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Transcript
Unit I Lecture 4
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,
Table 2.4 Genomes 3 (© Garland Science 2007)
Cloning in Saccharomyces
cerevisiae
• In 1996 the sequencing of the entire 12 Mb genome of S.
cerevisiae was completed and most, if not all, of the
genes have been identified.
• fungi are not naturally transformable and artificial means
have to be used for introducing foreign DNA.
• One method involves the use of spheroplasts (i.e. wallless cells) and was first developed for S. cerevisiae
(Hinnen et al. 1978).
• In this method, the cell wall is removed enzymically and
the resulting spheroplasts are fused with ethylene glycol
in the presence of DNA and CaCl2
Reasons for cloning DNA in S. cerevisiae
• At that time the primary purpose of cloning was
to understand what particular genes do in vivo
• to understand those cellular functions unique to
eukaryotes such as mitosis, meiosis, signal
transduction, obligate cellular differentiation
• offer a number of advantages, such as the ability
to glycosylate protein, the absence of
pyrogenic toxins, and in the case of the
methylotrophic yeast Pichia pastoris, the ability
to get very high yields of recombinant
proteins.
• ability to clone very large pieces of DNA
• Certain yeast vectors can accept inserts
greater than 1 Mb, much greater than
those found in BACs and PACs
• All contain unique target sites for a number of
restriction endonucleases.
• Secondly, they can all replicate in E. coli, often
at high copy number.
• all employ markers that can be selected readily
in yeast and which will often complement the
corresponding mutations in E. coli as well.
• The four most widely used markers are His3,
Leu2, Trp1, and Ura3.
Kinds of vectors have been
developed for use in S. cerevisiae
Four types of plasmid vector have been
developed:
• yeast episomal plasmids (YEps)
• yeast replicating plasmids (YRps)
• Yeast centromere plasmids (Ycps)
• yeast artificial chromosomes (YACs).
Yeast episomal plasmids
• YEps were first constructed by Beggs
(1978) by recombining an E. coli cloning
vector with the naturally occurring yeast 2
μm plasmid.
• This plasmid is 6.3 kb in size, has a copy
number of 50–100 per haploid cell and has
no known function. A representative
YEp is shown in Fig. 11.2.
Yeast replicating plasmids
• YRps were initially constructed by Struhl et al.
(1979).
• carry sequences that enable E. coli vectors to
replicate in yeast cells.
• sequences are known as ars autonomously
replicating sequences).
• An ars is quite different from a centromere:
• The ars acts as an origin of replication
• Centromer is involved in chromosome
segregation
Yeast centromere plasmids
• plasmids carrying an ars, most of the recombinants
were unstable in yeast
• plasmid-borne centromere sequences have the
same distinctive chromatin structure that occurs in
the centromere region of yeast chromosomes
(Bloom & Carbon 1982)
• YCps exhibit three characteristics of chromosomes
in yeast cells.
• First, they are mitotically stable in the absence of
selective pressure.
• Secondly, they segregate during meiosis in a
Mendelian manner.
• Finally, found at low copy number in the host cell.
Yeast artificial chromosomes
• All three autonomous plasmid vectors described
above are maintained in yeast as circular DNA
molecules, even the YCp vectors, which
possess yeast centromeres.
• Thus, none of these vectors resembles the
normal yeast chromosomes, which have a linear
structure.
• The ends of all yeast chromosomes, like
those of all other linear eukaryotic
chromosomes, have unique structures that are
called telomeres.
• Telomere structure has evolved as a device to
preserve the integrity of the ends of DNA
molecules, which often cannot be finished by the
conventional mechanisms of DNA replication.
• In 1982 Szostak & Blackburn developed the first
vector which could be maintained as a linear
molecule, thereby mimicking a chromosome,
by cloning yeast telomeres into a YRp.
• Such vectors are known as yeast artificial
chromosomes (YACs).
• The method for cloning large DNA sequences in
YACs developed by Burke et al. (1987) is shown
in next slide Fig 11.3.
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.
• Recombinogenic engineering can be used to move
genes from one vector to another
Promoter systems have been developed to
facilitate overexpression of recombinant
proteins in yeast
• The first overexpression systems developed
were for S. cerevisiae and used promoters from
genes encoding abundant glycolytic enzymes,
e.g. alcohol dehydrogenase (ADH1), PGK or
glyceraldehyde-3- phosphate dehydrogenase
(GAP).
• These are strong promoters and mRNA
transcribed from them can accumulate up to
5% of total.
• They were at first thought to be constitutive
but later were shown to be induced by
glucose
• The ideal promoter is one that is tightly
regulated so that the growth phase can be
separated from the induction phase.
• This minimizes the selection of non-expressing
cells and can permit the expression of proteins
normally toxic to the cell.
• ideal promoter will also have a high induction
ratio.
• One promoter which has these characteristics
and which is now the most widely used is that
from the GAL1 gene.
• Galactose regulation in yeast is now extremely well
studied and has become a model system for
eukaryotic transcriptional regulation
specialist multi-purpose vectors
have been developed for use in yeast
• incorporate the useful features found in the
corresponding E. coli vectors e.g. an f1 origin
to permit sequencing of inserts, production
of the cloned gene product as a purification
fusion
Heterologous proteins can be synthesized as
fusions for display on the cell surface of yeast
• used to detect protein–ligand interactions
• to select mutant proteins with altered
binding capacity
Figure 2.25 Genomes 3 (© Garland Science 2007)
Figure 2.25a Genomes 3 (© Garland Science 2007)
Figure 2.25b Genomes 3 (© Garland Science 2007)
Figure 2.26a Genomes 3 (© Garland Science 2007)
Figure 2.26b Genomes 3 (© Garland Science 2007)
Figure 2.27 Genomes 3 (© Garland Science 2007)
An E. coli-Yeast Shuttle Vector
BACs and PACs are vectors that can carry
much larger fragments of DNA than cosmids
because they do not have packaging
constraints
Figure 2.24 Genomes 3 (© Garland Science 2007)
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.