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Unit I Lecture 3
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,
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
DNA cloning with single-stranded
DNA vectors
• M13, f1, and fd are filamentous coliphages containing
a circular single-stranded DNA molecule.
• These coliphages have been developed as cloning
vectors, for they have a number of advantages over
other vectors, including the other two classes of vector
for E. coli: plasmids and phage λ
Filamentous bacteriophages have a number
of unique properties that make them suitable
as vectors
Unique properties of M13, f1, fd
• Contain a single-stranded circular DNA molecule, which
is 6407 (M13) or 6408 (fd) nucleotides long.
• The complete nucleotide sequences of fd and M13 are
available and they are 97% identical
• The filamentous phages only infect strains of enteric
bacteria harboring F pili. The adsorption site appears to
be the end of the F pilus
• Replication of phage DNA does not result in host-cell
lysis. Rather, infected cells continue to grow and divide,
albeit at a slower rate than uninfected cells, and extrude
virus particles.
• Up to 1000 phage particles may be released into the
medium per cell per generation
Vectors with single-stranded DNA
genomes have specialised uses
• Sequencing by the original dideoxy method required
single-stranded DNA
• oligonucleotide-directed mutagenesis
• probe preparation.
• can be purified and manipulated in vitro just like a
plasmid.
• the size of the phage particle is governed by the size of
the viral DNA and therefore there are no packaging
constraints
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.