restriction and modifying enzymes.pdf

B Tech BTE(2015-2019) UNIT I SBT1205 GENETIC ENGINEERING
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DNA MODIFYING ENZYMES
Restriction enzymes and DNA ligases represent the cutting and joining functions in DNA
manipulation. All other enzymes involved in genetic engineering fall under the broad
category of enzymes known as DNA modifying enzymes. These enzymes are involved in the
degradation, synthesis and alteration of the nucleic acids.
DNA ligase is an important cellular enzyme, as its function is to repair broken phosphodiester
bonds that may occur at random or as a consequence of DNA replication or recombination. In
genetic engineering it is used to seal discontinuities in the sugar—phosphate chains that arise
when recombinant DNA is made by joining DNA molecules from different sources. It can
therefore be thought of as molecular glue, which is used to stick pieces of DNA together.This
function is crucial to the success of many experiments, and DNA ligase is therefore a key
enzyme in genetic engineering.
The enzyme used most often in experiments is T4 DNA ligase, which is purified from E. coli
cells infected with bacteriophage T4.Although the enzyme is most efficient when sealing gaps in
fragments that are held together by cohesive ends, it will also join blunt-ended DNA molecules
together under appropriate conditionsT4 DNA Ligase catalyzes the formation of a
phosphodiester bond between juxtaposed 5'-phosphate and 3'-hydroxyl termini in duplex DNA or
RNA. The enzyme repairs single-strand nicks in duplex DNA, RNA, or DNA/RNA hybrids. It
also joins DNA fragments with either cohesive or blunt termini, but has no activity on single-
stranded nucleic acids. The T4 DNA Ligase requires ATP as a cofactor.
RESTRICTION ENDONUCLEASES
 Also called restriction enzymes
 1962: “molecular scissors” discovered in in bacteria
 E. coli bacteria have an enzymatic immune system that recognizes and destroys foreign
DNA
 3,000 enzymes have been identified, around 200 have unique properties, many are
purified and available commercially

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Named for bacterial genus, species, strain, and type
Example: EcoR1
Genus:Escherichia
Species:coli
Strain:R
Order discovered: 1
Enzymes recognize specific 4-8 bp sequences which are palindromic.
Some enzymes cut in a staggered fashion - “sticky ends”
EcoRI 5’…GAATTC…3’
3’…CTTAAG…5’
Some enzymes cut in a direct fashion – “blunt ends”
PvuII 5’…CAGCTG…3’
3’…GTCGAC…5’

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Uses of restriction enzymes
 RFLP analysis (Restriction Fragment Length Polymorphism)
 DNA sequencing
 DNA storage – libraries
 Transformation
 Large scale analysis – gene chips
TYPES OF DNA MODIFYING ENZYMES
Nuclease enzymes degrade nucleic acids by breaking the phosphodiester bond that holds
the nucleotides together. Restriction enzymes are good examples of endonucleases, which
cut within a DNA strand.A second group of nucleases, which degrade DNA from the

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termini of the molecule, are known as exonucleases. Apart from restriction enzymes,
there are four useful nucleases that are often used in genetic engineering. These are
 Bal 31
 exonuclease III (exonucleases),
 deoxyribonuclease I (DNase I)
 S1-nuclease (endonucleases).
These enzymes differ in their precise mode of action and provide the genetic engineer
with a variety of strategies for attacking DNA.
 Nuclease Bal 31 is a complex enzyme. Its primary activity is a fast-acting 3’ exonuclease,
which is coupled with a slow-acting endonuclease. When Bal 31 is present at a high
concentration these activities effectively shorten DNA molecules from both termini.
 Exonuclease III is a 3’ exonuclease that generates molecules with protruding 5’ termini.
 DNase I cuts either single-stranded or double-stranded DNA at essentially random sites.
 Nuclease S1 is specific for single-stranded RNA or DNA.
POLYMERASES
Polymerase enzymes synthesise copies of nucleic acid molecules and are used in many
genetic engineering procedures. When describing a polymerase enzyme, the terms ‘DNA-
dependent’ or ‘RNA-dependent’ may be used to indicate the type of nucleic acid template
that the enzyme uses.
DNA POLYMERASE I
The enzyme DNA polymerase I has, in addition to its polymerase function, 5’→3’ and
3’→5’ exonuclease activities. The enzyme catalyses a strand-replacement reaction, where
the 5’→3’ exonuclease function degrades the non-template strand as the polymerase
synthesises the new copy. A major use of this enzyme is in the nick translation procedure
for radiolabelling DNA.The 5’→3’ exonuclease function of DNA polymerase I can be

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removed by cleaving the enzyme to produce what is known as the Klenow fragment.
This retains the polymerase and 3’→5’ exonuclease activities. The Klenow fragment is
used where a single-stranded DNA molecule needs to be copied; because the 5’→3’
exonuclease function is missing, the enzyme cannot degrade the non-template strand of
dsDNA during synthesis of the new DNA. The 3’→5’ exonuclease activity is supressed
under the conditions normally used for the reaction. Major uses for the Klenow fragment
include radiolabelling by primed synthesis and DNA sequencing by the dideoxy method
in addition to the copying of single-stranded DNAs during the production of
recombinants.A modified form of DNA polymerase I called the Klenow fragment is a
useful polymerase that is used widely in a number of applications
Reverse transcriptase (RTase) is an RNA-dependent DNA polymerase, and therefore produces
a DNA strand from an RNA template.It has no associated exonuclease activity. The enzyme is
used mainly for copying mRNA molecules in the preparation of cDNA (complementary or
copy DNA) for cloning, although it will also act on DNA templates.Reverse transcriptase is a
key enzyme in the generation of cDNA; the enzyme is an RNA-dependent DNA polymerase,
which produces a DNA copy of an mRNA molecule.
ENZYMES THAT MODIFY THE ENDS OF DNA MOLECULES
The enzymes alkaline phosphatase, polynucleotide kinase, and terminal transferase act on the
termini of DNA molecules and provide important functions that are used in a variety of ways.
The phosphatase and kinase enzymes, as their names suggest, are involved in the removal or
addition of phosphate groups.
 Bacterial alkaline phosphatase (there is also a similar enzyme, calf intestinal alkaline
phosphatase) removes phosphate groups from the 5 ends of DNA, leaving a 5-OH group.
The enzyme is used to prevent unwanted ligation of DNA molecules, which can be a
problem in certain cloning procedures. It is also used prior to the addition of radioactive
phosphate to the 5 ends of DNAs by polynucleotide kinase.
 Terminal transferase (terminal deoxynucleotidyl transferase) repeatedly adds nucleotides
to any available 3 terminus. Although it works best on protruding 3 ends, conditions can
be adjusted so that blunt-ended or 3-recessed molecules may be utilised. The enzyme is

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mainly used to add homopolymer tails to DNA molecules prior to the construction of
recombinants
VECTORS
A cloning vector is a DNA molecule used as a vehicle to transfer foreign genetic material
Few of the generally used cloning vectors include:
1. Plasmids
2. Bacterophages
3. Cosmids
4. Yeast artifical chromosomes (YAC's)
5. Bacterial arifical chromosomes (BAC's)
Vectors for yeast and other fungi
The yeast Saccharomyces cerevisiae is one of the most important organisms in
biotechnology. Development of cloning vectors for yeast was stimulated by the discovery of a
plasmid that is present in most strains of S. cerevisiae. The 2 µm plasmid, as it is called, is one of
only a very limited number of plasmids found in eukaryotic cells.

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Why is it popularly used?
Yeast cells are much easier to grow and manipulate than plant and animal cells.
The cellular biochemistry and regulation of yeast are very like those of higher eukaryotes.
There are many yeast homologues of human genes, e.g. those involved in cell division. Thus
yeast can be a very good surrogate host for studying the structure and function of eukaryotic
gene products.
The different kind of cloning vector derived from yeast are:
1. 2 µm plasmid
2. yeast episomal plasmids (YEps)
3. Yeast integrative plasmids (YIps)
4. Yeast replicative plasmids (YRps)
5. yeast artificial chromosome (YAC)

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V e c t o rs f o r y e a s t a nd o t he r f ung i
2 µm plasmid
The 2 µm plasmid is an excellent basis for a cloning vector. It is 6 kb in size and has a high
copy number of between 70 and 200.
Replication makes use of a plasmid origin, several enzymes provided by the host cell, and the
proteins coded by the REP1 and REP2 genes carried by the plasmid.
However, all is not perfectly straightforward in using the 2 µm plasmid as a cloning vector.
First, there is the question of a selectable marker. For this purpose a normal yeast gene is used,
generally one that codes for an enzyme involved in amino acid biosynthesis. An example is the
gene LEU2, which codes for β-isopropyl-malate dehydrogenase, one of the enzymes involved in
the conversion of pyruvic acid to leucine.
In order to use LEU2 as a selectable marker, a special kind of host organism is needed. The host
must be an auxotrophic mutant that has a non-functional LEU2 gene.
Such a leu2− yeast is unable to synthesize leucine and can survive only if this amino acid is
supplied as a nutrient in the growth medium. Selection is possible because transformants contain
a plasmid-borne copy of the LEU2 gene, and so are able to grow in the absence of the amino
acid. In a cloning experiment, cells are plated out onto minimal medium, which contains no
added amino acids. Only transformed cells are able to survive and form colonies and thus are
easily differentiated.

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Yeast artificial chromosome (YAC) is a human-engineered DNA molecule used to clone DNA
sequences in yeast cells. YACs are often used in connection with the mapping and sequencing of
genomes. Segments of an organism's DNA, up to one million base pairs in length, can be
inserted into YACs. The YACs, with their inserted DNA, are then taken up by yeast cells. As the
yeast cells grow and divide, they amplify the YAC DNA, which can be isolated and used for
DNA mapping and sequencing. It is abbreviation is YAC.
They carry large amounts of DNA so a long distance map of the region can be obtained in
several steps. The repetitive rAegions may be 10-20 kb in length they are rarely longer than 50
kb. Thus a YAC with 100kb will contain some region that is single copy which can be used for
further steps in the walk.
Features of YACs
1. Large DNA (>100 kb) is ligated between two arms. Each arm ends with a yeast telomere
so that the product can be stabilized in the yeast cell. Interestingly, larger YACs are more
stable than shorter ones, which favors cloning of large stretches of DNA.
2. One arm contains an autonomous replication sequence (ARS), a centromere (CEN) and
aselectable marker (trp1). The other arm contains a second selectable marker (ura3).
3. Insertion of DNA into the cloning site inactivates a mutant expressed in the vector DNA
and red yeast colonies appear.
4. Transformants are identified as those red colonies which grow in a yeast cell that is
mutant for trp1 and ura3. This ensures that the cell has received an artificial chromosome
with both telomeres (because of complementation of the two mutants) and the artificial
chromosome contains insert DNA (because the cell is red).
Example : pYAC3
pYAC3 is essentially a pBR322 plasmid into which a number of yeast genes have been inserted.
Two of these genes, URA3 and TRP1, have been encountered already as the selectable markers
for YIp5 and YRp7, respectively. As in YRp7, the DNA fragment that carries TRP1 also

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contains an origin of replication, but in pYAC3 this fragment is extended even further to include
the sequence called CEN4, which is the DNA from the centromere region of chromosome 4. The
TRP1–origin–CEN4 fragment therefore contains two of the three components of the artificial
chromosome.
The third component, the telomeres, is provided by the two sequences called TEL.
These are not themselves complete telomere sequences, but once inside the yeast nucleus they
act as seeding sequences onto which telomeres will be built. This just leaves one other part of
pYAC3 that has not been mentioned: SUP4, which is the selectable marker into which new DNA
is inserted during the cloning experiment.

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B a c t e ri a l a rt i f i c i a l c hro mo s o me
A vector used to clone DNA fragments (100- to 300-kb insert size; average, 150 kb)
in Escherichia coli cells. Based on naturally occurring F-factor plasmid found in the
bacterium E. coli.
Bacterial Artificial Chromosomes (BAC) have been developed to hold much larger pieces of
DNA than a plasmid can. BAC vectors were originally created F’ plasmid present in few
bacterial plamids. it was found through studies that F’ plasmid were able hold up to a million
basepairs of DNA from nonself bacteria. In 1992, Hiroaki Shizuya took the parts of F’ that were
important, cleaned it up, and turned it into a vector.
BAC vectors are able to hold up to 350 kb of DNA and have all of the tools that a vector needs to
work properly, like replication origins, antibiotic resistance genes, and convenient places where
clone DNA can insert itself.[8]
By these vector that can hold larger pieces of DNA, the number of
clones required to cover the human could be reduced drastically from 1.8 billion to about 50
million.
Researchers have modified BAC vectors to become more convenient to use and more useful in
specialized situations. In addition to the antibiotic resistance gene that was added to identify
transfected bacteria, a gene was added that enabled the bacteria to turn the colourless substance
X-gal/IPTG blueSo it is possible to tell not only if a bacteria had been transfected (meaning
incorporated into the cell), but also if the bacteria was transfected with the vector containing
insert DNA or just the vector alone (remembering that if the vector has properly incorporated the
clone DNA, it will have lost its ability to change X-gal/IPTG blue). Modifications to BAC
vectors make them more specialized.

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Few advantages:
 The creation of BAC vectors has allowed researchers to do many things that they could
not do before and do them more quickly and more easily. [8]
 BACs have allowed researchers to look at microbial DNA without having to actually
grow the organisms[8]
 BAC vectors are also useful for studying pathogens, and are helpful in the development
of vaccines. [8]
BAC vectors are playing a tremendous role in discovering new and
powerful antibiotics in the environment.
 Discovering enzymes that are able to help clean up oil spills or help farmers breed
healthier farm animals or even process radioactive waste are just a few examples of what
Bacterial Artificial Chromosomes can do.
A ni ma l v i rus e s a s c l o ni ng v e c t o rs
The first eukaryotic DNA virus was SV40, for which a complete nucleotide sequence and a
detailed understanding of transcription were available. The genome of SV40 contains very little
non-essential DNA so it is necessary to insert the foreign gene in place of essential viral genes
and to propagate the recombinant genome in the presence of a helper virus. This virus is capable
of infecting several mammalian species, following a lytic cycle in some host and a lysogenic
cycle in others. The genome is 5.2kb in size and contains two sets of genes, the early genes,
expressed early in the infection cycle and coding for proteins involved in viral DNA replication,
and the late genes, coding for viral capsid proteins (Figure-18). However, all work using SV40
virions to propagate recombinant DNA molecules is severely constrained by the facts that the
viral genome is small, 5.24 kb, and that the packaging limits are strict. Such systems cannot,
therefore, be used for the analysis of most eukaryotic genes.

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Adenoviruses, are a group of viruses which enable larger fragments of DNA to be cloned than it
is possible with an SV40 vector. Adenoviruses are more difficult to handle because the genomes
are bigger.
Papillomaviruses, which also have a relatively high capacity for inserted DNA, have the
important advantage of enabling a stable transformed cell line to be obtained. Papillomavirus
transformed cells don't contain integrated viral DNA rather they contain between 50 and 300
copies of unintegrated, circular viral DNA although some proportion of these viral genomes
exists as concatamers and/or catenates. Bovine papillomavirus(BPV), which causes warts on
cattle, has an unusal infection cycle in mouse cells, taking the form of a multi copy plasmid with
about 100 molecules present per cell. It doesn't cause the death of the mouse cell, and BPV
molecules are passed to daughter cells on cell division. Shuttle vectors consisting of BPV and

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pBR322 sequences, and capable of replication in both mouse and bacterial cells, are therefore of
great value in animal cell biotechnology.
Retroviruses, though have single-stranded RNA genomes but provides perhaps the most
promising vector system of all. During the process of reverse transcription, sequences from the
termini of viral RNA are duplicated to generate long terminal repeats(LTRs). These long
terminal repeats contain both the promoter and the polyadenylation signal for the transcription of
viral mRNAs. The specificity of proviral DNA integration is also determined by the long
terminal repeats. Although retroviruses can integrate at many sites within the cellular genome,
integrative recombination always occurs at particular sites at the ends of the LTRs. The
sequences appropriately inserted between the two LTRs will be integrated intact which contrasts
sharply with the integration of papovavirus or adenovirus DNA, during which extensive
rearrangements of the integrated viral sequences are commonplace. A further great advantage of
retroviruses is that they are natural transducing viruses.

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Retroviral vectors are most frequently based upon the Moloney murine leukaemia virus (Mo-
MLV), which is an amphotrophic virus, capable of infecting both mouse cells, enabling vector
development in mouse models, & human cells, enabling human treatment. The viral genes (gag,
pol & env) are replaced with the transgene of interest & expressed on plasmids in the packaging
cell line. Because the non-essential genes lack the packaging sequence (psi) they are not included
in the virion particle. To prevent recombination resulting in replication competent retroviruses,
all regions of homology with the vector backbone should be removed & the non-essential genes
should be expressed by at least two transcriptional units.
The essential regions include the 5' & 3' LTRs & the packaging sequence lying downstream of
the 5' LTR. To aid identification of transformed cells selectable markers, such as neomycin &
beta galactosidase, can be included & transgenes expression can be improved with the addition
of internal ribosome sites. The available carrying capacity for retroviral vectors is approximately
7.5 kb, which is too small for some genes even if the cDNA is used.
A requirement for retroviral integration & expression of viral genes is that the target cells should
be dividing. This limits gene therapy to proliferating cells in vivo or ex vivo, whereby cells are
removed from the body, treated to stimulate replication & then transduced with the retroviral
vector, before being returned to the patient. When treating cancers in vivo, tumour cells are
preferentially targeted However, ex vivo cells can be more efficiently transduced, due to
exposure to higher virus titres & growth factors. Furthermore ex vivo treated tumour cells will
associate with the tumour mass & can direct tumouricidal effects.
Though transgene expression is usually adequate in vitro & initially in vivo, prolonged
expression is difficult to attain. Retroviruses are inactivated by c1 complement protein & an anti-
alpha galactosyl epitope antibody, both present in human sera. Transgene expression is also
reduced by inflammatory interferons, specifically IFN-alpha & IFN-gamma acting on viral
LTRs. As the retroviral genome integrates into the host genome it is most likely that the viral
LTR promoters are being inactivated, therefore one approach has been to use promoters for host
cell genes, such as tyrosine. Clearly this is an area where continued research is needed.

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Baculoviruses, enable large amounts of proteins to be obtained from genes cloned in insect cells.
One of the major proteins encoded by the virus genome is polyhedrin, which accumulates in very
large quantities in the nuclei of infected cells, since the gene has an extremely active promoter.
The same promoter can be used to drive the over expression of a foreign gene engineered into
the baculovirus genome, and large quantities of protein can be produced in infected insect cells
in culture. This method is being used increasingly for large-scale culture of proteins of animal
origin, since the insect cells can produce many of the post-translational modifications of animal
proteins which a bacterial expression system.
Expression vector
An expression vector, otherwise known as an expression construct, is usually a plasmid or
virus designed for protein expression in cells. The vector is used to introduce a specific gene into
a target cell, and can commandeer the cell's mechanism for protein synthesis to produce
the protein encoded by the gene. The plasmid is engineered to contain regulatory sequences that
act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the

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expression vector.[1]
The goal of a well-designed expression vector is the production of
significant amount of stablemessenger RNA, and therefore proteins. Expression vectors are basic
tools for biotechnology and the production of proteins. An example is insulin which is used for
medical treatments of diabetes.
Elements for expression
An expression vector must have elements necessary for protein expression. These may include a
strong promoter, the correct translation initiation sequence such as a ribosomal binding
site and start codon, a strong termination codon, and atranscription termination
sequence.[2]
There are differences in the machinery for protein synthesis between prokaryotes
and eukaryotes, therefore the expression vectors must have the elements for expression that is
appropriate for the chosen host. For example, prokaryotes expression vectors would have
a Shine-Dalgarno sequence at its translation initiation site for the binding of ribosomes, while
eukaryotes expression vectors would contain the Kozak consensus sequence.
The promoter initiates the transcription and is therefore the point of control for the expression of
the cloned gene. The promoters used in expression vector are normally inducible, meaning that
protein synthesis is only initiated when required by the introduction of an inducer such as IPTG.
Protein expression however may also be constitutive (i.e. protein is constantly expressed) in
some expression vectors. Low level of constitutive protein synthesis may occur even in
expression vectors with tightly controlled promoters.
Expression systems
Different organisms may be used to express a target protein, the expression vector used therefore
will have elements specific for use in the particular organism. The most commonly used
organism for protein expression is the bacterium Escherichia coli. However not all proteins can
be successfully expressed in E. coli, and other systems may therefore be used.

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Bacterial
An example of a bacterial expression vector is the pGEX-3x plasmid
The expression host of choice for the expression of many proteins is Escherichia coli as the
production of heterologous protein in E. coli is relatively simple and convenient, as well as being
rapid and cheap. A large number of E. coli expression plasmids are also available suitable for a
wide variety of needs. Other bacteria used for protein expression include Bacillus subtilis.
Most heterologous proteins are expressed in the cytoplasm of E. coli. However, not all proteins
formed may be soluble in the cytoplasm, and incorrectly folded proteins formed in cytoplasm
can form insoluble aggregates called inclusion body. Such insoluble proteins will require
refolding which can be an involved process and may not produce high yield. Where necessarily,
for example when the protein can only fold correctly in an oxidizing environment due to the
presence of disulphide bonds, the protein may be targeted to the periplasmic space by the use of
an N-terminalsignal sequence. Other more sophisticated systems are being developed; such
systems may allow for the expression of proteins previously thought impossible in E. coli, such
as glycosylated proteins.
The promoters used for these vector are usually based on the promoter of the lac operon or
the T7 promoter, and they are normally regulated by the lac operator. These promoters may also
be hybrids of different promoters, for example, the tac promoter is a hybrid
of trp and lac promoters.Note that most commonly used lac or lac-derived promoters are based
on the lacUV5 mutant which is insensitive to catabolite repression. This mutant allows for
expression of protein under the control of the lac promoter when the growth medium contains

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glucose since glucose would inhibit protein expression if wild-type lac promoter is
used.Presence of glucose nevertheless may still be used to reduce background expression
through residual inhibition in some systems.
Examples of E. coli expression vectors are the pGEX series of vectors where glutathione-S-
transferase is used as a fusion partner and protein expression is under the control of the tac
promoter, and the pET series of vectors which uses a T7 promoter.
It is possible to simultaneously express two or more different proteins in E. coli using different
plasmids. However, when 2 or more plasmids are used, each plasmid needs to use a different
antibiotic selection as well as a different origin of replication, otherwise the plasmids may not be
stably maintained. Many commonly-used plasmids are based on the ColE1replicon and are
therefore incompatible with each other; in order for a ColE1-based plasmid to coexist with
another in the same cell, the other would need to be of a different replicon, e.g. a p15A replicon-
based plasmid such as the pACYC series of plasmids. Another approach would be to use a single
two-cistron vector or design the coding sequences in tandem as a bi- or poly-cistronic construct.
Mammalian
Mammalian expression vectors offer considerable advantages for the expression of mammalian
proteins over bacterial expression systems - proper folding, post-translational modifications, and
relevant enzymatic activity. It may also be more desirable than other eukaryotic non-mammalian
systems whereby the proteins expressed may not contain the correct glycosylations. It is of
particular use in producing membrane-associating proteins that require chaperones for proper
folding and stability as well as containing numerous post-translational modifications. The
downside, however, is the low yield of product in comparison to prokaryotic vectors as well as
the costly nature of the techniques involved. Its complicated technology, and potential
contamination with animal viruses of mammalian cell expression have also placed a constraint
its use in large-scale industrial production.
Cultured mammalian cell lines such as the Chinese hamster ovary (CHO), HEK, HeLa, and COS
cell lines may be used to produce protein. Vectors are transfected into the cells and the DNA
may be integrated into the genome by homologous recombination in the case of stable
transfection, or the cells may be transiently transfected. Examples of mammalian expression
vectors include the adenoviral vectors, the pSV and the pCMV series of plasmid

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vectors, vaccinia andretroviral vectors, as well as baculovirus. The promoters
for cytomegalovirus (CMV) and SV40 are commonly used in mammalian expression vectors to
drive protein expression. Non-viral promoter, such as the elongation factor (EF)-1 promoter, is
also known.
Ti-plasmid
The virulent strains of A. tumefaciens harbor large plasmids (140–235 kbp) known as tumor-
inducing (Ti) plasmid involving elements like T-DNA, vir region, origin of replication, region
enabling conjugative transfer and o-cat region (required for catabolism of opines).
Ti-plasmid of Agrobacterium
T-DNA
It is a small, specific segment of the plasmid, about 24kb in size and found integrated in the plant
nuclear DNA at random site. This DNA segment is flanked by right and left borders.
The functions of T-DNA genes are listed
Gene Product Function
ocs Octopine synthase Opine synthesis
nos Nopaline synthase Opine synthesis
trns1 (iaaH, auxA) Tryptophan-2-mono-oxygenase Auxin synthesis
trns2 (iaaM, auxB) Indoleacetamide hydrolase Auxin synthesis
trnr (ipt, cyt) Isopentyltransferase Cytokinin synthesis

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frs Fructopine synthase Opine synthesis
mas Mannopine synthase Opine synthesis
ags Agropine synthase Opine synthesis
T- DNA:Border Sequences
 T-regions are defined by direct repeats known as T-DNA border sequences (Right and Left
Border i.e. RB and LB of 25 bp each).
 These are not transferred intact to the plant genome, but are involved in the transfer process.
 The RB is rather precise, but the LB can vary by about 100 nucleotides.
 Deletion of the RB repeat abolishes T-DNA transfer, but the LB seems to be non-essential. The
LB repeat has little transfer activity alone.
Disarmed Ti-plasmid derivatives as plant vectors
Ti plasmid is a natural vector for genetically engineering plant cells due to its ability to
transfer T-DNA from the bacterium to the plant genome. But wild-type Ti plasmids are
not suitable as vectors due to the presence of oncogenes in T-DNA that cause tumor
growth in the recipient plant cells. For efficient plant regeneration, vectors with disarmed
T-DNA are used by making it non-oncogenic by deleting all of its oncogenes. The
foreign DNA is inserted between the RB and LB and then integrated into the plant
genome without causing tumors.

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Structure of the Ti-plasmid pGV3850 with disarmed T-DNA.
 The creation of disarmed T-DNA is an important step forward, but the absence of tumor
formation makes it necessary to use an alternative method for the identification of
transformed plant cells. Opine production using pGV3850 was exploited as a screenable
phenotype, and the ocs and nosgenes are now widely used as screenable markers.
Drawbacks
Several drawbacks are associated with disarmed Ti- vector systems as discussed below;
 Necessity to carry out enzymatic assays on all potential transformants.
 Not convenient as experimental gene vectors due to large size.
 Difficulty in in vitro manipulation and
 Absence of unique restriction sites in the T-DNA.
Co- integrate vectors
Co-integrate vectors are the deletion derivatives of Ti-plasmids. The DNA to be introduced into
the plant transformation vector is sub cloned in a conventional Escherichia coli plasmid vector
for easy manipulation, producing a so-called intermediate vector. These vectors are incapable of
replication inA. tumefaciens and also lack conjugation functions. Transfer is achieved using a
‘triparental mating’ in which three bacterial strains are mixed together:
(i) An E. coli strain carrying a helper plasmid able to mobilize the intermediate vector in trans;
(ii) The E. coli strain carrying the recombinant intermediate vector;
(iii) A. tumefaciens carrying the Ti plasmid.
Conjugation between the two E. coli strains transfers the helper plasmid to the carrier of the
intermediate vector, which in turn is mobilized and transferred to the
recipient Agrobacterium.Homologous recombination between the T-DNA sequences of the Ti
plasmid and intermediate vector forms a large co- integrate plasmid resulting in the transfer of
recombinant T-DNA to the plant genome.

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Binary vector
 Binary vector was developed by Hoekma et al (1983) and Bevan in (1984).
 It utilizes the trans- acting functions of the vir genes of the Ti-plasmid and can act on any T-
DNA sequence present in the same cell.
 Binary vector contains transfer apparatus(the vir genes) and the disarmed T-DNA containing the
transgene on separate plasmids.
Advantages of Binary vector
 Small size due to the absence of border sequences needed to define T-DNA region and vir
region.
 Ease of manipulation

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