA Parts Using BioBrick Ends in E. coli. . . . . . . . 61 Olivia Ho-Shing, Kin H. Lau, William Vernon, Todd T. Eckdahl, and A. Malcolm Campbell 7 Assembling DNA Fragments by USER Fusion . . . . . . . . . . . . . . . . . . . . . . . . 77 Narayana Annaluru, Héloïse Muller, Sivaprakash Ramalingam, Karthikeyan Kandavelou, Viktoriya London, Sarah M. Richardson, Jessica S. Dymond, Eric M. Cooper, Joel S. Bader, Jef D. Boeke, and Srinivasan Chandrasegaran 8 Fusion PCR via Novel Overlap Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 Kamonchai Cha-aim, Hisashi Hoshida, Tomoaki Fukunaga, and Rinji Akada 9 Using Recombineering to Generate Point Mutations: The Oligonucleotide-Based “Hit and Fix” Method . . . . . . . . . . . . . . . . . . . . . 111 Suhwan Chang, Stacey Stauffer, and Shyam K. Sharan 10 Using Recombineering to Generate Point Mutations: galK-Based Positive–Negative Selection Method. . . . . . . . . . . . . . . . . . . . . . . 121 Kajal Biswas, Stacey Stauffer, and Shyam K. Sharan
- 13. viii Contents 11 Assembling Large DNA Segments in Yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 Héloïse Muller, Narayana Annaluru, Joy Wu Schwerzmann, Sarah M. Richardson, Jessica S. Dymond, Eric M. Cooper, Joel S. Bader, Jef D. Boeke, and Srinivasan Chandrasegaran 12 Recursive Construction of Perfect DNA Molecules and Libraries from Imperfect Oligonucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 Gregory Linshiz, Tuval Ben Yehezkel, and Ehud Shapiro 13 Cloning Whole Bacterial Genomes in Yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . 165 Gwynedd A. Benders 14 Production of Infectious Poliovirus from Synthetic Viral Genomes . . . . . . . . . 181 Jeronimo Cello and Steffen Mueller PART III SOFTWARE FOR GENE SYNTHESIS 15 In Silico Design of Functional DNA Constructs . . . . . . . . . . . . . . . . . . . . . . . 197 Alan Villalobos, Mark Welch, and Jeremy Minshull 16 Using DNAWorks in Designing Oligonucleotides for PCR-Based Gene Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215 David Hoover 17 De Novo Gene Synthesis Design Using TmPrime Software . . . . . . . . . . . . . . . 225 Mo-Huang Li, Marcus Bode, Mo Chao Huang, Wai Chye Cheong, and Li Shi Lim 18 Design-A-Gene with GeneDesign . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235 Sarah M. Richardson, Steffi Liu, Jef D. Boeke, and Joel S. Bader PART IV EDUCATION AND SECURITY 19 Leading a Successful iGEM Team . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251 Wayne Materi 20 The Build-a-Genome Course . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273 Eric M. Cooper, Helöise Müller, Srinivasan Chandrasegaran, Joel S. Bader, and Jef D. Boeke 21 DNA Synthesis Security . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285 Ali Nouri and Christopher F. Chyba Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297
- 14. ix Contributors RINJI AKADA • Department of Applied Molecular Bioscience, Yamaguchi University Graduate School of Medicine, Ube, Japan NARAYANA ANNALURU • Department of Environmental Health Sciences, Johns Hopkins University School of Public Health, Baltimore, MD, USA JOEL S. BADER • High Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore MD, USA; Whiting School of Engineering, Johns Hopkins University, Baltimore, MD, USA GWYNEDD A. BENDERS • J. Craig Venter Institute Inc., San Diego, CA, USA KAJAL BISWAS • Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD, USA MARCUS BODE • Institute of Bioengineering and Nanotechnology, The Nanos, Singapore JEF D. BOEKE • Department of Molecular Biology and Genetics, High Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA A. MALCOLM CAMPBELL • Department of Biology, Davidson College, Davidson, NC, USA; Genome Consortium for Active Teaching, Davidson, NC, USA JERONIMO CELLO • Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY, USA KAMONCHAI CHA-AIM • Department of Applied Molecular Bioscience, Yamaguchi University Graduate School of Medicine, Ube, Japan SRINIVASAN CHANDRASEGARAN • Department of Environmental Health Sciences, Johns Hopkins University School of Public Health, Baltimore, MD, USA SUHWAN CHANG • Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD, USA WAI CHYE CHEONG • Institute of Bioengineering and Nanotechnology, The Nanos, Singapore CHRISTOPHER F. CHYBA • Program on Science and Global Security, Woodrow Wilson School, Princeton University, Princeton, NJ, USA ERIC M. COOPER • High Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA JESSICA S. DYMOND • High Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA TODD T. ECKDAHL • Department of Biology, Missouri Western State University and Genome Consortium for Active Teaching., St. Joseph, MO, USA; Genome Consortium for Active Teaching, Davidson, NC, USA STEPHEN J. ELLEDGE • Department of Genetics, Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, USA; Division of Genetics, Department of Medicine, Brigham and Women’s Hospital, Boston, MA, USA TOMOAKI FUKUNAGA • Department of Applied Molecular Bioscience, Yamaguchi University Graduate School of Medicine, Ube, Japan
- 15. x Contributors DANIEL G. GIBSON • Department of Synthetic Biology, J. Craig Venter Institute, Inc., Rockville, MD, USA DAVID HOOVER • Scientific Computing Branch, Center for Information Technology, National Institutes of Health, Bethesda, MD, USA HISASHI HOSHIDA • Department of Applied Molecular Bioscience, Yamaguchi University Graduate School of Medicine, Ube, Japan OLIVIA HO-SHING • Department of Biology, Davidson College, Davidson, NC, USA MO CHAO HUANG • Institute of Bioengineering and Nanotechnology, The Nanos, Singapore KARTHIKEYAN KANDAVELOU • Pondicherry Biotech Private Limited, IT Park, Pondy Technopolis, Pillaichavady, Puducherry, India KIN H. LAU • Department of Biology, Davidson College, Davidson, NC, USA MAMIE Z. LI • Department of Genetics, Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, USA; Division of Genetics, Department of Medicine, Brigham and Women’s Hospital, Boston, MA, USA MO-HUANG LI • Institute of Bioengineering and Nanotechnology, The Nanos, Singapore LI SHI LIM • Institute of Bioengineering and Nanotechnology, The Nanos, Singapore GREGORY LINSHIZ • Department of Computer Science and Applied Mathematics, Weizmann Institute of Science, Rehovot, Israel; Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel STEFFI LIU • Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA VIKTORIYA LONDON • Department of Environmental Health Sciences, Johns Hopkins University School of Public Health, Baltimore, MD, USA JULIE A. MARCHAND • Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA, USA WAYNE MATERI • Carbonitum Energy Corporation, Edmonton, AB, Canada JEREMY MINSHULL • DNA2.0, Inc., Menlo Park, CA, USA STEFFEN MUELLER • Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY, USA HÉLOÏSE MULLER • Department of Environmental Health Sciences, Johns Hopkins University School of Public Health, Baltimore, MD, USA ALI NOURI • Program on Science and Global Security Woodrow Wilson School, Princeton University, Washington DC, USA JEAN PECCOUD • Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA, USA SIVAPRAKASH RAMALINGAM • Department of Environmental Health Sciences, Johns Hopkins University School of Public Health, Baltimore, MD, USA SARAH M. RICHARDSON • High Throughput Biology Center, Johns Hopkins University School of Public Health, Baltimore, MD, USA JOY WU SCHWERZMANN • Department of Environmental Health Sciences, Johns Hopkins University School of Public Health, Baltimore, MD, USA EHUD SHAPIRO • Department of Biological Chemistry, Weizman Institute of Science, Rehovot, Israel; Department of Computer Science and Applied Mathematics, Weizman Institute of Science, Rehovot, Israel SHYAM K. SHARAN • Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD, USA STACEY STAUFFER • Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD, USA
- 16. xi Contributors WILLIAM VERNON • Department of Biology, Missouri Western State University, St. Joseph, MO, USA ALAN VILLALOBOS • DNA2.0, Inc., Menlo Park, CA, USA MARK WELCH • DNA2.0, Inc., Menlo Park, CA, USA HONGYE YE • Institute of Bioengineering and Nanotechnology, The Nanos, Singapore TUVAL BEN YEHEZKEL • Department of Biological Chemistry, Weizman Institute of Science, Rehovot, Israel
- 18. Part I Assembly of Oligonucleotides in Synthons
- 20. 3 Jean Peccoud (ed.), Gene Synthesis: Methods and Protocols, Methods in Molecular Biology, vol. 852, DOI 10.1007/978-1-61779-564-0_1, © Springer Science+Business Media, LLC 2012 Chapter 1 Building Block Synthesis Using the Polymerase Chain Assembly Method Julie A. Marchand and Jean Peccoud Abstract De novo gene synthesis allows the creation of custom DNA molecules without the typical constraints of traditional cloning assembly: scars, restriction site incompatibility, and the quest to find all the desired parts to name a few. Moreover, with the help of computer-assisted design, the perfect DNA molecule can be created along with its matching sequence ready to download. The challenge is to build the physical DNA molecules that have been designed with the software. Although there are several DNA assembly methods, this section presents and describes a method using the polymerase chain assembly (PCA). Key words: Gene synthesis, Polymerase chain assembly, Building blocks, DNA fabrication, Computer-assisted design At its core, DNA fabrication relies on the synthesis of DNA oligomers at base level. The essential feature of DNA fabrication is that no naturally isolated DNA is used. Although clonal plasmid-based intermediates might exist during the assembly of a target DNA, every base originated as a phosphoramidite molecule at the beginning of the process. Today, all fabrication methods begin with solid-phase phosphoramidite chemistry to construct single- strandedDNAmoleculesthatarebetween10and100basepairs(bp) long, which are enzymatically assembled into larger molecules. This process is commonly referred to as “gene synthesis” and can be used to synthesize sequences up to 1 kilobase(kb)long. Still larger target DNA sequences require investigators to assemble partial products into the desired full-length construct. DNA of several kb in length can be enzymatically assembled from 1-kb DNA segments, whereas DNA of megabase length requires in vivo 1. Introduction
- 21. 4 J.A. Marchand and J. Peccoud recombination methods (Fig. 1). The details of DNA fabrication are therefore not monolithic and are distinct based on the size of the target DNA (1). Gene synthesis is the step during which oligonucleotides (oligos) are combined into DNA fragments of several hundred bases in length. Numerous protocols have been described and extensively reviewed, including polymerase chain assembly (PCA), thermodynamically balanced inside-out synthesis (TBIO) (2), and Fig. 1. PCA assembly of a DNA construct. A target sequence is shown in the top panel of this figure. The different color segments represent the oligos that are synthesized to build the construct. The pool of oligos is assembled in equimolar amounts and allowed to anneal. The annealed oligos are extended in the 3¢ direction until the end of their partner oligo is reached. The double-stranded DNA is melted and reannealed with extension products and any remaining oligos. Each extension reaction results in progressively longer products, and full-length products are eventually synthesized. At this step, the terminal oligos are added to the reaction, and full-length products from the previous reactions are amplified by PCR and subsequently cloned and sequenced. Figure reproduced with permission from ref. 1.
- 22. 5 1 Building Block Synthesis Using the Polymerase Chain Assembly Method ligase chain reaction (LCR) (1, 3). Here the PCA method (4) is used to synthesize 750-bp building blocks from the right arm of yeast chromosome 6. The right chromosome arm was first subdivided in 12 segments, each on average 12 kb, and then each segment was further subdivided in 15 building blocks of 750 bp each. The building blocks were further dissected into a set of about 12–13 oligonucleotides of about 60 bp in length. The building blocks were further synthesized from the oligonucleotides using the PCA method, cloned, and sent for sequencing. The primers are designed using the software GeneDesign, a web- based program for the design of synthetic genes (5). It consists of several modules that automate the tasks associated with the manip- ulation of synthetic sequences. The source code is from http:/ / github.com/notadoctor/GeneDesign/. 1. The primers were purchased from Integrated DNA Technologies, Inc. (IDT, Coralville, IA). They were ordered wet frozen (in water) in 96-well plates at a concentration of 60 μmol/L and a volume of 83.33 μl/well. 2. HotStarTaq master mix kit. 3. Nuclease-free water (not DEPC-treated). 4. 96-well PCR plates. 5. DNA 12000 kit (catalogue number 5067-1508 Agilent Technologies, Inc., Wilmington, DE). 6. Agilent Bioanalyzer (Agilent Technologies, Inc., Wilmington, DE). 7. 96-well block, 1 ml volume/well. 1. TOPO TA cloning® kit for sequencing (catalogue number K457501, Life technologies, Carlsbad, CA). 2. TOP10 chemically competent cells (catalogue number C404003, Life technologies, Carlsbad, CA). 3. Terrific agar plates supplemented with 100 μg/ml carbenicillin. 1. Luria broth (LB) medium supplemented with 10% glycerol and 50 μg/ml carbenicillin. 2. SOC medium: complement 1 L of LB medium with 10 ml of 1 M MgSO4 (10 mM final), 10 ml of 1 M MgCl2 (10 mM final), and 18 ml of 20% dextrose (0.36% w/vol). 3. Air-permeable sealing membrane. 2. Materials 2.1. Software 2.2. Gene Synthesis and Analysis 2.3. Cloning of Building Blocks 2.4. Bacterial Culture
- 23. 6 J.A. Marchand and J. Peccoud 4. Aluminum seal film. 5. 96-well culture plates. 6. Carbenicillin. The GeneDesign software (5) allows for breaking large sequences into several building blocks of about 750 bp, which, in turn, are further dissected into about 12–13 60-bp oligonucleotides used for the subsequent synthesis. The building blocks are synthesized manually here, but this can also be performed with a high-throughput liquid handling system. To create the DNA template for the build- ing blocks, this protocol uses the polymerase chain assembly (PCA) method in which oligonucleotides, present in equimolar quantity, span both strands of a DNA sequence, anneal through partial over- lap, and are extended in such a way that each are increased in length and can be extended further by hybridizing to other oligonucle- otides or products of subsequent extensions (1). Upon its creation, the full-length DNA template corresponding to a specific building block is amplified by polymerase chain reaction (PCR) using the two outermost oligonucleotides from the PCA step. Following the completion of the final amplification step, analysis of the PCR reaction by electrophoresis is used to visualize the presence and size of the generated building block. This final amplicon is then cloned in a TOPO® TA cloning vector and transformed. For each synthesized building block, an average of 12 clones are sent for sequencing in the form of liquid culture. To avoid template degra- dation, it is important to clone the amplicons immediately after the electrophoresis. 1. Using the GeneDesign software (see Subheading 2.1), the 750-bp building block sequences are entered in the program using option X (calibrate the Tm, length restriction sites added). This function will subdivide the sequences in several primers (usually 12–13 primers, but can be up to 20 for a longer building block), each with an average length of 60 bp, and will also deliver a list of primers. 2. Using the primer list, the primers are ordered from commercial suppliers as described in Subheading 2.2. 1. Using a multichannel pipette, the primers are diluted to 6 μM in nuclease-free water to a final volume of 100 μl in a second 96-well block to produce the working stock. From this 6 μM primer working stock, a template primer mix (TPM) and outer primer mix (OPM) are prepared. 3. Methods 3.1. Design of the Building Blocks 3.2. Building Block Synthesis
- 24. 7 1 Building Block Synthesis Using the Polymerase Chain Assembly Method 2. In the TPM, all primers must be present at a concentration of 300 nM (the primers must all be diluted by 1/20). These dilutions are prepared in a 0.5-ml reaction tube, and primer mixes are stored at −20°C when not in use. To prepare the TPM for a building block that consists of up to 20 primers, add 10 μl of each primer and if less than 20 primers are used, add nuclease-free water instead to yield a final volume of 200 μl; mix thoroughly. If more than 20 primers are used, add 10 μl of each primer, but no additional water. The primer con- centration should be around 250–275 nM. 3. In the OPM, the outer primers must be present at a concentra- tion of 3 μM (i.e., both primers are diluted by 1/2). Again, these dilutions are prepared in a 0.5-ml reaction tube in nuclease- free water, and primer mixes are stored at −20°C when not in use. To obtain the OPM, add 25 μl of the first and last primer and mix thoroughly to give a total volume of 50 μl. 4. The building block template synthesis is performed by poly- merase chain assembly (PCA) in a thermocycler. This reaction is also called templateless PCR; it has a final volume of 25 μl and contains the following reagents: 12.5 μl of HotStarTaq master mix 2× (containing the buffer, the dNTPS, and the Taq polymerase), 2.5 μl of TPM, and 10 μl of nuclease-free water. The assembly is performed using the following program: 95°C for 15 min, 55°C for 30 s, and 72°C for 1 min; then 25 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min; followed by 72°C for 3 min and 10°C forever (see Note 1). 5. The building block templates are then amplified by PCR using the OPM to create the finished PCR and thus the building blocks. This reaction has a final volume of 25 μl and contains the following reagents: 12.5 μl of HotStarTaq master mix 2× (the buffer, the dNTPS, and the Taq polymerase), 2.5 μl of templateless PCR reaction from step 4 above (diluted 1:5), 2 μl of OPM, and 10 μl of nuclease-free water. The amplifica- tion is performed with the following program: 95°C for 15 min, 55°C for 30 s, and 72°C for 1 min; 25 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min; followed by 72°C for 3 min and 10°C forever (see Note 1). 6. The building block synthesis is monitored with a microfluidic electrophoresis using the DNA 12000 kit and the Bioanalyzer instrument from Agilent according to the manufacturer’s instructions (Fig. 2). This analysis can also be performed with an agarose gel. 1. The building blocks are cloned in the TOPO® TA cloning vector pCR4® (see Subheading 2.3). All reagents are provided in the kit. The ligation reaction is composed of 2 μl of the PCR reac- tion from step 6 above (see Note 2), 1 μl of ultra salt solution, 3.3. Cloning of Building Blocks
- 25. 8 J.A. Marchand and J. Peccoud 2 μl of ultra pure water, and 1 μl of pCR4® TOPO® vector, given a final volume of 6 μl. The reagents are added in the specified order, and the reaction is incubated at room tempera- ture for 12 min (see Note 3). 2. The cloned building blocks are transformed into chemically competent bacterial cells E. coli TOP10 (see Note 4). The entire ligation reaction (6 μl) is added to 50 μl of chemically competent E. coli TOP10 cells and incubated on ice for 30 min, before being heat-shocked at 42°C for 30 s and placed back onto ice for 2 min. Then 400 μl of SOC medium without anti- biotics is added, and the cells are incubated at 37°C for 1 h. Finally, the cells are spread on terrific agar plates supplemented with carbenicillin at 100 μg/ml and incubated at 37°C for 18 h (see Note 6). The vector has a negative selection with E. coli lethal gene ccdB fused to LacZα fragment. Upon ligation of an insert, the LacZα-ccdB gene fusion expression is disrupted, and thus the cell survives. The host must not express the ccdA gene (see Note 5). 1. For each construct, 12 colonies are manually picked from the 18-h agar plates and inoculated in a 96-well culture plate con- taining 200 μl of LB broth supplemented with 10% glycerol and 50 μg/ml carbenicillin (see Note 6). The plate is sealed 3.4. Culturing of Clones for Sequencing Fig. 2. Gel electrophoresis for a selection of synthesized building blocks from segment 15 of the right arm of yeast chromo- some 6.These building blocks were synthesized using the PCA method, and a microfluidic electrophoresis was performed to monitor the success of the synthesis. For each building block, positive synthesis is demonstrated by the presence of a band with a molecular weight that corresponds to the associated building block.
- 26. 9 1 Building Block Synthesis Using the Polymerase Chain Assembly Method with an air-permeable membrane and incubated without agitation at 37°C for 18 h. 2. The 18-h plate is then used to inoculate two replicate 96-well culture plates. Using a multichannel pipette, 10 μl of the 18-h culture plate is transferred to each of the 96-well plates con- taining 190 μl of LB broth supplemented with 10% glycerol and 50 μg/ml carbenicillin. The plates are sealed with an air- permeable membrane and incubated without agitation at 37°C for exactly 12 h. The 18-h plate is sealed with aluminum seal and stored at −80°C. 3. Upon completion of the incubation time, the two 12-h plates are sealed with aluminum seal and stored at −80°C. One of the 12-h plates will be sent to an external company for sequencing, and the other is kept. 4. After the sequencing results are analyzed, the selected perfect clones are picked from their respective culture plates and grown in tubes containing 3 ml of LB broth supplemented with 10% glycerol and 100 μg/ml carbenicillin and incubated in an incu- bator shaker at 37°C and 250 RPM for exactly 12 h. The culture is then frozen as a glycerol stock in a cryogenic vial. The 96-well plates containing the incorrect clones are discarded. 1. The annealing temperature for PCA or finishing PCR reaction must be adjusted according to the Tm of the primers. 2. The volume of PCR reactions used for the ligation can range from 0.5 μl to 4 μl depending on the yield of the amplicons. 3. The building block can also be cloned by traditional ligation provided that restriction sites are added on the outermost primers, or by cloning systems such as Gateway® that has recombination sites added to the outmost primers. If a uracil- specific excision reagent (USER) fusion system is used, it is important to note that wild-type archaeal DNA polymerases are inhibited by the deoxyuracil. 4. The selection of the bacterial strain should be made according to both the strain genotype and the type of insert. For the expression of yeast parts, the E. coli TOP10 cells appear more suitable, and we have observed increased number of colonies and good growth with this strain. 5. Despite the presence of the ccdB lethal gene, we have observed some negative colonies that do not appear to be satellite colonies. Random screening or sending more clones for sequencing might be advisable. 4. Notes
- 27. 10 J.A. Marchand and J. Peccoud 6. This growth medium is recommended by Beckman Coulter Genomics where our group receives the sequencing service. The selective antibiotic is based on the resistance encoded by the vector of choice. References 1. Czar MJ, Anderson JC, Bader JS and Peccoud J. (2009) Gene synthesis demystified. Trends Biotechnol 27:63–72. 2. Xiong AS, Peng RH, Zhuang J, Gao F, Li Y, Cheng Z M and Yao, QH (2008) Chemical gene synthesis: strategies, softwares, error corrections, and applications. FEMS Microbiol 32:522–540. 3. Cello J, Paul AV and Wimmer E (2002) Chemical Synthesis of Poliovirus cDNA: Generation of Infectious Virus in the Absence of Natural Template. Science 297:1016–1018. 4. Dymond J, Scheifele L, Richardson S, Lee P, Chandrasegaran S, Bader J and Boeke JD (2009) Teaching Synthetic Biology, Bioinfor- matics, and Engineering to Undergraduates: The Interdisciplinary Build-a-Genome Course. Genetics 18:13–21. 5. Richardson SM, Wheelan SJ, Yarrington, RM and Boeke, JD (2006) GeneDesign: rapid, auto- mated design of multikilobase synthetic genes. Genome Res 16:550–6.
- 28. 11 Jean Peccoud (ed.), Gene Synthesis: Methods and Protocols, Methods in Molecular Biology, vol. 852, DOI 10.1007/978-1-61779-564-0_2, © Springer Science+Business Media, LLC 2012 Chapter 2 Oligonucleotide Assembly in Yeast to Produce Synthetic DNA Fragments Daniel G. Gibson Abstract The yeast Saccharomyces cerevisiae can take up and assemble at least 38 overlapping single-stranded oligonucleotides and a linear double-stranded vector in one transformation event. These oligonucleotides can overlap by as few as 20 bp and can be as long as 200 nucleotides in length to produce kilobase-sized synthetic DNA molecules. A protocol for designing the oligonucleotides to be assembled, transforming them into yeast, and confirming their assembly is described here. This straightforward scheme for assem- bling chemically synthesized oligonucleotides can be a useful tool for building synthetic DNA molecules. Key words: In vivo DNA assembly, Yeast transformation, Gene synthesis, Oligonucleotides, Synthetic biology Chemically synthesized oligonucleotides (oligos) are often joined into larger DNA fragments containing full-length genes. This was first demonstrated in 1970 when Khorana and colleagues synthe- sized the 77-nucleotide gene encoding a yeast alanine transfer RNA from 17 overlapping oligonucleotides (1). Since then, chemical oli- gonucleotide synthesis has improved tremendously (2), and a num- ber of in vitro enzymatic strategies are available for the assembly of oligos into larger constructs (3–5). It is now possible to produce genes, biosynthetic pathways, and even entire chromosomes from chemically synthesized DNA (6, 7). Because absolute control can be exerted over the sequence of chemically derived DNA mole- cules, genetic components can be exhaustively optimized. The capacity of the yeast Saccharomyces cerevisiae to take up and recombine DNA fragments has made it a model eukaryote for studying numerous cellular processes. This is mainly because DNA sequences can be genetically altered by transforming yeast with 1. Introduction
- 29. 12 D.G. Gibson either double-stranded (ds) DNA fragments (8) or single-stranded (ss) oligos (9). In addition, homologous recombination in yeast can be used to build DNA fragments from overlapping constituent parts. This was first demonstrated when a plasmid was constructed from two dsDNA fragments containing homologous ends (10). Two nonhomologous dsDNA fragments can also be bridged by single- stranded oligonucleotides that join the ends of the two fragments (11). Previously, we showed that six overlapping dsDNA fragments could be assembled by yeast into an entire Mycoplasma genitalium genome (6). Subsequently, this process was improved, and 25 over- lapping fragments, between 17 kb and 35 kb in length, were assem- bled at once into this genome (12). More recently, we reported on the synthesis of a 1.08-Mbp Mycoplasma mycoides genome, which was used to produce a cell controlled only by this synthetic genome (7). Using yeast recombination, the synthetic M. mycoides genome was assembled in three stages from 1,078 overlapping 1,080-bp DNA fragments that were each chemically synthesized. To exclusively use yeast in the production of whole genomes and large constructs of any reasonable sequence, what remained was the demonstration of the assembly of chemically synthesized oligonucleotides into appropriate dsDNA molecules, which we reported in 2009 (13). There we showed that yeast could take up and assemble at least 38 overlapping single-stranded oligonucle- otides and a linear double-stranded vector in one transformation event to produce ~1.2-kb dsDNA fragments. These oligonucle- otides can overlap by as few as 20 bp and can be as long as 200 nucleotides in length. A protocol for synthesizing kilobase-sized DNA fragments in yeast from a series of overlapping oligos is described here. 1. Yeast/E. coli shuttle vector [e.g., pRS313 (ATCC 77142), pRS314 (ATCC 77143), pRS315 (ATCC 77144), and pRS316 (ATCC 77145)]. 2. Primers for assembly vector amplification. 3. Overlapping synthetic oligonucleotides to be assembled. 4. High-fidelity polymerase chain reaction (PCR) amplification kit (e.g., Phusion® polymerase (New England BioLabs® , Inc. [NEB])). 5. Gel extraction kit (e.g., QIAquick Gel Extraction Kit, Qiagen). 6. Tris–EDTA buffer pH 8.0 (TE buffer). 7. DNA analysis software (e.g., Vector NTI® [Invitrogen], Clone Manager [Sci-Ed], and CLC Genomics Workbench [CLC bio]). 2. Materials 2.1. Design and Preparation of the Oligonucleotides and Assembly Vector
- 30. 13 2 Oligonucleotide Assembly in Yeast to Produce Synthetic DNA Fragments 1. 100× adenine hemisulfate solution: 1% (w/v) adenine hemisulfate. Autoclave or filter sterilize, and store at room temperature. 2. YPAD100 liquid medium: 2% (w/v) bacto peptone, 1% (w/v) bacto yeast extract, 2% (w/v) dextrose, 1× adenine hemisulfate solution. Autoclave or filter sterilize and store at 4°C. 3. YPAD100 agar plates: YPAD100 liquid medium plus 2% (w/v) bacto agar. Autoclave and store plates at 4°C. 4. Yeast strain to be transformed (e.g., VL6-48, ATCC Number MYA-3666). 5. Sterile water. 6. 1 M sorbitol. 7. Sorbitol/sodium phosphate/EDTA (SPE) solution: 1 M sor- bitol, 0.01 M sodium phosphate, 0.01 M Na2 EDTA (pH 7.5). Autoclave or filter sterilize and store at room temperature. 8. Beta-mercaptoethanol (BME), 14 M. 9. Zymolyase-20T solution: 10 mg/ml Zymolyase-20T (ICN Biochemicals, cat. no. 320921), 25% (w/v) glycerol, 50 mM Tris–HCl, pH 7.5. Aliquot 500 ml portions and store at −20°C. 10. Sorbitol/Tris–Cl/CaCl2 (STC) solution: 1 M sorbitol, 0.01 M Tris–HCl, pH 7.5, 0.01 M CaCl2 . Autoclave or filter sterilize and store at room temperature. 11. Transforming DNA. 12. PEG/CaCl2 solution: 20% (w/v) PEG 8000 (US Biological, cat. no. 19966), 10 mM CaCl2 , 10 mM Tris–HCl, pH 7.5. Store at room temperature for up to 2 weeks. 13. SOS solution: 1 M sorbitol, 6.5 mM CaCl2 , 0.25% bacto yeast extract, 0.5% bacto peptone. Autoclave or filter sterilize and store at room temperature. 14. Selective regeneration bottom plates: Supplement complete minimal (CM) dropout plates (see below) with 1 M sorbitol and 1× adenine hemisulfate solution. Autoclave and store plates at 4°C. 15. Selective regeneration top agar: Supplement CM dropout plates with 1 M sorbitol, 1× adenine hemisulfate solution, and bacto agar up to 3% (w/v). Autoclave and store at room temperature. 16. Sorbitol/DMSO solution (optional): 1 M sorbitol, 15% DMSO. Prepare fresh from sterile solutions. 1. Complete minimal (CM) dropout plates: 0.17% (w/v) yeast nitrogen base, 0.5% (w/v) ammonium sulfate, 2% (w/v) dex- trose, 2% (w/v) bacto agar, complete supplemental mixture. 2. Cell resuspension buffer (Qiagen buffer P1): 50 mM Tris–Cl (pH 8.0), 10 mM EDTA. Autoclave or filter sterilize and store at room temperature. 2.2. Yeast Transformation 2.3. Identifying Yeast Clones Containing the Assembled Products
- 31. 14 D.G. Gibson 3. Zymolyase-100T solution: 20 mg/ml Zymolyase-100T (US Biological, cat. no. Z1004), 50% (w/v) glycerol, 2.5% (w/v) glucose, 50 mM Tris–HCl (pH 7.5). Prepare from sterilized solutions and store at −20°C. 4. Beta-mercaptoethanol (BME), 14 M. 5. Alkaline-lysis solution (Qiagen solution P2): 200 mM NaOH, 1% SDS (w/v). Filter sterilize and store at room temperature. 6. Neutralization solution (Qiagen solution P3): 3 M potassium acetate, pH 5.5. Autoclave or filter sterilize and store at room temperature. 7. QIAprep Spin Miniprep Kit, Qiagen (optional). 8. Isopropanol. 9. Multiplex PCR screening kit (e.g., Qiagen Multiplex PCR kit). 10. PCR kit for screening yeast clones (e.g., Hot Start Phusion® polymerase, NEB). 11. Diagnostic primers to confirm the assembled product. 12. 70% ethanol. 13. TE buffer, pH 8.0. 14. Electrocompetent E. coli cells. 15. Electrocuvettes. 16. 14-ml round-bottom tubes. 17. SOC. 18. LB plates containing antibiotic. Yeast can take up and assemble at least 38 overlapping single- stranded oligonucleotides and a linear double-stranded vector in one transformation event to produce gene-sized fragments. These oligos can overlap by as few as 20 bp and can be as long as 200 nucleotides in length. Thus, gaps as long as 160 nucleotides can be filled by yeast. In this method, the oligos are assembled with a vector to form a circular product. The terminal oligos in the set contain 20 bp overlapping sequence to the ends of a yeast/E. coli shuttle vector and restriction sites to release the synthesized dsDNA fragment from the vector: 1. PCR amplify a yeast/E. coli shuttle vector (see Note 1). 2. Purify the PCR-amplified vector from an agarose gel following electrophoresis with a commercially available kit (e.g., QIAquick Gel Extraction Kit, Qiagen). 3. Methods 3.1. Design and Preparation of the Oligonucleotides and Assembly Vector
- 32. 15 2 Oligonucleotide Assembly in Yeast to Produce Synthetic DNA Fragments 3. Quantify the PCR product and dilute to 100 ng/ml in TE buffer. 4. Synthesize or purchase oligonucleotides (see Note 2). Oligonucleotides can range from 40 to 200 bases and overlap neighboring oligos by 20–30 bases (see Note 3). Terminal oli- gos should have 20 bases of sequence that overlap with the PCR-amplified assembly vector. If PCR-amplified pRS313 (described in Note 1) is chosen as the assembly vector, these terminal oligo sequences would be 5¢-caggtcgactctagaggatcx— xW—W-3¢ for the first oligo in the series and 5¢-gaattcgagctcg gtacccg x—xW—W-3¢ for the last oligo in the series, where x—x are restriction sites to release the assembled insert from the vector (e.g., NotI restriction site, gcggccgc), and W—W is new DNA sequence that is synthesized. See Fig. 1 for an exam- ple of how to design the overlapping oligos. 5. Adjust each oligo to 50 mM with TE buffer. 6. Combine equal volumes of oligonucleotides and dilute to a per-oligonucleotide concentration of 60–240 nM in TE buffer (see Note 4). 7. Combine 20 ml of the oligo pool with 2 ml of PCR-amplified vector (step 3) and use as the transforming DNA described in the transformation procedure below. To assemble genes and genome-sized fragments from overlapping DNA molecules, the yeast spheroplast transformation procedure is carried out. In this method, cells are treated with Zymolyase® to weaken the cell wall. These yeast spheroplasts are then made com- petent to take up the overlapping DNA fragments by treatment with polyethylene glycol (PEG) and CaCl2 . A slightly modified 3.2. Yeast Transformation Fig. 1. Overlapping oligonucleotide design for assembly into a yeast vector. (a) A 340-bp sequence, which includes 20 bp overlapping sequence to PCR-amplified pRS313 (nonbolded lowercase) and NotI restriction sites (bolded and underlined). Because 56 bp is used for assembly into and release from the vector, only 284 bp of unique sequence (uppercase) is synthesized. (b) The sequence shown in (a) can be synthesized from the eight 60-mer oligos shown, which contain 20-bp overlaps.
- 33. 16 D.G. Gibson protocol described by Kouprina and Larionov (14) is carried out. This procedure is optimized for use with the VL6-48 yeast strain (ATCC Number MYA-3666): 1. Streak a frozen glycerol stock containing the yeast strain onto a YPAD100 agar plate. Incubate the plate at 30°C for 2–3 days or until individual colonies appear. Store the plate at 4°C. 2. Inoculate a single colony into 50 ml YPAD100 medium (see Note 5). 3. Harvest the cells in a 50-ml tube at 1,600×g for 3 min once the cells reach an OD600 of 0.5–0.6 (~107 cells/ml) (see Note 6). 4. Resuspend cell pellets in 50 ml sterile water. Harvest the cells as in step 3. 5. Resuspend the cells in 20 ml 1 M sorbitol. Leave the cells on ice in a covered bucket at 4°C for 4 h. Alternatively, the cells can remain on ice for up to 24 h, and the transformation procedure can be continued from step 6 on the following day. 6. Invert the tube several times to resuspend the cells that have settled. Harvest the cells as in step 3. 7. Resuspend the cells in 20 ml SPE solution. Add 40 ml BME and invert to mix. Add 40 ml Zymolyase-20T solution and invert to mix (see Note 7). 8. Incubate for 40 min in a 30°C air incubator at 50 RPM with the tube on its side. Invert the tube three to four times halfway through the incubation (see Note 8). 9. Add 1 M sorbitol up to 50 ml. Invert to mix. 10. Harvest cells at 1,600×g for 5 min. Pour off the supernatant. 11. Resuspend the spheroplasts in 20 ml of 1 M sorbitol by pipetting up and down with a 25-ml pipette (see Note 9). Add 1 M sorbitol up to 50 ml. Invert to mix. 12. Harvest the yeast spheroplasts as in step 10 (see Note 10). 13. Resuspend the spheroplasts in 2.8 ml STC solution by pipetting up and down with a 5-ml pipette (see Note 11). 14. Incubate the spheroplasts at room temperature for 10 min. 15. Add 200 ml spheroplasts to the transforming DNA solution already contained in a microfuge tube (see Note 12). Mix the spheroplasts with the DNA by slowly adding them to the DNA while stirring at the same time. 16. Incubate the spheroplasts/DNA mixture at room temperature for 10 min. 17. Add 1 ml PEG/CaCl2 solution. Mix by inverting the tube ten times. 18. Incubate the tube at room temperature for 20 min. 19. Harvest the cells at 1,500×g for 8 min in a microfuge.
- 34. 17 2 Oligonucleotide Assembly in Yeast to Produce Synthetic DNA Fragments 20. Remove supernatant with a 1-ml pipette. 21. Add 800 ml SOS solution. Resuspend by pipetting up and down with a wide-bore 1-ml pipette tip. 22. Incubate the tube in a 30°C water bath for 30 min. 23. During the incubation in step 22, add 8 ml equilibrated selec- tive regeneration top agar to a 15-ml tube. Keep tube in a 55°C water bath. 24. Add cells to the 8-ml selective regeneration top agar, invert three times, and then pour onto a selective regeneration bottom plate (see Note 13). 25. Incubate the plate at 30°C for 3–4 days. Yeast clones containing full-length assemblies can be screened by PCR or restriction digestion following its transfer to E. coli. Because of the error rates associated with oligo synthesis, assembled inserts should also be sequenced. PCR screening and DNA sequencing reactions can be carried out with primers that anneal to the vector and point inward toward the insert. The primers M13F (5¢-tgtaaaac gacggccagt-3¢) and M13R (5¢-caggaaacagctatgacc-3¢) will anneal to many commonly used vectors, including the pRS313-316 vec- tor series described above. Alternatively, the plasmid DNA can be transferred from yeast to E. coli, where it can be extracted and ana- lyzed following restriction digestion or DNA sequencing. The pro- tocol described here will provide DNA of sufficient quality and quantity for PCR analysis and E. coli transformation. In this method, primary transformants are transferred and grown on selective plates as small patches. The cells are first treated with Zymolyase® to remove the cell wall, and then a standard alkaline-lysis procedure, as performed with E. coli, is carried out: 1. Use thin pipette tips (e.g., 10-ml tips) to transfer individual colonies to CM dropout plates in ~0.5 cm2 patches. 2. Incubate the plates overnight (16–24 h) at 30°C (see Note 14). 3. Add 250 ml cell resuspension buffer containing 0.25 ml BME and 2.5 ml Zymolyase-100T solution to a microfuge tube. 4. Use a 1-ml pipette tip to scrape cells from the yeast patch and combine them with the 250-ml buffer from step 3. 5. Vortex to resuspend the cells in this mixture. 6. Incubate at 37°C for 1 h. 7. Add 250 ml alkaline-lysis solution and invert the tube seven times. 8. Incubate the tube for 5 min at room temperature. 9. Add 250 ml cold neutralization solution and invert the tube seven times (see Note 15). 10. Incubate the tube on ice for 10 min. 3.3. Identifying Yeast Clones Containing the Assembled Products
- 35. 18 D.G. Gibson 11. Centrifuge the sample at 4°C for 10 min at 16,500×g in a microfuge. 12. Pour supernatant into a fresh tube containing 700 ml isopropanol. 13. Invert the tube ten times to mix. 14. Incubate the sample at room temperature for 10 min. 15. Centrifuge at 16,500×g for 10 min. 16. Pour off the isopropanol. 17. Wash the DNA pellet with 1 ml 70% ethanol. 18. Centrifuge at 16,500×g for 5 min. 19. Pour off the 70% ethanol. 20. Spin again briefly to bring the ethanol to the bottom of the tube. 21. Remove the excess ethanol by pipetting or aspirating. 22. Allow the DNA pellet to air dry for 5 min. 23. Resuspend the DNA pellet in 50 ml TE buffer (see Note 16). If an E. coli clone is not used, proceed to step 31. 24. Electroporate 3 ml DNA from step 23 into E. coli cells (see Note 17). 25. Recover cells at 37°C for 1.5 h in 1 ml SOC medium in 14-ml round-bottom tubes. 26. Plate cells onto LB medium containing the appropriate antibi- otic (for the pRS313-316 vector series, use 100 mg/ml carbeni- cillin or ampicillin). 27. Incubate the plates at 37°C for 12–18 h. 28. Grow individual colonies in 1 ml LB medium+100 mg/ml car- benicillin (see Note 18). 29. Extract plasmid DNA using a commercially available miniprep kit (e.g., QIAprep Spin Miniprep Kit, Qiagen) (see Note 19). 30. Analyze the restriction patterns of the plasmid DNA on an agarose gel following electrophoresis (see Note 20). 31. Sequence both strands of the insert DNA. For the pRS313- 316 vector series, the M13F and M13R primers can be used. Standard Sanger sequencing reactions can be carried out on a 3100 sequencer (Applied Biosystems). 32. Align trace files with the reference sequence (see Note 21). 1. The pRS313 vector has been demonstrated to work well for assembling oligonucleotides in yeast. This vector can be linear- ized by restriction digestion with Bam HI then extracted from 4. Notes
- 36. 19 2 Oligonucleotide Assembly in Yeast to Produce Synthetic DNA Fragments an agarose gel following electrophoresis. This linearized vector can then be PCR-amplified with a forward primer having the sequence 5¢-gatcctctagagtcgacctgcaggaattcgatatcaagcttatcg-3¢ and a reverse primer having the sequence 5¢-cgggtaccgagctcga- attcggagctccaattcgccctat-3¢ where pRS313-specific sequence is bolded. The gel purification of the Bam HI restriction frag- ments and its PCR amplification help reduce the background of undesired vector-only clones following yeast transformation. 2. The oligos can be synthesized without modifications and with standard desalting. 3. Shorter oligonucleotides, such as 60-mers, can be used to avoid secondary structures and increased error rates that may occur with longer oligos. 4. A 60-nM concentration for each oligo typically works well for oligos up to 90 bases. However, for oligos that are more than 90 bases, a concentration of 200 nM for each oligo is recommended. If longer oligos are to be used (e.g., ³90-mers), a high-fidelity synthesis process and/or additional oligo purifi- cation (e.g., polyacrylamide gel electrophoresis purification, PAGE purification) should be considered to reduce the errors commonly associated with longer oligos. 5. This amount of culture can be used for up to 14 transformations. Inoculate a larger culture volume if more transformations are desired. To ensure that a logarithmic phase culture is ready to be processed the following day, a second culture that is a 1/5 dilution of the first can also be inoculated. 6. Cultures from a freshly streaked plate of yeast will typically be ready within 12–16 h. However, cultures from plates that are more than 1 month old may take as long as 24 h to reach an OD600 of 0.5. 7. Yeast spheroplasts are more fragile than cells with an intact cell wall. To avoid a reduction in transformation efficiency, the yeast should be handled with care once the Zymolyase® solu- tion is added. For example, yeast spheroplasts should not be vortexed, and pipetting should be carried out with wide-bore pipette tips. 8. During the 40-min incubation (step 8), prewarm the selective regeneration bottom plates at 37°C, melt (by microwave), and then equilibrate the selective regeneration top agar to 55°C. 9. It is normal for it to take 2–3 min to completely resuspend the yeast spheroplasts. Yeast spheroplasts are more difficult to resuspend than yeast cells with intact cell walls. 10. Optional: At this point, yeast spheroplasts can be resuspended in sorbitol/DMSO solution and stored at −80°C for later use if a seven to ten times reduction in transformation efficiency is acceptable. If this route is chosen, resuspend the yeast
- 37. 20 D.G. Gibson spheroplasts in 2.8 ml sorbitol/DMSO solution, aliquot 200 ml samples to 14 microfuge tubes, and then freeze yeast sphero- plasts in a dry ice/ethanol bath and store aliquots at −80°C. 11. Optional: The yeast transformation procedure can be carried out from this point using previously frozen yeast spheroplasts (see Note 10). If this route is chosen, thaw spheroplasts on ice, harvest them at 1,500×g for 8 min in a microfuge, and then resuspend them in 200 ml STC. 12. The volume of the transforming DNA solution should not exceed 40 ml. 13. This step must be done quickly to ensure that the top agar does not solidify prior to being poured onto the plate. 14. Alternatively, single colonies can be inoculated into 0.5 ml CM dropout liquid medium and grown overnight with agitation at 30°C. If this route is chosen, harvest the cells in a microfuge tube by centrifugation at 16,500×g for 30 s, remove the super- natant, wash the cells with 1 ml sterile water, harvest the cells as above, and then proceed with step 3. 15. Alternatively, at this step, the QIAprep Spin Miniprep Kit (Qiagen) can be used. In this case, 350 ml buffer N3 (Qiagen) is added to the sample, and the procedure is carried out as described in the instructions provided in the kit. 16. If an E. coli clone is not used, this DNA may be used as tem- plate in PCR reactions in order to screen for full-length assem- blies (e.g., with the M13 F and M13 R primer set). These PCR products may then be sequenced. 17. The EPI300™ (Epicentre) electrocompetent E. coli cells work well with this procedure. Combine 3 ml DNA with 30 ml of these cells in a 1-mm cuvette (BioRad) and electroporate the cells at 1,200 V, 25 mF, and 200 W using a Gene Pulser Xcell electroporation system (BioRad). 18. This transformation usually results in hundreds to thousands of E. coli clones. It is usually only necessary to pick one or two E. coli clones because the DNA was derived from a single yeast clone, and thus, most colonies will contain the same plasmid DNA sequence. 19. Alternatively, the method described in steps 3–23 can be car- ried out with E. coli cell pellets, with two exceptions: (1) The Zymolyase® solution and BME do not need to be added to the resuspension buffer, and the 1-h incubation step does not need to be carried out; and (2) the DNA pellet (step 23) should be suspended in TE buffer containing 0.1 mg/ml RNAse A and incubated at 37°C for 30 min. 20. If NotI restriction sites were designed into the assembly strategy, the NotI restriction enzyme can be used to release the insert to determine if a full-length assembly is present.
- 38. 21 2 Oligonucleotide Assembly in Yeast to Produce Synthetic DNA Fragments 21. ClustalW Multiple alignment (15), contained within the BioEdit Sequence Alignment Editor software can be used for this purpose. Acknowledgments The author would like to thank the Synthetic Biology Group at JCVI for the helpful discussions and Synthetic Genomics, Inc. for funding this work. References 1. Agarwal, K. L., Buchi, H., Caruthers, M. H., Gupta, N., Khorana, H. G., Kleppe, K., Kumar, A., Ohtsuka, E., Rajbhandary, U. L., Van de Sande, J. H., Sgaramella, V., Weber, H., and Yamada, T. (1970) Total synthesis of the gene for an alanine transfer ribonucleic acid from yeast, Nature 227, 27–34. 2. Reese, C. B. (2005) Oligo- and poly-nucleotides: 50 years of chemical synthesis, Org. Biomol. Chem. 3, 3851–3868. 3. Xiong, A. S., Peng, R. H., Zhuang, J., Gao, F., Li, Y., Cheng, Z. M., and Yao, Q. H. (2008) Chemical gene synthesis: strategies, softwares, error corrections, and applications, FEMS Microbiol. Rev. 32, 522–540. 4. Xiong, A. S., Peng, R. H., Zhuang, J., Liu, J. G., Gao, F., Chen, J. M., Cheng, Z. M., and Yao, Q. H. (2008) Non-polymerase-cycling- assembly-based chemical gene synthesis: strate- gies, methods, and progress, Biotechnol. Adv. 26, 121–134. 5. Czar, M. J., Anderson, J. C., Bader, J. S., and Peccoud, J. (2009) Gene synthesis demystified, Trends. Biotechnol., 27(2):63–72. 6. Gibson, D. G., Benders, G. A., Andrews- Pfannkoch, C., Denisova, E. A., Baden-Tillson, H., Zaveri, J., Stockwell, T. B., Brownley, A., Thomas, D. W., Algire, M. A., Merryman, C., Young, L., Noskov, V. N., Glass, J. I., Venter, J. C., Hutchison, C. A., 3rd, and Smith, H. O. (2008) Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome, Science 319, 1215–1220. 7. Gibson, D. G., Glass, J. I., Lartigue, C., Noskov, V. N., Chuang, R. Y., Algire, M. A., Benders, G. A., Montague, M. G., Ma, L., Moodie, M. M., Merryman, C., Vashee, S., Krishnakumar, R., Assad-Garcia, N., Andrews- Pfannkoch, C., Denisova, E. A., Young, L., Qi, Z. Q., Segall-Shapiro, T. H., Calvey, C. H., Parmar, P. P., Hutchison, C. A., 3rd, Smith, H. O., and Venter, J. C. (2010) Creation of a bacterial cell controlled by a chemically synthesized genome, Science 329, 52–56. 8. Orr-Weaver, T. L., Szostak, J. W., and Rothstein, R. J. (1981) Yeast transformation: a model sys- tem for the study of recombination, Proc. Natl. Acad. Sci. USA 78, 6354–6358. 9. Moerschell, R. P., Tsunasawa, S., and Sherman, F. (1988) Transformation of yeast with syn- thetic oligonucleotides, Proc. Natl. Acad. Sci. USA 85, 524–528. 10. Ma, H., Kunes, S., Schatz, P. J., and Botstein, D. (1987) Plasmid construction by homologous recombination in yeast, Gene 58, 201–216. 11. Raymond, C. K., Sims, E. H., and Olson, M. V. (2002) Linker-mediated recombinational sub- cloning of large DNA fragments using yeast, Genome Res. 12, 190–197. 12. Gibson, D. G., Benders, G. A., Axelrod, K. C., Zaveri, J., Algire, M. A., Moodie, M., Montague, M. G., Venter, J. C., Smith, H. O., and Hutchison, C. A., 3rd. (2008) One-step assembly in yeast of 25 overlapping DNA frag- ments to form a complete synthetic Mycoplasma genitalium genome, Proc. Natl. Acad. Sci. USA 105, 20404–20409. 13. Gibson, D. G. (2009) Synthesis of DNA frag- ments in yeast by one-step assembly of overlap- ping oligonucleotides, Nucleic Acids Res. 37, 6984–6990. 14. Kouprina, N., and Larionov, V. (2008) Selective isolation of genomic loci from complex genomes by transformation-associated recombination cloning in the yeast Saccharomyces cerevisiae, Nat Protoc. 3, 371–377. 15. Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, posi- tion-specific gap penalties and weight matrix choice, Nucleic Acids Res. 22, 4673–4680.
- 40. 23 Jean Peccoud (ed.), Gene Synthesis: Methods and Protocols, Methods in Molecular Biology, vol. 852, DOI 10.1007/978-1-61779-564-0_3, © Springer Science+Business Media, LLC 2012 Chapter 3 TopDown Real-Time Gene Synthesis Mo Chao Huang, Wai Chye Cheong, Hongye Ye, and Mo-Huang Li Abstract This chapter introduces a simple, cost-effective TopDown one-step gene synthesis method, which is suitable for the sequence assembly of fairly long DNA. This method can be distinguished from conventional gene synthesis methods by two key features: (1) the melting temperature of the outer primers is designed to be ~8°C lower than that of the assembly oligonucleotides, and (2) different annealing temperatures are utilized to selectively control the efficiencies of oligonucleotide assembly and full-length template amplifi- cation. This method eliminates the interference between polymerase chain reactions (PCR) assembly and amplification in one-step gene synthesis. Additionally, the TopDown gene synthesis has been combined with the LCGreen I DNA fluorescence dye in a real-time gene synthesis approach for investigating the stepwise efficiency and kinetics of PCR-based gene synthesis. The obtained real-time fluorescence signals are compared with gel electrophoresis results to optimize gene synthesis conditions. Key words: TopDown gene synthesis, PCR, Real-time gene synthesis, De novo gene synthesis, LCGreen I, Assembly efficiency De novo gene synthesis is a powerful molecular tool for creating man-made DNA sequences. This technology has broad applica- tions for protein engineering (1, 2), development of artificial gene networks (3, 4), and creation of synthetic genomes (5, 6). Current gene synthesis methods include ligase chain reaction (LCR) (7) and polymerase chain reaction (PCR) assembly (8), which both rely on the use of overlapping oligonucleotides to construct genes. Various PCR-based methods that have been reported include the thermodynamically balanced inside-out (TBIO) method (9), suc- cessive PCR (10), dual asymmetrical PCR (DA-PCR) (11), overlap extension PCR (OE-PCR) (12, 13), PCR-based two-step DNA synthesis (8, 10, 14), and one-step gene synthesis (15). Although the PCR assembly method has been commonly used for de novo gene synthesis, there is a lack of a universal synthesis 1. Introduction
- 41. 24 M.C. Huang et al. method and capability in accurately predicting the gene synthesis. Herein, we present a simple, cost-effective TopDown one-step gene synthesis approach (16) which can selectively control the effi- ciencies of oligonucleotide assembly and full-length template amplification of relatively long genes. This method utilizes a com- puter program to design the outer primers with the melting tem- perature ~8°C lower than that of the assembly oligonucleotides to minimize the interference between the PCR assembly and amplifi- cation in the one-step gene synthesis. The overlapping gene syn- thesis is performed in one PCR mixture with two annealing temperature segments for oligonucleotide assembly and full-length template amplification, respectively. The outer primers are sub- jected to an elevated annealing condition during the assembly pro- cess,whichpreventsmispairingamongprimersandoligonucleotides (Fig. 1). The assembly process automatically switches to a prefer- ential full-length amplification as the full-length template emerges. This greatly improves the assembly efficiency of the PCR process as Fig. 1. Schematic illustration of TopDown one-step gene synthesis.This approach combines PCR assembly and amplification into a single stage by employing different annealing temperatures for assembly and amplification.The melting temperatures of the inner oligos (To ) and outer primers (Tp ) are designed so that To −Tp ³8°C to minimize potential interfer- ence during PCR.
- 42. 25 3 TopDown Real-Time Gene Synthesis compared to the conventional one-step and two-step gene synthesis processes. Furthermore, the TD one-step method is combined with real- time fluorescence analysis to investigate the gene synthesis process. Comparing the real-time fluorescence signals with gel electrophore- sis results allows to optimize the gene synthesis conditions. The effects of the concentrations of oligonucleotides and of outer primer, the stringency of annealing temperature, and the number of PCR cycles can so be analyzed and the PCR conditions optimized. 1. 100 mM Oligonucleotides (desalted without additional purifi- cation, Research Biolabs, Singapore) (see Note 1). 2. 100 mM Forward and reverse primers (Research Biolabs, Singapore) (see Note 1). 3. 25 mM MgSO4 (see Note 2). 4. dNTP mixture (containing 25 mM dATP, 25 mM dGTP, 25 mM dCTP, and 25 mM dTTP) (see Note 2). 5. High-fidelity KOD Hot Start DNA polymerase (1.0 U/ml) and 10× KOD buffer (Novagen) (see Note 3). 6. 10 mg/ml Bovine serum albumin (BSA). 7. 10× LCGreen I (Idaho Technology Inc.) (see Note 4). 8. Deionized distilled water. 9. Agarose gel powder. 10. 10× TBE buffer. 11. 50 ng/ml 100 bp DNA ladder. 12. 6× DNA loading dye. 1. Computer software to design oligonucleotides (e.g., TmPrime and DNAWorks). 2. Vortex mixer. 3. Gel electrophoresis apparatus, digital electrophoresis power supply. 4. Real-time PCR thermocycler, such as LightCycler® 1.5 (Roche), CFX96 (Bio-Rad), or ABI 7300/7500 (Applied Biosystems). 5. Gel imaging system such as Typhoon 9200 imager or Gel Doc XR. 6. Microcentrifuge. 7. LightCycler® centrifuge adapters (Roche). 2. Materials 2.1. Reagents 2.2. Equipment
- 43. 26 M.C. Huang et al. The DNA sequence to be synthesized can be designed manually or using computer software. We strongly recommend using computer software [e.g., DNAWorks (http:/ /helixweb.nih.gov/dnaworks) (17) or TmPrime (http:/ /prime.ibn.a-star.edu.sg/) (18)] to design the gene sequence. These computer programs allow for the con- struction of oligonucleotides with a uniform melting temperature which increases the yield of the assembled full-length DNA prod- uct of the PCR gene assembly. Additionally, these programs also analyze the potential for mishybridization and secondary structures among the oligonucleotides, which you may want to check before conducting the gene assembly. For DNA with high sequence repeats, the PCR-based gene synthesis may not be the best choice, and the LCR-based approach is more effective for these challeng- ing DNAs (19). We use TmPrime to design the gene sequence. Figure 2 illustrates all the parameters needed to generate the oligonucleotide set when using TmPrime. Most of the parameters are self-explanatory. For instance, the user is asked to provide gene information, gene assem- bly buffer condition, oligonucleotide and outer primer concentra- tions, optional parameters for long DNA assembly, and parameters for mispriming analysis. The software will report the melting tem- peratures, oligonucleotide sequences, potential formation of sec- ondary structures, and statistical information for the oligo sets of each pool in a PDF file (see Note 5). To ensure successful TopDown gene synthesis, oligos are designed to have a melting temperature that is ~8°C higher than that of the outer primers to minimize the competition between PCR assembly and PCR amplification of the assembled product in the one-step gene synthesis (see Note 6). The sequence of the human calcium-binding protein A4 promoter (S100A4, 752 bp; chr1:1503312036–1503311284) has been selected here as the target DNA for demonstration. The average melting temperature of the designed oligonucleotides is 66°C and consists of a pool of 30 oligos ranging in length from 41 nucle- otides (nt) to 66 nt. The primers that anneal to the target gene can be designed using IDT SciTools (http:/ /www.idtdna.com/SciTools/SciTools.aspx) (20) and TmPrime with an outer primer concentration of 300– 400 nM. It is important that the primers are designed to anneal only to the outermost 3¢ and 5¢ ends of the target gene. 1. Set up the master mix containing the inner oligos: add 2 ml (100 mM) of each of the oligos to a 600-ml microfuge tube, and add deionized distilled water to a final volume of 200 ml. 3. Methods 3.1. Reagent Setup: Designing the DNA of Interest 3.1.1. Design of Oligonucleotides and Outer Primers 3.1.2. Designing Gene-Specific Primers 3.2. Real-Time TopDown Gene Synthesis
- 44. 27 3 TopDown Real-Time Gene Synthesis Fig. 2. Web interface for TmPrime. TmPrime is implemented in functional modules, each module reflecting a different aspect of the oligonucleotide design process with the interface elements organized in a coherently grouped fashion.
- 45. 28 M.C. Huang et al. The final concentration of the oligo master mix is 1 mM. Then pipette 10 ml of the master mix into a 200-ml PCR tube, and add deionized distilled water to 40 ml. The concentration of the diluted master mix is 250 nM. The master mix should be stored at −20°C to prevent degradation of the oligos. 2. Set up the master mix of the gene-specific outer primers: add 5 ml (100 mM) of each outer primer into a 600-ml microfuge tube, and add deionized distilled water to 50 ml. Mix the con- tents of the tube by flicking and then pulse vortex in a vortex mixer. The final concentration of the primer master mix is 10 mM. 3. Prepare the TopDown gene synthesis mixture: add the PCR reaction components below to a thin-walled 200-ml PCR tube and mix the reaction mixture by flicking and spinning briefly. Pipette 20 ml of this reaction mixture into the reaction capillary of a LightCycler® 1.5 real-time thermal cycling machine. Cap the reaction capillary manually or using the LightCycler® capping tool. Throughput this procedure, all reaction solu- tions should be stored and handled on ice. Component Amount Final amount/concentration dNTP (100 mM) 4 ml 4 mM Oligo mix (250 nM) 2 ml 10 nM Primer mix (10 mM) 2 ml 400 nM MgSO4 (25 mM) 8 ml 4 mM 10× KOD buffer 5 ml 1× KOD Hot Start polymerase 1 ml 1 U BSA (10 mg/ml) 2.5 ml 0.5 mg/ml 10× LCGreen I 10 ml 1× ddH2 O 15.5 ml Total volume 50 ml 4. Gently insert the capillary into LightCycler® centrifuge adapt- ers. Transfer the centrifuge adapters into a standard microcen- trifuge and briefly spin for 3–5 s at 500–1,000 rpm. Check and ensure that the reaction mix fills the capillary. (Note: repeat the centrifugation step if the reaction mix fails to fill the length of capillary or large air bubbles are found within the capillary as this will degrade signal detection during real-time PCR.) Remove the reaction capillary from the centrifuge adapters and gently insert it into the LightCycler® sample carousel. (Note: take note of the position of capillary on carousel.) You can use a different real-time PCR thermal cycler as long as the
- 46. 29 3 TopDown Real-Time Gene Synthesis thermal cycler can detect the LCGreen I (optimum excitation 440–470 nm, optimum emission 470–520 nm). 5. Carry out the real-time TopDown gene synthesis in a Roche LightCycler® 1.5 (or another real-time thermocycler) with the following thermal cycling conditions: 2 min initial denatur- ation at 95°C; 15 cycles of 95°C for 5 s, 65–70°C (according to the Tm of inner oligos) for 60 s, 72°C for 30 s; followed by 15 cycles of 95°C for 5 s, 50–55°C (according the Tm of outer primers) for 60 s, 72°C for 30 s; followed by a final extension step at 72°C for 10 min (see Note 7). 1. Dilute the 10× TBE buffer with deionized water to the final concentration of 0.5×. 2. Prepare a 1.5% agarose gel solution by adding 3 g of agarose power to 200 ml of 0.5× TBE buffer using a 500-ml beaker or plastic bottle. 3. Heat the beaker in a microwave oven on medium power for 3–5 min until the solution is boiling. This step ensures that the agarose powder is fully dissolved. 4. Cool the solution to 50–60°C prior to gel casting. Cast the gel to the gel tray and wait for ~1 h until the gel is solidified. 5. Prepare DNA samples: mix 5 ml of the assembled product (from step 5, Subheading 3.2) with 1 ml of loading dye. For real-time gene synthesis, the assembly mixture already contains LCGreen I; thus, no additional LCGreen is needed. 6. Prepare 100 bp DNA ladder: mix 5 ml of 100 bp DNA ladder with 1 ml of LCGreen I. 7. Load the DNA ladder and DNA samples to the wells of cast gel. 8. Perform gel electrophoreses at 60 V for 60 min. 9. Scan the gel image with the Typhoon 9200 image scanner or any type of gel imaging system with emission filter for LCGreen I (optimum excitation 440–470 nm, optimum emis- sion 470–520 nm). 1. The optimum oligo and outer primer concentrations are 10–20 nM and 0.3–0.4 mM, respectively for TopDown gene synthesis (Figs. 3 and 4). 2. dNTP and Mg2+ concentration: the dNTPs concentration has been increased from 0.2 mM each as used in standard PCR to 1 mM each for TopDown gene synthesis to prevent the deple- tion of dNTPs. The Mg2+ concentration has been empirically 3.3. Agarose Gel Electrophoresis 4. Notes
- 47. 30 M.C. Huang et al. Fig. 3. Effect of oligo concentration of gene synthesis. The oligonucleotide concentration is critical for successful gene synthesis. S100A4 (752 bp) was synthesized using various oligonucleotide concentrations ranging from 5 to 80 nM and annealing temperatures of 67°C (first 20 cycles) and 49°C (next 20 cycles). (a) Fluorescence intensity versus cycle number plot for different oligonucleotide concentrations: 5 nM (open diamond), 7 nM (open square), 10 nM (open triangle), 13 nM (plus sign), 17 nM (multiplication sign), 20 nM (open circle), 40 nM (filled circle), 64 nM (filled triangle), and 80 nM (filled square). (b) Corresponding agarose gel (1.5%) electrophoresis results. The increasing slope of the fluorescence intensity during the early cycles and again around cycle number 21 indicates the efficiency of the assembly and amplification process, respectively. optimized (with an optimum of 4 mM) based on the concen- tration of dNTPs that can chelate Mg2+ , thereby affecting poly- merase activity (21, 22). 3. Choice of DNA polymerase: KOD Hot Start polymerase is rec- ommended for TopDown gene synthesis as we have observed that this polymerase outperforms Taq and Pfu polymerases. 4. Choice of DNA fluorescence dye: LCGreen I, which has a sim- ilar fluorescence spectrum to SYBR Green I, which is com- monly used in real-time PCR (23), is more suitable for studying
- 48. 31 3 TopDown Real-Time Gene Synthesis Fig. 4. Effect of outer primer concentration on gene synthesis. S100A4 (752 bp) is suc- cessfully synthesized with different primer concentrations ranging from 60 nM to 1 mM, as indicated by the sharp, narrow gel band of the desired size. (a) Fluorescence intensity versus cycle number plot for outer primers’ concentrations of 60 nM (open diamond ), 120 nM (open square), 200 nM (open triangle), 300 nM (multiplication sign), 400 nM (plus sign), and 1 mM (open circle). The inset shows the fluorescence signal for the first 20 cycles. (b) Corresponding agarose gel (1.5%) gel electrophoresis results.
- 49. 32 M.C. Huang et al. Fig. 5. Effect of annealing temperature on gene synthesis. S100A4 (752 bp) was synthe- sized using different assembly annealing temperatures ranging from 58 to 70°C for the first 20 cycles, followed by another 20 cycles at annealing temperature of 49°C. (a) Fluorescence intensity versus cycle number plot for different annealing temperatures: 58°C (open diamond), 60°C (open square), 62°C (open triangle), 65°C (multiplication sign), 67°C (plus sign), and 70°C (open circle). The inset shows the 15 midcycles (cycles 13–27). (b) Agarose gel (1.5%) electrophoresis results. A higher yield of gene synthesis was obtained with a stringent assembly annealing temperature (>67°C).
- 50. 33 3 TopDown Real-Time Gene Synthesis real-time gene synthesis. SYBR Green I binds preferentially to long DNA fragments (24) and can redistribute from short DNA fragments to large DNA fragments during thermal cycling, which makes it difficult to analyze the observed fluo- rescence signal, as the assembly mixture contains dsDNA of various sizes. The optimum concentration of LCGreen I is 1× for TopDown gene synthesis with 10–20 nM of oligos. 5. TmPrime gene design program is being optimized continu- ously. Hence, the average melting temperature and oligonucle- otide sequences may be different from the provided data. 6. We recommend designing the oligos and outer primers with the following conditions: (1) design outer primers (Tm =50–55°C) and inner oligos (Tm ~65°C) with distinct melting temperatures (i.e., DTm ³8°C); (2) oligos and outer primers concentration of 10 nm and 400 nM, respectively; and (3) 50 mM of Na+ /K+ , 4 mM of Mg2+ , and 4 mM of dNTPs. 7. Optimize the assembly cycle numbers: the number of PCR cycles influences the quality and quantity of PCR-based gene synthesis. The fluorescence curve (see 10-nM curve in Fig. 3) suggests that the assembly and amplification processes reach the plateau at around cycle 15 and cycle 35. Thus, the opti- mum number of PCR cycles is 30–15 cycles each for the assem- bly and the amplification reaction. The amplification efficiency of the PCR reaction decreases after it reaches the plateau. Additional PCR cycling will favor nonspecific annealing of the full-length product to either randomly assembled fragments or to itself. Additionally, we recommend conducting TopDown synthesis with an assembly annealing temperature that is 2–5°C higher than the average melting temperature of the constructed oligos. Such a stringent annealing condition usually leads to a better yield of the full-length DNA product (Fig. 5). The assembly efficiency of PCR gene synthesis depends on the gene length and sequence content. So far, the maximum gene length that we have successfully constructed using the TopDown approach is ~1.6 kb from a pool of 60 oligonucleotides. References 1. He M, Stoevesandt O, Palmer EA, Khan F, Ericsson O, and Taussig MJ (2008) Printing protein arrays from DNA arrays. Nat. Methods 5:175–177. 2. Cox JC, Lape J, Sayed MA and Hellinga HW (2007) Protein fabrication automation. Protein Sc. 16:379–390. 3. Sprinzak D, and Elowitz MB (2005) Reconstruction of genetic circuits. Nature 438: 443–448. 4. Basu S, Gerchman Y, Collins,CH. Arnold,FH and Weiss RA (2005) Synthetic multicellular system for programmed pattern formation. Nature 434:1130–1134. 5. Smith HO, Hutchison CA III, Pfannkoch C and Venter JC (2003) Generating a synthetic genome by whole genome assembly: FX174 bacteriophage from synthetic oligonucle- otides. Proc Natl Acad Sci USA 100: 15440–15445.
- 51. 34 M.C. Huang et al. 6. Gibson DG, Benders GA, Andrews-Pfannkoch C, Denisova EA, Baden-Tillson H, Zaveri J, Stockwell TB, Brownley A, Thomas DW, Algire MA, Merryman C, Young L, Noskov VN, Glass JI, Venter JC, Hutchison CA.III and Smith HO (2008) Complete chemical synthesis, assembly, and cloning of a Mycoplasma genita- lium genome. Science 319:1215–1220. 7. Au LC, Yang FY, Yang WJ, Lo SH and Kao,CF (1998) Gene synthesis by a LCR-based approach: High-level production of leptin-L54 using synthetic gene in Escherichia coli. Biochem Biophys Res.Commun 248:200–203. 8. Stemmer WP, Crameri A, Ha KD, Brennan TM and Heyneker HL (1995) Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene 164:49–53. 9. Gao X, Yo P, Keith A, Ragan TJ and Harris TK (2003) Thermodynamically balanced inside- out (TBIO) PCR-based gene synthesis: A novel method of primer design for high-fidelity assembly of longer gene sequences. Nucleic Acids Res 31:,e143. 10. Xiong A-S, Yao Q-H, Peng R-H, Li X, Fan H-Q, Cheng Z-M and Li Y (2004) A simple, rapid, high-fidelity and cost-effective PCR- based two-step DNA synthesis method for long gene sequences. Nucleic Acids Res 32:e98. 11. Sandhu GS, Aleff RA and Kline, BC (1992) Dual asymmetric PCR: One-step construction of synthetic genes. Biotechniques 12:14–16. 12. Toung L and Dong,Q.(2004) Two-step total gene synthesis method. Nucleic Acids Res 32:e59. 13. Prodromou C and Pearl L (1992) Recursive PCR: A novel technique for total gene synthe- sis. Protein Eng 5:827–829. 14. Xiong A-S, Yao Q-H, Peng R-H, Duan H, Li X, Fan H-Q, Cheng Z-M and Li Y (2006) PCR- basedaccuratesynthesisoflongDNAsequences. Nat Protoc 1:791–797. 15. Wu G, Wolf JB, Ibrahim AF, Vadasz S, Gunasinghe M and Freeland SJ (2006) Simplified gene synthesis: A one-step approach to PCR-based gene construction. J. Biotechnol 124: 496–503. 16. Ye H, Huang MC, Li M-H and Ying JY (2009) Experimental analysis of gene assembly with TopDown one-step real-time gene synthesis. Nucleic Acids Res 37:e51. 17. Hoover DM and Lubkowski J (2002) DNAWorks: An automated method for design- ing oligonucleotides for PCR-based gene syn- thesis. Nucleic Acids Res 30:e43. 18. Bode M, Khor,S, Ye H, Li M-H and Ying JY (2009) TmPrime: fast, flexible oligonucleotide design software for gene synthesis. Nucleic Acids Res 37:W214–W221. 19. Bang D and Church GM (2008) Gene synthe- sis by circular assembly amplification. Nat Methods 5:37–39. 20. Owczarzy R. Tataurov AV, Wu,Y, Manthey,JA, McQuisten KA, Almabrazi HG et al. (2008) IDT SciTools: a suite for analysis and design of nucleic acid oligomers. Nucleic Acids Res 36:W163–W169. 21. Ely JJ, Reeves-Daniel A, Campbell ML, Kohler S and Stone WH (1998) Influence of magne- sium ion concentration and PCR amplification conditions on cross-species PCR. Biotechniques 25:38–40. 22. von Ahsen N, Wittwer CT and Schütz E. (2001) Oligonucleotide melting temperatures under PCR conditions: Nearest-neighbor cor- rections for Mg2+ , deoxynucleotide triphos- phate, and dimethyl sulfoxide concentrations with comparison to alternative empirical for- mulas. Clin Chem 47:1956–1961. 23. WittwerCT,ReedGH,Gundry,CN,Vandersteen JG and Pryor RJ (2003) High-resolution geno- typing by amplicon melting analysis using LCGreen. Clin Chem 49:853–860. 24. Giglio S, Monis PT and Saint CP (2003) Demonstration of preferential binding of SYBR Green I to specific DNA fragments in real-time multiplex PCR. Nucleic Acids Res.31:e136.
- 52. 35 Jean Peccoud (ed.), Gene Synthesis: Methods and Protocols, Methods in Molecular Biology, vol. 852, DOI 10.1007/978-1-61779-564-0_4, © Springer Science+Business Media, LLC 2012 Chapter 4 De Novo DNA Synthesis Using Single-Molecule PCR Tuval Ben Yehezkel, Gregory Linshiz, and Ehud Shapiro Abstract The throughput of DNA reading (i.e., sequencing) has dramatically increased recently owing to the incorporation of in vitro clonal amplification. The throughput of DNA writing (i.e., synthesis) is trailing behind, with cloning and sequencing constituting the main bottleneck. To overcome this bottleneck, an in vitro alternative for in vivo DNA cloning needs to be integrated into DNA synthesis methods. Here, we show how a new single-molecule PCR (smPCR)-based procedure can be employed as a general substitute for in vivo cloning, thereby allowing for the first time in vitro DNA synthesis. We integrated this rapid and high fidelity in vitro procedure into our previously described recursive DNA synthesis and error correction procedure and used it to efficiently construct and error-correct a 1.8-kb DNA molecule from synthetic unpurified oligonucleotides, entirely in vitro. Although we demonstrate incorporating smPCR in a particular method, the approach is general and can be used, in principle, in conjunction with other DNA synthesis methods as well. Key words: DNA synthesis, In vitro cloning, In vivo cloning, DNA error correction, Single-molecule PCR, Synthetic biology The broad availability of synthetic DNA oligonucleotides enabled the development of many powerful applications in biotechnology. Longer synthetic DNA molecules and libraries (generated from assembly of these oligonucleotides) in the 0.5–5 kb range are now becoming increasingly available owing to newly developed synthesis and error correction methods (1–7). The wide availability of such molecules, in great need since the advent of synthetic biology and modern genetic engineering, is expected to enable the routine creation of new genetic material, as well as offer an alternative to obtaining DNA from natural sources. 1. Introduction
- 53. 36 T.B. Yehezkel et al. Unfortunately, the synthetic DNA oligonucleotides (oligos) used as building blocks for the generation of the longer constructs are error-prone. Such errors accumulate linearly with the length of the constructed molecule and result in an exponential decrease in the fraction of error-free molecules. Hence, an exponentially increasing number of molecules have to be screened, i.e., cloned into a host organism and sequenced, in order to obtain ever longer error-free molecules. In order to mitigate this effect, a two-step assembly process (4, 7) is often used, in which fragments in the 500–1,000 bp range are first screened via cloning and sequencing before the error-free clones are synthesized. In vivo cloning (1–7) is time consuming, labor intensive, and difficult to scale up and automate. These limitations combined with the sheer number of clones that needs to be screened to obtain long error-free synthetic DNA make the cloning phase a bottleneck in de novo DNA synthesis and prevent synthetic DNA from being routinely produced in a fast, cheap, and high-throughput manner. Reducing the number of clones required to obtain an error-free molecule is the subject of intensive ongoing research (1, 2, 4, 6), also recently addressed by us (5) with the development of a method that we believe relieves much of this burden. In this chapter, we address the second major issue, namely, replacing the time consuming and labor intensive in vivo cloning procedure that is associated with synthetic DNA synthesis with a faster and less laborious in vitro cloning procedure. Since its introduction, the polymerase chain reaction (PCR) (8) has been implemented in a myriad of variations, one of which is PCR on a single DNA template molecule (9), which essentially creates a PCR “clone.” Single-molecule PCR (smPCR) is a faster, cheaper, scalable, and automatable alternative to traditional in vivo cloning. Its standard application in molecular biology has been nonsystematic, most commonly used for the amplification of single molecules for sequencing, genotyping, or downstream translation purposes (8–12). Recently, it has been systematically integrated into high-throughput DNA sequencing (13, 14). High-throughput DNA synthesis technologies can also benefit from smPCR, as demonstrated here for the use of smPCR in the context of our recently introduced DNA synthesis procedure (5), which combines recursive synthesis and error correction. In this chapter, we show that in vitro cloning based on smPCR can be used as a practical alternative to conventional in vivo cloning. In particular, we show the successful construction of a 1.8-kb-long DNA molecule from synthetic unpurified oligos using our recursive synthesis and error correction procedure combined with smPCR. As a control, we also constructed the same molecule using conventional in vivo cloning, and the results are compared below.
- 54. 37 4 De Novo DNA Synthesis Using Single-Molecule PCR 1. T4 polynucleotide kinase (NEB, Ipswich, MA, USA). 2. Thermo-Start DNA polymerase (ABgene). 3. Lambda exonuclease (Epicenter). 1. Hot-start Accusure (BioLINE, Taunton, MA, USA). 2. Taq polymerase (ABgene, UK). Oligonucleotides for all experiments were ordered from commercial providers (Sigma Genosys & IDT) with standard desalting. QIAGEN’s QIAquick 96-well PCR purification kit and QIAGEN’s MinElute PCR purification kit. pGEM-T Easy Vector System and JM109 competent cells from PROMEGA. 1. Recursive (or other type of de novo) construction of the molecule from synthetic unpurified oligos (see Fig. 1), as specified below (see Subheading 3.4) and in ref. 5. 2. “Adaptor PCR” for the insertion of the CA primer sequence and the random bar-coding nucleotides (see Fig. 2) on templates from step 1. This is done using the PCR protocol and the primers specified in (15). Alternatively, these sequences can also be included as part of the original target sequence to avoid the additional PCR. 3. Early termination of the PCR of step 2 within the twofold exponential amplification phase (as shown in Fig. 3) to prevent heterodimer formation (see Figs. 4 and 5). 4. Optical density (OD) measurement and dilution of the PCR from step 3 according to the graphs depicted in Fig. 6. Alternatively, the real-time PCR-assisted calibration experiment (described in Subheading 3.7) can also be used to determine the required dilution instead of measuring the OD. 5. smPCRs using the CA primer and templates from the dilution prepared in step 4. The number of reactions prepared is deter- mined according to the required number of clones as shown in Fig. 6, and the error rate as described in Figs. 7–9. The PCR 2. Materials 2.1. Core Recursive DNA Construction 2.2. smPCR 2.3. Chemical Oligonucleotide Synthesis 2.4. DNA Purification Kits 2.5. Cloning System 3. Methods 3.1. Summary of the Procedure
- 55. Other documents randomly have different content
- 56. CHAPTER XXVIII BELFAST TO THE CAUSEWAY THE Lough of Belfast has a reputation for beauty almost as great as that of the Bay of Dublin; but though, on the day I left Belfast for Larne, the morning was fine, and the sky clear and blue above, an envious mist lay on the water, which hid all its beauties from the dozen of passengers on the Larne coach. All we could see were ghostly-looking silhouettes of ships gliding here and there through the clouds; and I am sure the coachman’s remark was quite correct, that it was a pity the day was so misty. I found myself, before I was aware, entrapped into a theological controversy with two grave gentlemen outside the coach—another fog, which did not subside much before we reached Carrickfergus. The road from the Ulster capital to that little town seemed meanwhile to be extremely lively; cars and omnibuses passed thickly peopled. For some miles along the road is a string of handsome country-houses, belonging to the rich citizens of the town; and we passed by neat-looking churches and chapels, factories and rows of cottages clustered round them, like villages of old at the foot of feudal castles. Furthermore it was hard to see, for the mist which lay on the water had enveloped the mountains too, and we only had a glimpse or two of smiling comfortable fields and gardens. Carrickfergus rejoices in a real romantic-looking castle jutting bravely into the sea, and famous as a background for a picture. It is of use for little else now, luckily, nor has it been put to any real warlike purposes since the day when honest Thurot stormed, took, and evacuated it. Let any romancer who is in want of a hero peruse the second volume, or it may be the third, of the Annual Register, where the adventures of that gallant fellow are related. He was a gentleman, a genius, and, to crown all, a smuggler. He lived for some time in Ireland, and in England, in disguise; he had love-passages and romantic adventures; he landed a body of his countrymen on these shores, and died in the third volume, after a battle gallantly fought on both sides, but in which victory rested with the British arms. What can a novelist want more? William III. also landed here; and as for the rest, ‘M’Skimin, the
- 57. accurate and laborious historian of the town, informs us that the founding of the castle is lost in the depths of antiquity.’ It is pleasant to give a little historic glance at a place as one passes through. The above facts may be relied on as coming from Messrs. Curry’s excellent new Guide-book, with the exception of the history of Mons. Thurot, which is ‘private information,’ drawn years ago from the scarce work previously mentioned. By the way, another excellent companion to the traveller in Ireland is the collection of the Irish Penny Magazine, which may be purchased for a guinea, and contains a mass of information regarding the customs and places of the country. Willis’s work is amusing, as everything is, written by that lively author, and the engravings accompanying it as unfaithful as any ever made. Meanwhile, asking pardon for this double digression, which has been made while the guard-coachman is delivering his mail-bags—while the landlady stands looking on in the sun, her hands folded a little below the waist—while a company of tall burly troops from the castle has passed by, ‘surrounded’ by a very mean, mealy-faced, uneasy-looking little subaltern —while the poor, epileptic idiot of the town, wallowing and grinning in the road, and snorting out supplications for a halfpenny, has tottered away in possession of the coin;—meanwhile, fresh horses are brought out, and the small boy who acts behind the coach, makes an unequal and disagreeable tootooing on a horn kept to warn sleepy carmen and celebrate triumphal entries into and exits from cities. As the mist clears up, the country shows round about wild but friendly; at one place we passed a village where a crowd of well-dressed people were collected at an auction of farm-furniture, and many more figures might be seen coming over the fields and issuing from the mist. The owner of the carts and machines is going to emigrate to America. Presently we come to the demesne of Red Hall, ‘through which is a pretty drive of upwards of a mile in length: it contains a rocky glen, the bed of a mountain stream—which is perfectly dry, except in winter—and the woods about it are picturesque, and it is occasionally the resort of summer-parties of pleasure.’ Nothing can be more just than the first part of the description, and there is very little doubt that the latter paragraph is equally faithful;—with which we come to Larne, a ‘most thriving town,’ the same authority says, but a most dirty and narrow-streeted and ill-built one. Some of the houses reminded one of the south, as thus— A benevolent fellow-passenger said that the window was ‘a convanience’; and here, after a drive of nineteen miles upon a comfortable
- 58. coach, we were transferred with the mail-bags to a comfortable car that makes the journey to Ballycastle. There is no harm in saying that there was a very pretty smiling buxom young lass for a travelling companion; and somehow, to a lonely person, the landscape always looks prettier in such society. The ‘Antrim coast road,’ which we now, after a few miles, begin to follow, besides being one of the most noble and gallant works of art that is to be seen in any country, is likewise a route highly picturesque and romantic; the sea spreading wide before the spectator’s eyes upon one side of the route;—the tall cliffs of limestone rising abruptly above him on the other. There are in the map of Curry’s Guide-book points indicating castle and abbey ruins in the vicinity of Glenarm; and the little place looked so comfortable as we abruptly came upon it round a rock, that I was glad to have an excuse for staying, and felt an extreme curiosity with regard to the abbey and the castle. The abbey only exists in the unromantic shape of a wall; the castle, however, far from being a ruin, is an antique in the most complete order— an old castle repaired so as to look like new, and increased by modern wings, towers, gables, and terraces, so extremely old that the whole forms a grand and imposing-looking baronial edifice, towering above the little town which it seems to protect, and with which it is connected by a bridge and a severe-looking armed tower and gate. In the town is a town-house, with a campanile in the Italian taste, and a school or chapel opposite, in the Early English; so that the inhabitants can enjoy a considerable architectural variety. A grave-looking church, with a beautiful steeple, stands amid some trees hard by a second handsome bridge and the little quay; and here, too, was perched a poor little wandering theatre (gallery 1d., pit 2d.), and proposing that night to play ‘Bombastes Furioso, and the Comic Bally of Glenarm in an Uproar.’ I heard the thumping of the drum in the evening; but, as at Roundwood, nobody patronised the poor players: at nine o’clock there was not a single taper lighted under their awning, and my heart (perhaps it is too susceptible) bled for Fusbos. The severe gate of the castle was opened by a kind, good-natured old porteress, instead of a rough gallowglass with a battle-axe and yellow shirt (more fitting guardian of so stern a postern), and the old dame insisted upon my making an application to see the grounds of the castle, which request was very kindly granted, and afforded a delightful half-hour’s walk. The
- 59. grounds are beautiful, and excellently kept; the trees in their autumn livery of red, yellow, and brown, except some stout ones that keep to their green summer clothes, and the laurels and their like, who wear pretty much the same dress all the year round. The birds were singing with most astonishing vehemence in the dark glistening shrubberies; but the only sound in the walks was that of the rakes pulling together the falling leaves. There was of these walks one especially, flanked towards the river by a turreted wall covered with ivy, and having on the one side a row of lime-trees that had turned quite yellow, while opposite them was a green slope, and a quaint terrace-stair, and a long range of fantastic gables, towers, and chimneys;— there was, I say, one of these walks which Mr. Cattermole would hit off with a few strokes of his gallant pencil, and which I could fancy to be frequented by some of those long-trained, tender, gentle-looking young beauties whom Mr. Stone loves to design. Here they come talking of love in a tone that is between a sigh and a whisper, and gliding in rustling shot silks over the fallen leaves. There seemed to be a good deal of stir in the little port, where, says the Guide-book, a couple of hundred vessels take in cargoes annually of the produce of the district. Stone and lime are the chief articles exported, of which the cliffs for miles give an unfailing supply; and, as one travels the mountains at night, the kilns may be seen lighted up in the lonely places, and flaring red in the darkness. If the road from Larne to Glenarm is beautiful, the coast route from the latter place to Cushendall is still more so; and, except peerless Westport, I have seen nothing in Ireland so picturesque as this noble line of coast- scenery. The new road, luckily, is not yet completed, and the lover of natural beauties had better hasten to the spot in time, ere, by flattening and improving the road, and leading it along the sea-shore, half the magnificent prospects are shut out, now visible from along the mountainous old road; which, according to the good old fashion, gallantly takes all the hills in its course, disdaining to turn them. At three miles’ distance, near the village of Cairlough, Glenarm looks more beautiful than when you are close upon it; and, as the car travels on to the stupendous Garron Head, the traveller, looking back, has a view of the whole line of coast southward as far as Isle Magee, with its bays and white villages, and tall precipitous cliffs, green, white, and grey. Eyes left, you may look with wonder at the mountains rising above, or presently at the pretty park and grounds of Drumnasole.
- 60. Here, near the woods of Nappan, which are dressed in ten thousand colours —ash-leaves turned yellow, nut-trees red, birch-leaves brown, lime-leaves speckled over with black spots (marks of a disease which they will never get over)—stands a school-house that looks like a French château, having probably been a villa in former days, and discharges, as we pass, a cluster of fair-haired children that begin running madly down the hill, their fair hair streaming behind them. Down the hill goes the car madly too, and you wonder and bless your stars that the horse does not fall, or crush the children that are running before, or you that are sitting behind. Every now and then, at a trip of the horse, a disguised lady’s-maid, with a canary-bird in her lap and a vast anxiety about her best bonnet in the bandbox, begins to scream; at which the car-boy grins, and rattles down the hill only the quicker. The road, which almost always skirts the hillside, has been torn sheer through the rock here and there; and immense work of levelling, shovelling, picking, blasting, filling, is going on along the whole line. As I was looking up a vast cliff, decorated with patches of green here and there at its summit, and at its base, where the sea had beaten until now, with long, thin, waving grass, that I told a grocer, my neighbour, was like mermaids’ hair (though he did not in the least coincide in the simile)—as I was looking up the hill, admiring two goats that were browsing on a little patch of green, and two sheep perched yet higher (I had never seen such agility in mutton) —as, I say once more, I was looking at these phenomena, the grocer nudges me and says, ‘Look on to this side—that’s Scotland yon,’ If ever this book reaches a second edition, a sonnet shall be inserted in this place, describing the author’s feelings on his first view of Scotland. Meanwhile, the Scotch mountains remain undisturbed, looking blue and solemn far away in the placid sea. Rounding Garron Head, we come upon the inlet which is called Red Bay, the shores and sides of which are of red clay, that has taken the place of limestone, and towards which, between two noble ranges of mountains, stretches a long green plain, forming, together with the hills that protect it and the sea that washes it, one of the most beautiful landscapes of this most beautiful country. A fair writer, whom the Guide-book quotes, breaks out into strains of admiration in speaking of this district; calls it ‘Switzerland in miniature,’ celebrates its mountains of Glenariff and Lurgethan, and lauds, in terms of equal admiration, the rivers, waterfalls, and other natural beauties that lie within the glen.
- 61. The writer’s enthusiasm regarding this tract of country is quite warranted, nor can any praise in admiration of it be too high; but alas! in calling a place ‘Switzerland in miniature,’ do we describe it? In joining together cataracts, valleys, rushing streams, and blue mountains, with all the emphasis and picturesqueness of which type is capable, we cannot get near to a copy of Nature’s sublime countenance; and the writer can’t hope to describe such grand sights so as to make them visible to the fireside reader, but can only, to the best of his taste and experience, warn the future traveller where he may look out for objects to admire. I think this sentiment has been repeated a score of times in this journal; but it comes upon one at every new display of beauty and magnificence, such as here the Almighty in His bounty has set before us; and every such scene seems to warn one, that it is not made to talk about too much, but to think of, and love, and be grateful for. Rounding this beautiful bay and valley, we passed by some caves that penetrate deep into the red rock, and are inhabited—one by a blacksmith, whose forge was blazing in the dark; one by cattle; and one by an old woman that has sold whisky here for time out of mind. The road then passes under an arch cut in the rock by the same spirited individual who has cleared away many of the difficulties in the route to Glenarm, and beside a conical hill, where for some time previous have been visible the ruins of the ‘ancient ould castle’ of Red Bay. At a distance, it looks very grand upon its height; but on coming close it has dwindled down to a mere wall, and not a high one. Hence, quickly we reach Cushendall, where the grocer’s family are on the look-out for him; the driver begins to blow his little bugle, and the disguised lady’s-maid begins to smooth her bonnet and hair. At this place a good dinner of fresh whiting, broiled bacon, and small beer was served up to me for the sum of eightpence, while the lady’s-maid in question took her tea. ‘This town is full of Papists,’ said her ladyship, with an extremely genteel air; and, either in consequence of this, or because she ate up one of the fish, which she had clearly no right to, a disagreement arose between us, and we did not exchange another word for the rest of the journey. The road led us for fourteen miles by wild mountains, and across a fine aqueduct to Ballycastle; but it was dark as we left Cushendall, and it was difficult to see more in the grey evening but that the country was savage and lonely, except where the kilns were lighted up here and there in the hills, and a shining river might be seen winding in the dark ravines. Not
- 62. far from Ballycastle lies a little old ruin, called the Abbey of Bonamargy: by it the Margy river runs into the sea, upon which you come suddenly; and on the shore are some tall buildings and factories, that looked as well in the moonlight as if they had not been in ruins; and hence a fine avenue of limes leads to Ballycastle. They must have been planted at the time recorded in the Guide-book, when a mine was discovered near the town, and the works and warehouses on the quay erected. At present, the place has little trade, and half a dozen carts with apples, potatoes, dried fish, and turf, seem to contain the commerce of the market. The picturesque sort of vehicle which is here designed, is said to be going much out of fashion in the country, the solid wheels giving place to those common to the rest of Europe. A fine and edifying conversation took place between the designer and the owner of the vehicle. ‘Stand still for a minute, you and the car, and I will give you twopence!’ ‘What do you want to do with it?’ says the latter. ‘To draw it.’ ‘To draw it?’ says he, with a wild look of surprise, ‘and is it you’ll draw it?’ ‘I mean, I want to take a picture of it; you know what a picture is?’ ‘No, I don’t.’ ‘Here’s one,’ says I, showing him a book. ‘Oh, faith, sir,’ says the carman, drawing back rather alarmed, ‘I’m no scholar!’ And he concluded by saying, ‘Will you buy the turf, or will you not? by which straightforward question he showed himself to be a real practical man of sense; and, as he got an unsatisfactory reply to this query, he forthwith gave a lash to his pony, and declined to wait a minute longer. As for the twopence, he certainly accepted that handsome sum, and put it into his pocket, but with an air of extreme wonder at the transaction, and of contempt for the giver, which very likely was perfectly justifiable. I have seen men despised in genteel companies with not half so good a cause. In respect to the fine arts, I am bound to say that the people in the south and west showed much more curiosity and interest with regard to a sketch and its progress than has been shown by the badauds of the north; the former looking on by dozens, and exclaiming, ‘That’s Frank Mahony’s house!’ or, ‘Look at Biddy Mullins and the child!’ or ‘He’s taking off the chimney now!’ as the case may be; whereas, sketching in the north, I have collected no such spectators, the people not taking the slightest notice of the transaction.
- 63. The little town of Ballycastle does not contain much to occupy the traveller: behind the church stands a ruined old mansion with round turrets, that must have been a stately tower in former days. The town is more modern, but almost as dismal as the tower. A little street behind it slides off into a potato-field—the peaceful barrier of the place; and hence I could see the tall rock of Bengore, with the sea beyond it, and a pleasing landscape stretching towards it. Dr. Hamilton’s elegant and learned book has an awful picture of yonder head of Bengore; and hard by it the Guide-book says is a coal-mine, where Mr. Barrow found a globular stone hammer, which, he infers, was used in the coal-mine before weapons of iron were invented. The former writer insinuates that the mine must have been worked more than a thousand years ago, ‘before the turbulent chaos of events that succeeded the eighth century.’ Shall I go and see a coal-mine that may have been worked a thousand years since? Why go see it? says idleness. To be able to say that I have seen it. Sheridan’s advice to his son here came into my mind;[32] and I shall reserve a description of the mine, and an antiquarian dissertation regarding it, for publication elsewhere. Ballycastle must not be left without recording the fact that one of the snuggest inns in the country is kept by the postmaster there; who has also a stable full of good horses for travellers who take his little inn on the way to the Giant’s Causeway. The road to the Causeway is bleak, wild, and hilly. The cabins along the road are scarcely better than those of Kerry, the inmates as ragged, and more fierce and dark-looking. I never was so pestered by juvenile beggars as in the dismal village of Ballintoy. A crowd of them rushed after the car, calling for money in a fierce manner, as if it was their right; dogs as fierce as the children came yelling after the vehicle; and the faces which scowled
- 64. out of the black cabins were not a whit more good-humoured. We passed by one or two more clumps of cabins, with their turf and corn-stacks lying together at the foot of the hills; placed there for the convenience of the children, doubtless, who can thus accompany the car either way, and shriek out their ‘Bonny gantleman, gie us a hap’ny.’ A couple of churches, one with a pair of its pinnacles blown off, stood in the dismal open country, and a gentleman’s house here and there: there were no trees about them, but a brown grass round about—hills rising and falling in front, and the sea beyond. The occasional view of the coast was noble; wild Bengore towering eastwards as we went along; Raghery Island before us, in the steep rocks and caves of which Bruce took shelter when driven from yonder Scottish coast, that one sees stretching blue in the north-east. I think this wild gloomy tract through which one passes is a good prelude for what is to be the great sight of the day, and got my mind to a proper state of awe by the time we were near the journey’s end; and turning away shorewards by the fine house of Sir Francis Macnaghten, went towards a lone handsome inn, that stands close to the Causeway. The landlord at Ballycastle had lent me Hamilton’s book, to read on the road; but I had not time then to read more than half a dozen pages of it. They described how the author, a clergyman distinguished as a man of science, had been thrust out of a friend’s house by the frightened servants one wild night, and butchered by some White Boys, who were waiting outside, and called for his blood. I had been told at Belfast that there was a corpse in the inn: was it there now? It had driven off, the car-boy said, ‘in a handsome hearse and four to Dublin the whole way.’ It was gone, but I thought the house looked as if the ghost was there. See, yonder are the black rocks stretching to Portrush; how leaden and grey the sea looks! how grey and leaden the sky! You hear the waters roaring evermore, as they have done since the beginning of the world. The car drives up with a dismal grinding noise of the wheels to the big lone house; there’s no smoke in the chimneys; the doors are locked; three savage-looking men rush after the car: are they the men who took out Mr. Hamilton—took him out and butchered him in the moonlight? Is everybody, I wonder, dead in that big house? Will they let us in before those men are up? Out comes a pretty smiling girl, with a curtsey, just as the savages are at the car, and you are ushered into a very comfortable room; and the men turn out to be guides. Well, thank Heaven
- 65. it’s no worse! I had fifteen pounds still left; and, when desperate, have no doubt should fight like a lion.
- 66. CHAPTER XXIX THE GIANT’S CAUSEWAY—COLERAINE—PORTRUSH THE traveller no sooner issues from the inn by a back door, which he is informed will lead him straight to the Causeway, than the guides pounce upon him, with a dozen rough boatmen who are likewise lying in wait; and a crew of shrill beggar-boys, with boxes of spars, ready to tear him and each other to pieces seemingly, yell and bawl incessantly round him. ‘I’m the guide Miss Henry recommends,’ shouts one. ‘I’m Mr. Macdonald’s guide,’ pushes in another. ‘This way,’ roars a third, and drags his prey down a precipice; the rest of them clambering and quarrelling after. I had no friends: I was perfectly helpless. I wanted to walk down to the shore by myself, but they would not let me, and I had nothing for it but to yield myself into the hands of the guide who had seized me, who hurried me down the steep to a little wild bay, flanked on each side by rugged cliffs and rocks, against which the waters came tumbling, frothing, and roaring furiously. Upon some of these black rocks two or three boats were lying: four men seized a boat, pushed it shouting into the water, and ravished me into it. We had slid between two rocks, where the channel came gurgling in; we were up one swelling wave that came in a huge advancing body ten feet above us, and were plunging madly down another (the descent causes a sensation in the lower regions of the stomach which it is not at all necessary here to describe), before I had leisure to ask myself why the deuce I was in that boat, with four rowers hurrooing and bounding madly from one huge liquid mountain to another—four rowers whom I was bound to pay. I say, the query came qualmishly across me why the devil I was there, and why not walking calmly on the shore.
- 67. A ROW TO THE GIANT’S CAUSEWAY The guide began pouring his professional jargon into my ears. ‘Every one of them bays,’ says he, ‘has a name (take my place, and the spray won’t come over you): that is Port Noffer, and the next, Port na Gange; them rocks is the Stookawns (for every rock has his name as well as every bay); and yonder—give way, my boys,—hurray, we’re over it now; has it wet you much, sir?—that’s the little cave; it goes five hundred feet under ground, and the boats goes in it easy of a calm day.’ ‘Is it a fine day or a rough one now?’ said I; the internal disturbance going on with more severity than ever. ‘It’s betwixt and between; or, I may say, neither one nor the other. Sit up, sir; look at the entrance of the cave: don’t be afraid, sir; never has an accident happened in any one of these boats, and the most delicate ladies has rode in them on rougher days than this. Now, boys, pull to the big cave; that, sir, is six hundred and sixty yards in length, though some says it goes for miles inland, where the people sleeping in their houses hears the waters roaring under them.’ The water was tossing and tumbling into the mouth of the little cave. I looked,—for the guide would not let me alone till I did,—and saw what might be expected: a black hole of some forty feet high, into which it was no more possible to see than into a millstone. ‘For Heaven’s sake, sir,’ says I, ‘if you’ve no particular wish to see the mouth of the big cave, put about and let us see the Causeway and get ashore.’ This was done, the guide meanwhile telling some story of a ship of the Spanish Armada having fired her guns at two peaks of rock, then visible, which the crew mistook for
- 68. chimney-pots—what benighted fools these Spanish Armadilloes must have been—it is easier to see a rock than a chimney-pot; it is easy to know that chimney-pots do not grow on rocks:—but where, if you please, is the Causeway? ‘That’s the Causeway before you,’ says the guide. ‘Which?’ ‘That pier which you see jutting out into the bay right ahead.’ ‘Mon Dieu! and have I travelled a hundred and fifty miles to see that?’ I declare, upon my conscience, the barge moored at Hungerford Market is a more majestic object, and seems to occupy as must space. As for telling a man that the Causeway is merely a part of the sight; that he is there for the purpose of examining the surrounding scenery; that if he looks to the westward he will see Portrush and Donegal Head before him; that the cliffs immediately in his front are green in some places, black in others, interspersed with blotches of brown and streaks of verdure;—what is all this to a lonely individual lying sick in a boat, between two immense waves that only give him momentary glimpses of the land in question, to show that it is frightfully near, and yet you are an hour from it? They won’t let you go away—that cursed guide will tell out his stock of legends and stories. The boatmen insist upon your looking at boxes of ‘specimens,’ which you must buy of them; they laugh as you grow paler and paler; they offer you more and more ‘specimens’; even the dirty lad who pulls number three, and is not allowed by his comrades to speak, puts in his oar, and hands you over a piece of Irish diamond (it looks like half-sucked alicompayne), and scorns you. ‘Hurray, lads, now for it, give way!’ how the oars do hurtle in the rullocks, as the boat goes up an aqueous mountain, and then down into one of those cursed maritime valleys where there is no rest as on shore! At last, after they had pulled me enough about, and sold me all the boxes of specimens, I was permitted to land at the spot whence we set out, and whence, though we had been rowing for an hour, we had never been above five hundred yards distant. Let all cockneys take warning from this; let the solitary one caught issuing from the back door of the hotel, shout at once to the boatmen to be gone—that he will have none of them. Let him, at any rate, go first down to the water to determine whether it be smooth enough to allow him to take any decent pleasure by riding on its surface. For after all, it must be remembered that it is pleasure we come for—that we are not
- 69. obliged to take those boats.—Well, well! I paid ten shillings for mine, and ten minutes before would cheerfully have paid five pounds to be allowed to quit it; it was no hard bargain after all. As for the boxes of spar and specimens, I at once, being on terra firma, broke my promise, and said I would see them all —— first. It is wrong to swear, I know; but sometimes it relieves one so much! The first act on shore was to make a sacrifice to Sanctissima Tellus; offering up to her a neat and becoming Taglioni coat, bought for a guinea in Covent Garden only three months back. I sprawled on my back on the smoothest of rocks that is, and tore the elbows to pieces: the guide picked me up; the boatmen did not stir, for they had had their will of me; the guide alone picked me up, I say, and bade me follow him. We went across a boggy ground in one of the little bays, round which rise the green walls of the cliff, terminated on either side by a black crag, and the line of the shore washed by the poluphlosboiotic, nay, the poluphlosboiotatotic sea. Two beggars stepped over the bog after us, howling for money, and each holding up a cursed box of specimens. No oaths, threats, entreaties, would drive this vermin away; for some time the whole scene had been spoilt by the incessant and abominable jargon of them, the boatmen, and the guides. I was obliged to give them money to be left in quiet, and if, as no doubt will be the case, the Giant’s Causeway shall be a still greater resort of travellers than ever, the county must put policemen on the rocks to keep the beggars away, or fling them in the water when they appear. And now, by force of money, having got rid of the sea and land beggars, you are at liberty to examine at your leisure the wonders of the place. There is not the least need for a guide to attend the stranger, unless the latter have a mind to listen to a parcel of legends, which may be well from the mouth of a wild simple peasant who believes in his tales, but are odious from a dullard who narrates them at the rate of sixpence a lie. Fee him and the other beggars, and at last you are left tranquil to look at the strange scene with your own eyes, and enjoy your own thoughts at leisure. That is, if the thoughts awakened by such a scene may be called enjoyment; but for me, I confess, they are too near akin to fear to be pleasant; and I don’t know that I would desire to change that sensation of awe and terror which the hour’s walk occasioned, for a greater familiarity with this wild, sad, lonely place. The solitude is awful. I can’t understand
- 70. how those chattering guides dare to lift up their voices here, and cry for money. It looks like the beginning of the world, somehow: the sea looks older than in other places, the hills and rocks strange, and formed differently from other rocks and hills—as those vast dubious monsters were formed who possessed the earth before man. The hilltops are shattered into a thousand cragged fantastical shapes; the water comes swelling into scores of little strange creeks, or goes off with a leap, roaring into those mysterious caves yonder, which penetrate who knows how far into our common world. The savage rock-sides are painted of a hundred colours. Does the sun ever shine here? When the world was moulded and fashioned out of formless chaos, this must have been the bit over—a remnant of chaos! Think of that!—it is a tailor’s simile. Well, I am a cockney: I wish I were in Pall Mall! Yonder is a kelp-burner: a lurid smoke from his burning kelp rises up to the leaden sky, and he looks as naked and fierce as Cain. Bubbling up out of the rocks at the very brim of the sea rises a little crystal spring: how comes it there? and there is an old grey hag beside it, who has been there for hundreds and hundreds of years, and there sits and sells whisky at the extremity of creation! How do you dare to sell whisky there, old woman? Did you serve old Saturn with a glass when he lay along the Causeway here? In reply, she says, she has no change for a shilling: she never has; but her whisky is good. This is not a description of the Giant’s Causeway (as some clever critic will remark), but of a Londoner there, who is by no means so interesting an object as the natural curiosity in question. That single hint is sufficient; I have not a word more to say. ‘If,’ says he, ‘you cannot describe the scene lying before us—if you cannot state from your personal observation that the number of basaltic pillars composing the Causeway has been computed at about forty thousand, which vary in diameter, their surface presenting the appearance of a tesselated pavement of polygonal stones—that each pillar is formed of several distinct joints, the concave end of the one being accurately fitted into the concave of the next, and the length of the joints varying from five feet to four inches—that although the pillars are polygonal, there is but one of three sides in the whole forty thousand (think of that!), but three of nine sides, and that it may be safely computed that ninety-nine out of one hundred pillars have either five, six, or seven sides;
- 71. —if you cannot state something useful, you had much better, sir, retire and get your dinner.’ Never was summons more gladly obeyed. The dinner must be ready by this time; so, remain you, and look on at the awful scene, and copy it down in words if you can. If at the end of the trial you are dissatisfied with your skill as a painter, and find that the biggest of your words cannot render the hues and vastness of that tremendous swelling sea—of those lean solitary crags standing rigid along the shore, where they have been watching the ocean ever since it was made—of those grey towers of Dunluce standing upon a leaden rock, and looking as if some old, old princess, of old, old fairy times, were dragon-guarded within—of yon flat stretches of sand where the Scotch and Irish mermaids hold conference—come away too, and prate no more about the scene! There is that in nature, dear Jenkins, which passes even our powers. We can feel the beauty of a magnificent landscape, perhaps; but we can describe a leg of mutton and turnips better. Come, then, this scene is for our betters to depict. If Mr. Tennyson were to come hither for a month, and brood over the place, he might, in some of those lofty heroic lines which the author of the ‘Morte d’Arthur’ knows how to pile up, convey to the reader a sense of this gigantic desolate scene. What! you too are a poet? Well then, Jenkins, stay! but believe me, you had best take my advice, and come off. The worthy landlady made her appearance with the politest of bows and an apology,—for what does the reader think a lady should apologise in the most lonely rude spot in the world?—because a plain servant-woman was about to bring in the dinner, the waiter being absent on leave at Coleraine! O heaven and earth! where will the genteel end? I replied philosophically that I did not care twopence for the plainness or beauty of the waiter, but that it was the dinner I looked to, the frying whereof made a great noise in the huge lonely house; and it must be said, that though the lady was plain, the repast was exceedingly good. ‘I have expended my little all,’ says the landlady, stepping in with a speech after dinner, ‘in the building of this establishment; and though to a man its profits may appear small, to such a being as I am it will bring, I trust, a sufficient return’; and on my asking her why she took the place, she replied that she had always, from her earliest youth, a fancy to dwell in that spot, and had accordingly realised her wish by building this hotel—this mausoleum. In spite of the bright fire, and the
- 72. good dinner, and the good wine, it was impossible to feel comfortable in the place; and when the car-wheels were heard, I jumped up with joy to take my departure and forget the awful lonely shore, that wild, dismal, genteel inn. A ride over a wide gusty country, in a grey, misty, half-moonlight, the loss of a wheel at Bushmills, and the escape from a tumble, were the delightful varieties after the late awful occurrences. ‘Such a being’ as I am, would die of loneliness in that hotel; and so let all brother cockneys be warned. Some time before we came to it, we saw the long line of mist that lay above the Bann, and coming through a dirty suburb of low cottages, passed down a broad street with gas and lamps in it (thank Heaven, there are people once more!), and at length drove up in state, across a gas-pipe, in a market-place, before an hotel in the town of Coleraine, famous for linen and for Beautiful Kitty, who must be old and ugly now, for it’s a good five-and- thirty years since she broke her pitcher, according to Mr. Moore’s account of her. The scene as we entered the Diamond was rather a lively one—a score of little stalls were brilliant with lights; the people were thronging in the place making their Saturday bargains; the town clock began to toll nine; and hark! faithful to a minute, the horn of the Derry mail was heard tootooing, and four commercial gentlemen, with Scotch accents, rushed into the hotel at the same time with myself. Among the beauties of Coleraine may be mentioned the price of beef, which a gentleman told me may be had for fourpence a pound; and I saw him purchase an excellent codfish for a shilling. I am bound, too, to state, for the benefit of aspiring Radicals, what two Conservative citizens of the place stated to me, viz.:—that though there were two Conservative candidates then canvassing the town, on account of a vacancy in the representation, the voters were so truly liberal that they would elect any person of any other political creed, who would simply bring money enough to purchase their votes. There are 220 voters, it appears; of whom it is not, however, necessary to ‘argue’ with more than fifty, who alone are open to conviction; but as parties are pretty equally balanced, the votes of the quinquagint, of course, carry an immense weight with them. Well, this is all discussed calmly standing on an inn steps, with a jolly landlord and a professional man of the town to give the information. So, Heaven bless us, the ways of London are beginning to be known even here. Gentility has already taken up her seat in the Giant’s Causeway, where she apologises for
- 73. the plainness of her look; and, lo! here is bribery as bold as in the most civilised places—hundreds and hundreds of miles away from St. Stephen’s and Pall Mall. I wonder, in that little island of Raghery, so wild and lonely, whether civilisation is beginning to dawn upon them?—whether they bribe and are genteel? But for the rough sea of yesterday, I think I would have fled thither to make the trial. The town of Coleraine, with a number of cabin suburbs belonging to it, lies picturesquely grouped on the Bann river; and the whole of the little city was echoing with psalms as I walked through it on the Sunday morning. The piety of the people seems remarkable; some of the inns even will not receive travellers on Sunday; and this is written in an hotel, of which every room is provided with a Testament, containing an injunction on the part of the landlord to consider this world itself as only a passing abode. Is it well that Boniface should furnish his guest with Bibles as well as bills, and sometimes shut his door on a traveller, who has no other choice but to read it on a Sunday? I heard of a gentleman arriving from shipboard at Kilrush on a Sunday, when the pious hotel-keeper refused him admittance; and some more tales, which to go into would require the introduction of private names and circumstances, but would tend to show that the Protestant of the north is as much priest-ridden as the Catholic of the south;—priest and old- woman-ridden, for there are certain expounders of doctrine in our Church, who are not, I believe, to be found in the Church of Rome; and woe betide the stranger who comes to settle in these parts, if his ‘seriousness’ be not satisfactory to the heads (with false fronts to most of them) of the congregations. Look at that little snug harbour of Portrush; a hideous new castle standing on a rock protects it on one side, a snug row of gentlemen’s cottages curve round the shore facing northwards, a bath-house, an hotel, more smart houses, face the beach westward, defended by another mound of rocks. In the centre of the little town stands a new-built church; and the whole place has an air of comfort and neatness which is seldom seen in Ireland. One would fancy that all the tenants of these pretty snug habitations, sheltered in this nook far away from the world, have nothing to do but to be happy, and spend their little comfortable means in snug little hospitalities among one another, and kind little charities among the poor. What does a man in active life ask for more than to retire to such a competence, to such a snug nook of the world; and there repose with a stock
- 74. of healthy children round the fireside, a friend within call, and the means of decent hospitality wherewith to treat him? Let any one meditating this pleasant sort of retreat, and charmed with the look of this or that place as peculiarly suited to his purpose, take a special care to understand his neighbourhood first, before he commit himself by lease-signing or house-buying. It is not sufficient that you should be honest, kind-hearted, hospitable, of good family—what are your opinions upon religious subjects? Are they such as agree with the notions of old Lady This, or Mrs. That, who are the patronesses of the village? If not, woe betide you! you will be shunned by the rest of the society, thwarted in your attempts to do good, whispered against over evangelical bohea and serious muffins. Lady This will inform every new arrival that you are a reprobate, and lost; and Mrs. That will consign you and your daughters, and your wife (a worthy woman, but, alas! united to that sad worldly man!) to damnation. The clergyman who partakes of the muffins and bohea before mentioned, will very possibly preach sermons against you from the pulpit: this was not done at Portstewart to my knowledge, but I have had the pleasure of sitting under a minister in Ireland who insulted the very patron who gave him his living, discoursing upon the sinfulness of partridge-shooting, and threatening hell-fire as the last ‘meet’ for fox-hunters; until the squire, one of the best and most charitable resident landlords in Ireland, was absolutely driven out of the church where his fathers had worshipped for hundreds of years, by the insults of this howling evangelical inquisitor. So much as this I did not hear at Portstewart; but I was told that at yonder neat-looking bath-house a dying woman was denied a bath on a Sunday. By a clause of the lease by which the bath-owner rents his establishment, he is forbidden to give baths to any one on the Sunday. The landlord of the inn, forsooth, shuts his gates on the same day, and his conscience on week-days will not allow him to supply his guests with whisky or ardent spirits. I was told by my friend, that because he refused to subscribe for some fancy charity, he received a letter to state that ‘he spent more in one dinner than in charity in the course of the year.’ My worthy friend did not care to contradict the statement, as why should a man deign to meddle with such a lie? But think how all the fishes, and all the pieces of meat, and all the people who went in and out of his snug cottage by the seaside must have been watched by the serious round about! The sea is not more constant roaring there, than scandal is whispering. How happy I felt,
- 75. while hearing these histories (demure heads in crimped caps peering over the blinds at us as we walked on the beach), to think I am a cockney, and don’t know the name of the man who lives next door to me! I have heard various stories, of course from persons of various ways of thinking, charging their opponents with hypocrisy, and proving the charge by statements clearly showing that the priests, the preachers, or the professing religionists in question, belied their professions wofully by their practice. But in matters of religion, hypocrisy is so awful a charge to make against a man, that I think it is almost unfair to mention even in the cases in which it is proven, and which,—as, pray God, they are but exceptional,—a person should be very careful of mentioning, lest they be considered to apply generally. Tartuffe has been always a disgusting play to me to see, in spite of its sense and its wit; and so, instead of printing, here or elsewhere, a few stories of the Tartuffe kind which I have heard in Ireland, the best way will be to try and forget them. It is an awful thing to say of any man walking under God’s sun by the side of us, ‘You are a hypocrite, lying as you use the Most Sacred Name, knowing that you lie while you use it.’ Let it be the privilege of any sect that is so minded, to imagine that there is perdition in store for all the rest of God’s creatures who do not think with them; but the easy countercharge of hypocrisy, which the world has been in the habit of making in its turn, is surely just as fatal and bigoted an accusation as any that the sects make against the world. What has this disquisition to do à propos of a walk on the beach at Portstewart? Why, it may be made here as well as in other parts of Ireland, or elsewhere as well, perhaps, as here. It is the most priest-ridden of countries; Catholic clergymen lord it over their ragged flocks, as Protestant preachers, lay and clerical, over their more genteel co-religionists. Bound to inculcate peace and goodwill, their whole life is one of enmity and distrust. Walking away from the little bay and the disquisition which has somehow been raging there, we went across some wild dreary highlands to the neighbouring little town of Portrush, where is a neat town and houses, and a harbour, and a new church too, so like the last-named place that I thought for a moment we had only made a round, and were back again at Portstewart. Some gentlemen of the place, and my guide, who had a neighbourly liking for it, showed me the new church, and seemed to be well pleased with the edifice; which is, indeed, a neat and convenient one, of a rather irregular Gothic. The best thing about the church, I think, was the
- 76. history of it. The old church had lain some miles off, in the most inconvenient part of the parish, whereupon the clergyman and some of the gentry had raised a subscription in order to build the present church. The expenses had exceeded the estimates, or the subscriptions had fallen short of the sums necessary; and the church, in consequence, was opened with a debt on it, which the rector and two more of the gentry had taken on their shoulders. The living is a small one; the other two gentlemen going bail for the edifice not so rich as to think light of the payment of a couple of hundred pounds beyond their previous subscriptions—the lists are therefore still open; and the clergyman expressed himself perfectly satisfied either that he would be reimbursed one day or other, or that he would be able to make out the payment of the money for which he stood engaged. Most of the Roman Catholic churches that I have seen through the country have been built in this way,—begun when money enough was levied for constructing the foundation, elevated by degrees as fresh subscriptions came in, and finished—by the way, I don’t think I have seen one finished— but there is something noble in the spirit (however certain economists may cavil at it) that leads people to commence these pious undertakings with the firm trust that ‘Heaven will provide.’ Eastwards from Portrush, we came upon a beautiful level sand which leads to the White Rocks, a famous place of resort for the frequenters of the neighbouring watering-places. Here are caves, and for a considerable distance a view of the wild and gloomy Antrim coast as far as Bengore. Midway, jutting into the sea (and I was glad it was so far off), was the Causeway; and nearer, the grey towers of Dunluce. Looking north, were the blue Scotch hills and the neighbouring Raghery Island. Nearer Portrush are two rocky islands, called the Skerries, of which a sportsman of our party vaunted the capabilities, regretting that my stay was not longer, so that I might land and shoot a few ducks there. This unlucky lateness of the season struck me also as a most afflicting circumstance. He said also that fish were caught off the island—not fish good to eat, but very strong at pulling, eager of biting, and affording a great deal of sport. And so we turned our backs once more upon the Giant’s Causeway, and the grim coast on which it lies; and as my taste in life leads me to prefer looking at the smiling fresh face of a young cheerful beauty, rather than at the fierce countenance and high features of a fierce
- 77. dishevelled Meg Merrilies, I must say again that I was glad to turn my back on that severe part of the Antrim coast, and my steps towards Derry.
- 78. CHAPTER XXX PEG OF LIMAVADDY BETWEEN Coleraine and Derry there is a daily car (besides one or two occasional queer-looking coaches), and I had this vehicle, with an intelligent driver, and a horse with a hideous raw on his shoulder, entirely to myself for the five-and-twenty miles of our journey. The cabins of Coleraine are not parted with in a hurry, and we crossed the bridge, and went up and down the hills of one of the suburban streets, the Ban flowing picturesquely to our left; a large Catholic chapel, the before-mentioned cabins, and farther on, some neat-looking houses and plantations, to our right. Then we began ascending wide lonely hills, pools of bog shining here and there amongst them, with birds, both black and white, both geese and crows, on the hunt. Some of the stubble was already ploughed up, but by the side of most cottages you saw a black potato-field that it was time to dig now, for the weather was changing and the winds beginning to roar. Woods, whenever we passed them, were flinging round eddies of mustard-coloured leaves; the white trunks of lime and ash trees beginning to look very bare. Then we stopped to give the raw-backed horse water; then we trotted down a hill with a noble bleak prospect of Lough Foyle and the surrounding mountains before us, until we reached the town of Newtown Limavaddy, where the raw-backed horse was exchanged for another not much more agreeable in his appearance, though, like his comrade, not slow on the road. Newtown Limavaddy is the third town in the county of Londonderry. It comprises three well-built streets, the others are inferior; it is, however, respectably inhabited; all this may be true, as the well-informed Guide-book avers, but I am bound to say that I was thinking of something else as we drove through the town, having fallen eternally in love during the ten minutes of our stay. Yes, Peggy of Limavaddy, if Barrow and Inglis have gone to Connemara to fall in love with the Misses Flynn, let us be allowed to come to Ulster and offer a tribute of praise at your feet—at your stockingless feet, O Margaret! Do you remember the October day (‘twas the first day of the hard weather), when the way-worn traveller entered your
- 79. inn? But the circumstances of this passion had better be chronicled in deathless verse.
- 80. PEG OF LIMAVADDY RIDING from Coleraine (Famed for lovely Kitty), Came a cockney bound Unto Derry city; Weary was his soul, Shivering and sad he Bump’d along the road Leads to Limavaddy. Mountains stretch’d around, Gloomy was their tinting, And the horse’s hoofs Made a dismal clinting; Wind upon the heath Howling was and piping, On the heath and bog, Black with many a snipe in: ‘Mid the bogs of black, Silver pools were flashing, Crows upon their sides Picking were and splashing. Cockney on the car Closer folds his plaidy, Grumbling at the road Leads to Limavaddy. Through the crashing woods Autumn brawl’d and bluster’d, Tossing round about Leaves the hue of mustard; Yonder lay Lough Foyle, Which a storm was whipping, Covering with mist Lake, and shores, and shipping. Up and down the hill (Nothing could be bolder), Horse went with a raw, Bleeding on his shoulder. ‘Where are horses changed?’ Said I to the laddy
- 81. Driving on the box: ‘Sir, at Limavaddy.’ Limavaddy inn’s But a humble baithouse, Where you may procure Whisky and potatoes; Landlord at the door Gives a smiling welcome To the shivering wights Who to his hotel come. Landlady within Sits and knits a stocking, With a wary foot Baby’s cradle rocking. To the chimney nook, Having found admittance, There I watch a pup Playing with two kittens; (Playing round the fire, Which of blazing turf is, Roaring to the pot Which bubbles with the murphies); And the cradled babe Fond the mother nursed it, Singing it a song As she twists the worsted! Up and down the stair Two more young ones patter (Twins were never seen Dirtier nor fatter); Both have mottled legs, Both have snubby noses, Both have—Here the host Kindly interposes: ‘Sure you must be froze With the sleet and hail, sir, So will you have some punch, Or will you have some ale, sir?’ Presently a maid
- 82. Enters with the liquor, (Half a pint of ale Frothing in a beaker). Gods! I didn’t know What my beating heart meant, Hebe’s self I thought Enter’d the apartment. As she came she smiled, And the smile bewitching, On my word and honour, Lighted all the kitchen! With a curtsey neat Greeting the new-comer, Lovely, smiling Peg Offers me the rummer; But my trembling hand Up the beaker tilted, And the glass of ale Every drop I spilt it; Spilt it every drop (Dames, who read my volumes, Pardon such a word) On my whatd’yecall’ems! Such a silver peal! In the meadows listening, You who’ve heard the bells Ringing to a christening; You who ever heard Caradori pretty, Smiling like an angel Singing ‘Giovinetti,’ Fancy Peggy’s laugh, Sweet, and clear, and cheerful, At my pantaloons With half-a-pint of beer full!
- 83. Witnessing the sight Of that dire disaster, Out began to laugh Missis, maid, and master; Such a merry peal, ‘Specially Miss Peg’s was (As the glass of ale Trickling down my legs was), That the joyful sound Of that ringing laughter Echoed in my ears Many a long day after. When the laugh was done. Peg, the pretty hussy, Moved about the room Wonderfully busy; Now she looks to see If the kettle keep hot, Now she rubs the spoons, Now she cleans the teapot: Now she sets the cups Trimly and secure, Now she scours a pot, And so it was I drew her. Thus it was I drew her Scouring of a kettle,[33] (Faith! her blushing cheeks Redden’d on the metal!) Ah! but ‘tis in vain That I try to sketch it;
- 84. The pot perhaps is like, But Peggy’s face is wretched. No: the best of lead, And of Indian rubber, Never could depict That sweet kettle-scrubber! See her as she moves! Scarce the ground she touches, Airy as a fay, Graceful as a duchess; Bare her rounded arm, Bare her little leg is, Vestris never show’d Ankles like to Peggy’s; Braided is her hair, Soft her look and modest, Slim her little waist Comfortably boddiced. This I do declare, Happy is the laddy Who the heart can share Of Peg of Limavaddy; Married if she were, Blest would be the daddy Of the children fair Of Peg of Limavaddy; Beauty is not rare In the land of Paddy, Fair beyond compare Is Peg of Limavaddy. Citizen or squire, Tory, Whig, or Radical would all desire Peg of Limavaddy. Had I Homer’s fire, Or that of Sergeant Taddy, Meetly I’d admire Peg of Limavaddy. And till I expire, Or till I grow mad, I
- 85. Will sing unto my lyre Peg of Limavaddy!
- 86. CHAPTER XXXI TEMPLEMOYLE—DERRY FROM Newtown Limavaddy to Derry, the traveller has many wild and noble prospects of Lough Foyle and the plains and mountains round it, and of scenes which may possibly in this country be still more agreeable to him— of smiling cultivation, and comfortable well-built villages, such as are only too rare in Ireland. Of a great part of this district, the London Companies are landlords—the best of landlords, too, according to the report I could gather; and their good stewardship shows itself especially in the neat villages of Muff and Ballikelly, through both of which I passed. In Ballikelly, besides numerous simple, stout, brick-built dwellings for the peasantry, with their shining windows and trim garden-plots, is a Presbyterian meeting-house, so well-built, substantial and handsome, so different from the lean, pretentious, sham-Gothic ecclesiastical edifices which have been erected of late years in Ireland, that it can’t fail to strike the tourist who has made architecture his study or his pleasure. The gentlemen’s seats in the district are numerous and handsome; and the whole movement along the road betokened cheerfulness and prosperous activity. As the carman had no other passengers but myself, he made no objection to carry me a couple of miles out of his way, through the village of Muff, belonging to the Grocers of London (and so handsomely and comfortably built by them as to cause all cockneys to exclaim, ‘Well done our side!’), and thence to a very interesting institution, which was established some fifteen years since in the neighbourhood—the Agricultural Seminary of Templemoyle. It lies on a hill in a pretty wooded country, and is most curiously secluded from the world by the tortuousness of the road which approaches it. Of course it is not my business to report upon the agricultural system practised there, or to discourse on the state of the land or the crops; the best testimony on this subject is the fact, that the Institution hired, at a small rental, a tract of land, which was reclaimed and farmed, and that of this farm the landlord has now taken possession, leaving the young farmers to
- 87. labour on a new tract of land, for which they pay five times as much rent as for their former holding. But though a person versed in agriculture could give a far more satisfactory account of the place than one to whom such pursuits are quite unfamiliar, there is a great deal about the establishment which any citizen can remark on; and he must be a very difficult cockney indeed who won’t be pleased here. After winding in and out, and up and down, and round about the eminence on which the house stands, we at last found an entrance to it, by a courtyard, neat, well-built, and spacious, where are the stables and numerous offices of the farm. The scholars were at dinner off a comfortable meal of boiled beef, potatoes, and cabbages, when I arrived; a master was reading a book of history to them; and silence, it appears, is preserved during the dinner. Seventy scholars were here assembled, some young, and some expanded into six feet and whiskers—all, however, are made to maintain exactly the same discipline, whether whiskered or not. The ‘head farmer’ of the school, Mr. Campbell, a very intelligent Scotch gentleman, was good enough to conduct me over the place and the farm, and to give a history of the establishment and the course pursued there. The Seminary was founded in 1827, by the North-West of Ireland Society, by members of which and others about three thousand pounds were subscribed, and the buildings of the school erected. These are spacious, simple, and comfortable; there is a good stone house, with airy dormitories, schoolrooms, etc., and large and convenient offices. The establishment had, at first, some difficulties to contend with, and for some time did not number more than thirty pupils. At present, there are seventy scholars, paying ten pounds a year, with which sum, and the labour of the pupils on the farm, and the produce of it, the school is entirely supported. The reader will, perhaps, like to see an extract from the Report of the school, which contains mere details regarding it. ‘TEMPLEMOYLE WORK AND SCHOOL TABLE ‘From 20th March to 23rd September ‘Boys divided into two classes, A and B Hours. At work. At school. 5½ All rise. 6—8 ....................... A ................... B
- 88. 8—9 Breakfast. 9—1 ....................... A ................... B 1—2 Dinner and recreation. 2—6 ....................... B ................... A 6—7 Recreation. 7—9 Prepare lessons for next day. 9 To bed. ‘On Tuesday B commences work in the morning and A at school, and so on alternate days. ‘Each class is again subdivided into three divisions, over each of which is placed a monitor, selected from the steadiest and best-informed boys; he receives the Head Farmer’s directions as to the work to be done, and superintends his party while performing it. ‘In winter the time of labour is shortened according to the length of the day, and the hours at school increased. ‘In wet days, when the boys cannot work out, all are required to attend school. ‘Dietary ‘Breakfast.—Eleven ounces of oatmeal made in stirabout, one pint of sweet milk. ‘Dinner.—Sunday—Three-quarters of a pound of beef stewed with pepper and onions, or one-half pound of corned beef with cabbage, and three and one-half pounds of potatoes. ‘Monday—One-half pound of pickled beef, three and a half pounds of potatoes, one pint of buttermilk. ‘Tuesday—Broth made of one-half pound of beef, with leeks, cabbage, and parsley, and three and a half pounds of potatoes. ‘Wednesday—Two ounces of butter, eight ounces of oatmeal made into bread, three and one-half pounds of potatoes, and one pint of sweet milk. ‘Thursday—Half a pound of pickled pork, with cabbage or turnips, and three and a half pounds of potatoes. ‘Friday—Two ounces of butter, eight ounces wheat meal made into bread, one pint of sweet milk or fresh buttermilk, three and a half pounds of potatoes.
- 89. Welcome to our website – the perfect destination for book lovers and knowledge seekers. We believe that every book holds a new world, offering opportunities for learning, discovery, and personal growth. That’s why we are dedicated to bringing you a diverse collection of books, ranging from classic literature and specialized publications to self-development guides and children's books. More than just a book-buying platform, we strive to be a bridge connecting you with timeless cultural and intellectual values. With an elegant, user-friendly interface and a smart search system, you can quickly find the books that best suit your interests. Additionally, our special promotions and home delivery services help you save time and fully enjoy the joy of reading. Join us on a journey of knowledge exploration, passion nurturing, and personal growth every day! ebookbell.com