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Unit I Lecture 2
B. Tech. (Biotechnology) III Year
V th Semester
EBT-501, Genetic Engineering
EBT 501, Genetic Engineering
Unit I
Gene cloning -concept and basic steps; application of bacteria and viruses in genetic engineering; Molecular biology of E. coli and
bacteriophages in the context of their use in genetic engineering, Cloning vectors: Plasmid cloning vector PBR322, Vectors
for cloning large piece of DNA; –Bacteriophage-l and other phage vectors; Cosmids, Phagemids; YAC and BAC vectors,
Model vectors for eukaryotes – Viruses,
Unit II
Restriction modification, enzymes used in recombinant DNA technology endonucleases, ligases and other enzymes useful in gene
cloning, PCR technology for gene/DNA detection, cDNA, Use of Agrobacterium for genetic engineering in plants; Gene libraries; Use
of marker genes. Cloning of foreign genes: DNA delivery methods -physical methods and biological methods, Genetic transformation
of prokaryotes: Transferring DNA into E. coli –Chemical induction and Electroporation,
Unit III
Gene library: Construction cDNA library and genomic library, Screening of gene libraries – screening by DNA hybridization,
immunological assay and protein activity, Marker genes: Selectable markers and Screenable markers, nonantibiotic markers, Gene
expression in prokaryotes: Tissue specific promoter, wound inducible promoters, Strong and regulatable promoters; increasing
protein production; Fusion proteins; Translation expression vectors; DNA integration into bacterial genome; Increasing secretions;
Metabolic load, Recombinant protein production in yeast: Saccharomyces cerevisiae expression systems; Mammalian cell
expression vectors: Selectable markers;
Unit IV
Origins of organismal cloning in developmental biology research on frogs; nuclear transfer procedures and the cloning of sheep
(Dolly) & other mammals; applications in conservation; therapeutic vs. reproductive cloning; ethical issues and the prospects for
human cloning; Two-vector expression system; two-gene expression vector, Directed mutagenesis; transposon mutagenesis, Gene
targeting, Site specific recombination
Unit V
General principles of cell signaling, Extracellular signal molecule and their receptors, Operation of signaling molecules over various
distances, Sharing of signal information, Cellular response to specific combinations of extracellular signal molecules; Different
response by different cells to same extracellular signal molecule, NO signaling by binding to an enzyme inside target cell, Nuclear
receptor; Ion channel linked, G-protein- linked and enzyme-linked receptors, Relay of signal by activated cell surface receptors via
intracellular signaling proteins, Intracellular signaling proteins as molecular switches, Interaction between modular binding domain
and signaling proteins, Remembering the effect of some signal by cells.
Unit I
• Gene cloning -concept and basic steps
• Application of bacteria and viruses in genetic engineering
• Molecular biology of E. coli and bacteriophages in the
context of their use in genetic engineering
• Cloning vectors: Plasmid cloning vector pBR322,
• Vectors for cloning large piece of DNA
– Bacteriophage-l and other phage vectors
– Cosmids
– phasmids
– Phagemids
– BAC vectors
– PAC vectors
– YAC vectors
• Model vectors for eukaryotes - Viruses,
Bacteriophage Vectors- phage 
• Most bacteriophage cloning vectors have been
constructed from the phage  chromosome.
• phage  is extensively studied virus of E. coli
• The DNA of phage λ, in the form in which it is isolated
from the phage particle, is a linear duplex molecule of
about 48.5 kbp.
• The entire DNA sequence has been determined
•
At each end are short single-stranded 5′ projections
of 12 nucleotides, which are complementary in
sequence and by which the DNA adopts a circular
structure when it is injected into its host cell, i.e. λ
DNA naturally has cohesive termini, which associate
to form the cos site.
phage 
• It is possible to insert foreign DNA into the chromosome
of phage-λ derivative and, in some cases, foreign genes
can be expressed efficiently via λ promoters.
• The central one-third (about 15 kb) of the  chromosome
contains genes required for lysogeny but not for lytic
growth.
• This portion of the chromosome can be excised and
replaced with foreign DNA.
• The foreign DNA inserted must be 10-15 kb.
• It is possible to insert foreign DNA into the chromosome
of phage-λ derivative and, in some cases, foreign genes
can be expressed efficiently via λ promoters.
• Functionally related genes of phage λ are clustered
together on the map, except for the two positive
regulatory genes N and Q.
• Genes on the left of the conventional linear map (Fig.
4.10) code for head and tail proteins of the phage particle.
•
Genes of the central region are concerned with
recombination (e.g. red) and the process of
lysogenization, in which the circularized chromosome is
inserted into its host chromosome and stably replicated
along with it as a prophage.
• Much of this central region, including these genes, is not
essential for phage growth and can be deleted or
replaced without seriously impairing the infectious growth
cycle.
phage 
• To the right of the central region are genes concerned
with regulation and prophage immunity to superinfection
(N, cro, cI),
• followed by DNA synthesis (O, P),
• late function regulation (Q), and
• host cell lysis (S, R).
• Next Figure illustrates the Map of the λ chromosome,
showing the physical position of some genes on the fulllength DNA of wild-type bacteriophage λ.
• Clusters of functionally related genes are indicated.
• The central one-third (about 15 kb) of the  chromosome
contains genes required for lysogeny but not for lytic
growth.
• This portion of the chromosome can be excised and
replaced with foreign DNA.
phage 
• The lytic cycle, λ transcription occurs in three temporal
stages: early, middle, and late.
• Basically, early gene transcription establishes the lytic
cycle (in competition with lysogeny), middle gene
products replicate and recombine the DNA, and late
gene products package this DNA into mature phage
particles.
Cosmid Vectors
• Hybrids between
plasmids and the phage 
chromosome.
• Replicate autonomously
in E. coli.
• Can be packaged in vitro
into phage  heads.
• Accept inserts of 35-45
kb.
There are two basic types of phage lamda vectors:
insertional vectors and replacement
vectors
• Wild-type λ DNA contains several target sites for most of
the commonly used restriction endonucleases and so is
not itself suitable as a vector.
• Derivatives of the wild-type phage have therefore been
produced that either have a single target site at which
foreign DNA can be inserted (insertional vectors) or have
a pair of sites defining a fragment that can be removed
(stuffer) and replaced by foreign DNA (replacement
vectors).
• Since phage λ can accommodate only about 5% more
than its normal complement of DNA, vector derivatives
are constructed with deletions to increase the space
within the genome. The shortest λ DNA molecules that
produce plaques of nearly normal size are 25% deleted
Cosmids are plasmids that can be
packaged into bacteriophage lamda
particles
• Concatemers of unit-length λ DNA molecules can be
efficiently packaged if the cos sites, substrates for the
packaging-dependent cleavage, are 37–52 kb apart (75–
105% the size of λ+ DNA).
• Only a small region in the proximity of the cos site is
required for recognition by the packaging system
• Plasmids have been constructed which contain a
fragment of λ DNA including the cos site.
• These plasmids have been termed cosmids and can be
used as gene-cloning vectors in conjunction with the in
vitro packaging system.
• With a cosmid vector of 5 kb, we demand the insertion of
32–47 kb of foreign DNA – much more than a phage-λ
vector can accommodate.
• Note that, after packaging in vitro, the particle is used to
infect a suitable host. The recombinant cosmid DNA is
injected and circularizes like phage DNA but replicates
as a normal plasmid without the expression of any
phage functions.
• Transformed cells are selected on the basis of a vector drug
resistance marker.
• Cosmids provide an efficient means of cloning large
pieces of foreign DNA (50kb). Because of their capacity
for large fragments of DNA, cosmids are particularly
attractive vectors for constructing libraries of eukaryotic
genome fragments.
• Partial digestion with a restriction endonuclease provides
suitably large fragments
Aims for using Phage vectors
• rapid and efficient genomic or complementary DNA
(cDNA) library construction
• increase the capacity for foreign DNA fragments,
preferably for fragments generated by any one of several
restriction enzymes to devise methods for positively
selecting recombinant formation.
• To allow RNA probes to be conveniently prepared by
transcription of the foreign DNA insert; this facilitates the
screening of libraries in chromosome walking procedures.
An example of a vector with this property is λZAP
• To develop vectors for the insertion of eukaryotic cDNA
such that expression of the cDNA, in the form of a fusion
polypeptide with β- galactosidase, is driven in E. coli; this
form of expression vector is useful in antibody screening.
An example of such a vector is λgt11
BACs and PACs
• Bacterial artificial chromosomes (BACs) have
been constructed from bacterial fertility (F)
factors.
• Bacteriophage P1 artificial chromosomes
(PACs) have been constructed from
bacteriophage P1 chromosomes.
• BACs and PACs accept 150-300 kb inserts and
are less complex than YACs.
Phagemid Vectors
• Contain components from phage chromosomes
and plasmids.
• Replicate in E. coli as double-stranded plasmids.
• Addition of a helper phage causes the phagemid
to switch to the phage mode of replication,
resulting in the packaging of single-stranded
DNA into phage heads.
The Life Cycle of Bacteriophage
M13
Phagemids pUC118 and pUC119
Replication as Double-Stranded
Plasmids
Replication as Single-Stranded
Phage DNA
BACs and PACs are vectors that can carry
much larger fragments of DNA than cosmids
because they do not have packaging
constraints
• Phage P1 is a temperate bacteriophage which has been
extensively used for genetic analysis of Escherichia coli
because it can mediate generalized transduction.
• Sternberg and co-workers have developed a P1 vector
system which has a capacity for DNA fragments as
large as 100 kb.
• Thus the capacity is about twice that of cosmid
clones but less than that of yeast artificial chromosome
(YAC) clones.
• P1 vector contains a packaging site (pac) which is
necessary for in vitro packaging of recombinant
molecules into phage particles.
•
The vectors contain two loxP sites. These are the sites
recognized by the phage recombinase, the product of
the phage cre gene, and which lead to circularization of
the packaged DNA after it has been injected into an E.
coli host expressing the recombinase (Fig. 5.4).
• Clones are maintained in E. coli as low-copy-number
plasmids by selection for a vector kanamycin-resistance
marker.
• A high copy number can be induced by exploitation of the P1
lytic replicon (Sternberg 1990).
• This P1 system has been used to construct genomic
libraries of mouse, human, fission yeast, and Drosophila
Eukaryotic and Shuttle Vectors
• Because different organisms use different
origins of replication and regulatory signals,
different cloning vectors must be used in
different species.
• Special cloning vectors can replicate in other
prokaryotes and in eukaryotes.
• Shuttle vectors can replicate in E. coli and in
another species.
An E. coli-Yeast Shuttle Vector
Yeast Artificial Chromosomes (YACs)
• Genetically engineered yeast minichromosomes.
• Accept foreign DNA inserts of 200-500 kb.
• Contain a yeast origin of replication, yeast centromere,
two yeast telomeres, a selectable marker, and a
polycloning site.
The PAC Mammalian Shuttle
Vector pJCPAC-Mam1
Episomes
• An episome is a genetic element that is
not essential to the host and that can
either replicate autonomously or be
integrated into the bacterial chromosome.
• Integration depends on the presence of IS
elements.