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MSC I Sem I Paper IV Unit III

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MSC I Sem I Paper IV Unit III

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suyash singh
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© © All Rights Reserved
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MSc I Sem I

PSB104 RECOMBINANT DNA


TECHNOLOGY

Leela Chauhan
Recombinant DNA Technology
Vectors

 In general, the word “cloning” means the creation of a perfect replica


 Long before attempts were made to clone an entire organism,
researchers learned how to reproduce desired regions or fragments of
the genome, a process that is referred to as molecular cloning.
 Cloning small fragments of the genome allows for the manipulation and
study of specific genes (and their protein products), or noncoding
regions in isolation.
 A plasmid (also called a vector) is a small circular DNA molecule that
replicates independently of the chromosomal DNA of microorganisms
such as E. coli. In cloning, the plasmid molecules can be used to provide
a “folder” in which to insert a desired DNA fragment. Plasmids are
usually introduced into a bacterial host for proliferation. In the bacterial
context, the fragment of DNA from the human genome (or the genome
of another organism that is being studied) is referred to as foreign DNA,
or a transgene, to differentiate it from the DNA of the bacterium, which
is called the host DNA.
 Plasmids occur naturally in bacterial populations (such as Escherichia
coli) and have genes that can contribute favorable traits to the
organism, such as antibiotic resistance (the ability to be unaffected by
antibiotics). Plasmids have been repurposed and engineered as vectors
for molecular cloning and the large-scale production of important
reagents, such as insulin and human growth hormone. An important
feature of plasmid vectors is the ease with which a foreign DNA
fragment can be introduced via the multiple cloning site (MCS). The MCS
is a short DNA sequence containing multiple sites that can be cut with
different commonly available restriction endonucleases
 Restriction endonucleases recognize specific DNA sequences and cut
them in a predictable manner; they are naturally produced by bacteria
as a defense mechanism against foreign DNA. Many restriction
endonucleases make staggered cuts in the two strands of DNA, such
that the cut ends have a 2- or 4-base single-stranded overhang. Because
these overhangs are capable of annealing with complementary
overhangs, these are called “sticky ends.” Addition of an enzyme called
DNA ligase permanently joins the DNA fragments via phosphodiester
bonds. In this way, any DNA fragment generated by restriction
endonuclease cleavage can be spliced between the two ends of a
plasmid DNA that has been cut with the same restriction endonuclease
Recombinant DNA Molecules
 Plasmids with foreign DNA inserted into them are called recombinant
DNA molecules because they are created artificially and do not occur in
nature. They are also called chimeric molecules because the origin of
different parts of the molecules can be traced back to different species
of biological organisms or even to chemical synthesis. Proteins that are
expressed from recombinant DNA molecules are called recombinant
proteins. Not all recombinant plasmids are capable of expressing genes.
The recombinant DNA may need to be moved into a different vector (or
host) that is better designed for gene expression. Plasmids may also be
engineered to express proteins only when stimulated by certain
environmental factors, so that scientists can control the expression of
the recombinant proteins.
SV - 40
 Simian virus 40 (SV40) belongs to polyomavirus family, the family of
viruses that induce tumours in animals
 Simian virus 40 was first isolated from monkeys hence so named. SV40
was one of the first genetic elements to be studied by genetic
engineering techniques and has been used extensively as a vector for
transferring genes into eukaryotic cells.
 SV40 is a naked icosahedral virus with a diameter of 45 nm. Its capsid
contains 72 protein subunits
 When the virus enters the host cell, its genome migrates to the nucleus.
SV40 genome replicates inside the nucleus of the cell, but the capsid
protein are synthesized in the cytoplasm and migrate inside the nucleus
where, finally, the assembly of the virus take place.
Vaccinia
 VV is a poxvirus that was clinically used as vaccine for smallpox. The
genome of VV is completely sequenced and facilitates the creation of
recombinant viral vectors that could carry up to 25 kb of foreign DNA
 VV is a very large, complex, and enveloped virus that contains a linear
and dsDNA genome of about 180–190 kbp, with internal terminal
repeats, and hairpin loops. The life cycle of the VV occurs in the
cytoplasm and so it is a non integrative vector.
 An advantage of using vaccinia-derived vectors is that the vaccinia
vectors may carry until 25 kb of foreign DNA without the need for viral
deletions.27 Vaccinia vectors present other advantages as a broad host
range that permits the infections of primary cultures and many different
cell lines, cytoplasmic replication, or the fact that the viral genome does
not splice its primary transcripts.
Baculovirus
 Baculoviruses, which belong to the family Baculoviridae, are a group of
DNA viruses that infect insects. These viruses are very small, rod-shaped
structures, composing of a protein coat containing a small amount of
DNA which encodes components for further viral reproduction.
 Baculoviruses use unique mechanisms to survive and proliferate in their
environment. When baculoviruses are outside a host, waiting to be
ingested, they survive within occlusion bodies. These are bodies of
protein which envelop and protect the virions (the form of the virus
when external to a host cell) until they are ingested by a host.
 The occlusion bodies are stable in the environment, but when they
are ingested by an insect, the high pH of the insect gut causes the
breakdown of the protein and the release of the virions.
 The virions enter cells and self-replicate, hijacking the machinery of
the host cell to produce DNA and protein, forming more viruses.
After replication has occurred, the final step is the production of a
protein called polyhedrin. This protein crystallises in the cell and
surrounds the virions, forming an occlusion body ready for
dispersal.
 The virions produced by an occlusion body are called occlusion-
derived virions (ODV). Another type of virion that the baculoviruses
produce are called budding virions (BV) which are responsible for
the cell to cell, systemic infection within insects.
Retroviral vectors
 Retrovirus vectors are RNA-based vectors belonging to the family of
retroviridae.
 The genome of the retrovirus vector is a 7 to 10 kb single stranded RNA
containing long terminal repeats (LTR)
 Retroviruses enter the cells via cell fusion of the envelope protein with
the cell membrane.
 Retroviral vector-mediated gene transfer has been central to the
development of gene therapy. Retroviruses have several distinct
advantages over other vectors, especially when permanent gene
transfer is the preferred outcome. The most important advantage that
retroviral vectors offer is their ability to transform their single stranded
RNA genome into a double stranded DNA molecule that stably
integrates into the target cell genome. This means that retroviral
vectors can be used to permanently modify the host cell nuclear
genome
BACTERIAL ARTIFICIAL CHROMOSOME
 A bacterial artificial chromosome (BAC) is an engineered DNA
molecule used to clone DNA sequences in bacterial cells (for
example, E. coli). BACs are often used in connection with DNA
sequencing. Segments of an organism's DNA, ranging from
100,000 to about 300,000 base pairs, can be inserted into BACs.
The BACs, with their inserted DNA, are then taken up by bacterial
cells. As the bacterial cells grow and divide, they amplify the BAC
DNA, which can then be isolated and used in sequencing DNA.
 A large piece of DNA can be engineered in a fashion that allows it be
propagated as a circular artificial chromosome in bacteria--so-called
bacterial artificial chromosome, or BAC. Each BAC is a DNA clone
containing roughly 100 to 300 thousand base pairs of cloned DNA.
Because the BAC is much smaller than the endogenous bacterial
chromosome, it is straightforward to purify the BAC DNA away from the
rest of the bacteria cell's DNA, and thus have the cloned DNA in a
purified form. This and other powerful features of BACs have made them
extremely useful for mapping and sequencing mammalian genomes.
 The BAC is based on a plasmid in Escherichia coli that is termed the F
(for fertility) plasmid. The F plasmid (or F factor) contains information
that makes possible the process called conjugation .
 A BAC contains the conjugation promoting genetic information as well as
stretch of DNA that is destined for incorporation into the bacterium. The
foreign DNA (e.g., portion of human genome) is flanked by sequences
that mark the boundaries of the insert. The sequences are referred to as
sequence tag connectors. When the BAC becomes incorporated into the
genome of Escherichia coli the sequence tag connectors act as markers
to identify the inserted foreign DNA.
 The most dramatic recent example of the power of BACs is their use by
The Institute for Genomic Research (TIGR ) in the technique of shotgun
cloning that was employed in the sequence determination of the human
genome
YEAST ARTIFICIAL CHROMOSOME

 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.
 YAC vectors allows the insertion of DNA fragments up to 100-500 kb. A
YAC vector includes
 TEL: telomere (TEL) is located at each end of chromosome. It protects
the linear DNA from degradation by nucleases.
 CEN: The centromere which is the attachment site for mitotic spindle
 ARS: Replication origin sequences which are specific DNA sequences
that allows the DNA replication machinery to assemble on the DNA and
move at the replication forks.
 A and B: YAC contains two selectable markers that allow the easy
isolation of yeast cells that have taken up the artificial chromosome.
 URA3-involved in uracil biosynthesis
 TRP1 -involved in tryptophan biosynthesis
 Recognition site for the two restriction enzymes EcoRI and BamHI.
 Ampr – an ampicillin resistance gene allows positive selection by
ampicillin resistance in bacteria
 A YAC is built using an initial circular DNA plasmid, which is typically cut
into a linear DNA molecule using restriction enzymes; DNA ligase is then
used to ligate a DNA sequence or gene of interest into the linearized
DNA, forming a single large, circular piece of DNA
 Advantages
 Yeast expression vectors, such as YACs, YIps (yeast integrating
plasmids), and YEps (yeast episomal plasmids) can be used to express
eukaryotic proteins that require posttranslational modification.
 YACs vectors are able to insert large fragments of DNA and can be
utilized to clone and assemble the entire genomes of an organism.
 the inserted sequences can be cloned and physically mapped using a
process called chromosome walking
 Study expression of entire mammalian genes
 Not used too much anymore, but important for historical reasons. One of
the very early ways that scientists--geneticists--manipulated large
pieces of DNA...very important to be able to isolate and reproduce large
pieces of DNA for the purposes of isolating disease genes. So what
scientists did was they co-opted the parts of a yeast chromosome, which
are important for packing that DNA, and replicating the DNA, and they
took out all the yeast sequences in the middle and they put in human
sequences or some other sequence, and then stuck it back in the yeast
cell, and the yeast cell didn't know the difference and replicated the
human DNA instead, and so served as a Trojan horse for the genetics
researchers to be able to replicate, and therefore study, the human and
other organism DNA that they were interested in.
 Disadvantages
 YACs are significantly less stable than BACs, producing “chimeric
effects”
 Difficult to isolate YACs from host yeast genome
 Have low copy number
 Slow growth rate
 Poor transformation efficiency
HIGH AND LOW COPY NUMBER OF PLASMIDS

 The plasmid copy number is the number of copies of a given plasmid in


a cell. To ensure survival and thus the continued propagation of the
plasmid, they must regulate their copy number. If a plasmid has too high
of a copy number, they may excessively burden their host by occupying
too much cellular machinery and using too much energy. On the other
hand, too low of a copy number may result in the plasmid not being
present in all of their host's progeny. Plasmids may be either high copy
number plasmids or low copy number plasmids; the regulation
mechanisms between these two types are often significantly different.
Biotechnology applications may involve engineering plasmids to allow a
very high copy number.
 High copy number plasmids, also called
relaxed plasmids, require a system to ensure
that replication is inhibited once the number of
plasmids in the cell reaches a certain threshold.
Relaxed plasmids are generally regulated
through one of two mechanisms: antisense RNA
or iteron binding groups
Glyphosate tolerance
Glyphosate-tolerant crops
 Glyphosate herbicide kills plants by blocking the EPSPS enzyme,
an enzyme involved in the biosynthesis of aromatic amino acids,
vitamins and many secondary plant metabolites. There are
several ways by which crops can be modified to be glyphosate-
tolerant. One strategy is to incorporate a soil bacterium gene that
produces a glyphosate tolerant form of EPSPS. Another way is to
incorporate a different soil bacterium gene that produces a
glyphosate degrading enzyme.
 Glyphosate herbicide-resistant crop plants, introduced commercially in
1994, now represent approximately 85% of the land area devoted to
transgenic crops. Herbicide resistance in commercial glyphosate-
resistant crops is due to expression of a variant form of a bacterial 5-
enolpyruvylshikimate-3-phosphate synthase with a significantly
decreased binding affinity for glyphosate at the target site of the
enzyme. As a result of widespread and recurrent glyphosate use, often
as the only herbicide used for weed management, increasing numbers
of weedy species have evolved resistance to glyphosate. Weed
resistance is most often due to changes in herbicide translocation
patterns, presumed to be through the activity of an as yet unidentified
membrane transporter in plants.
 Search identified a single non-target gene that, when overexpressed in
E. coli and Pseudomonas, confers high-level glyphosate resistance. The
gene, yhhS, encodes a predicted membrane transporter of the major
facilitator superfamily involved in drug efflux.
 An alternative mode of glyphosate resistance in E. coli is due to
reduced accumulation of glyphosate in cells that overexpress this
membrane transporter and discuss the implications for potential
alternative resistance mechanisms in other organisms such as plants
 TSR often involves mutations in genes encoding the protein targets of
herbicides, affecting the binding of the herbicide either at or near
catalytic domains or in regions affecting access to them.
 Detoxification of glyphosate
 Detoxification of the glyphosate molecule is another strategy that has been
employed to confer glyphosate resistance. Soil microorganisms can
metabolizes glyphosate by two different routes i) cleavage of the carbon-
phosphorus bond, resulting in the formation of phosphate and sarcosine (the
C-P lyase pathway) e.g., by Pseudomonas sp. PG2982; ii) oxidative cleavage
of the C-N bond on the carboxyl side catalyzed by glyphosate oxidoreductase
(GOX), resulting in the formation of aminomethylphosphonic acid (AMPA) and
glyoxylate (the AMPA pathway). Neither of these mechanisms has been
shown to occur in higher plants to a significant degree. The C-P lyase
pathway requires an unknown number of genes and the activity has not been
reconstituted in vitro, casting doubt on the ability to create the activity in
transgenic plants. The AMPA pathway appears to be the predominant route
for degradation of glyphosate in soil by a number of Gram-positive and Gram-
negative bacteria. Most recently, a glycine oxidase from B. subtilis was also
demonstrated to metabolize glyphosate into AMPA and glyoxylate, but using
a reaction mechanism different from GOX.
Nif genes
Transfer of nif genes
 Nitrogen is one of the most important nutrients for plant growth. To
enhance crop productivity, chemical nitrogen fertilizer is commonly
applied in agriculture. Biological nitrogen fixation, the conversion of
atmospheric N2 to NH3, is an important source of nitrogen input in
agriculture and represents a promising substitute for chemical nitrogen
fertilizers.). Thus, many biologists hope to reconstitute a nitrogenase
biosynthetic pathway in a eukaryotic host, with the final aim of developing
N2‐fixing cereal crops. With the advent of synthetic biology and a deep
understanding of the fundamental genetic determinants necessary to
sustain nitrogen fixation in bacteria, much progress has been made toward
this goal. Transfer of native and refactored nif (nitrogen fixation) genes to
non‐diazotrophs has been attempted in model bacteria, yeast, and plants.
Specifically, nif genes from Klebsiella oxytoca, Azotobacter vinelandii, and
Paenibacillus polymyxa have been successfully transferred and expressed
in Escherichia coli, Saccharomyces cerevisiae, and even in the tobacco
plant. These advances have laid the groundwork to enable cereal crops to
“fix” nitrogen themselves to sustain their growth and yield.

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