Transcript File

Unit I Lecture 1
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
Phagemids
YAC and BAC vectors
• Model vectors for eukaryotes - Viruses,
What is Gene Manipulation?
Creation and cloning of r-DNA
• r-DNA
– Artificially created DNA molecule which bring together
DNA Sequences not usually found together in nature
• Gene Manipulation
– Any, of variety of sophisticated techniques for creation
of r-DNA and (in many cases) its subsequent
introduction in living cell
• Cloning
– Propagation of r-DNA inside a particular host, so that
many copies of same sequence are produced
Cloning Overview
Four main steps in cloning:
•Insert synthesis
•Restriction enzyme digest
•Ligation
•Transformation
+
Plasmid
(vector)
Insert
(your gene)
Functional
construct
Vectors
Plasmid Vectors
Bacteriophage Vectors
Cosmid Vectors
Shuttle Vectors
Eukaryotic Vectors
Yeast Artificial Chromosomes (YACs)
Bacterial artificial chromosomes (BACs)
Bacteriophage P1 artificial chromosomes
(PACs)
• Plasmid Vectors
– pBR322, pSC101, pUC 18/19, pGEM3Z
– Yeast Plasmid vectors (Yep, YRp, YCp, Yip)
• Bacteriophage Vectors
– λ phage vectors, phage M13 vectors,
• Cosmid Vectors
– pJB8
• Phagemid Vectors
– pBluescriptSK(+/-)
• Phasmid Vector
– Phasmid λ ZAP vector
• Artificial Chromosome Vectors
– Bacterial artificial chromosomes (BACs)
• pBeloBAC11
– Fosmid vectors
– Yeast Artificial Chromosomes (YACs)
• YAC3
– Bacteriophage P1 artificial chromosomes (PACs)
•
•
•
•
Eukaryotic Vectors
Shuttle Vectors
Animal vectors
Plant vectors
Vectors
Plasmid Vectors
• Circular, double-stranded circular DNA
molecules present in bacteria.
• Range from 1 kb to over 200 kb.
• Replicate autonomously.
• Many carry antibiotic-resistance genes, which
can be used as selectable markers.
• Many useful cloning vectors were derived from
plasmid pBR322.
Plasmids
• Plasmids are widely used as cloning
vehicles
• Plasmids are widely distributed throughout
the prokaryotes
Plasmids are replicons which are stably
inherited in an extrachromosomal state
• Plasmids to which phenotypic traits have
not yet been ascribed are called cryptic
plasmids
• Most plasmids exist as double-stranded
circular DNA molecules
• If both strands of DNA are intact circles the
molecules are described as covalently
closed circles or CCC DNA
• If only one strand is intact, then the
molecules are described as open circles or
OC DNA
• Addition of an intercalating agent, such as
ethidium bromide, to supercoiled DNA
causes the plasmid to unwind.
• If excess ethidium bromide is added, the
plasmid will rewind in the opposite direction
• Linear plasmids have been found in a
variety of bacteria, e.g. Streptomyces sp.
and Borrelia burgdorferi.
• To prevent nuclease digestion, the ends of
linear plasmids need to be protected, and
two general mechanisms have evolved.
• Either there are repeated sequences ending
in a terminal DNA hairpin loop (Borrelia) or
• the ends are protected by covalent
attachment of a protein (Streptomyces).
• Two major types –
• conjugative or non-conjugative – depending
upon whether or not they carry a set of
transfer genes, called the tra genes,
which promote bacterial conjugation
• On the basis of their being maintained as multiple
copies per cell (relaxed plasmids) or as a limited
number of copies per cell (stringent plasmids)
copy number of a plasmid is determined by
regulating the initiation of plasmid replication
• regulation by antisense RNA and regulation by
binding of essential proteins to repeated
sequences called iterons
Plasmid Vectors
• A plasmid is a genetic element that can replicate
independently of the main chromosome in an
extrachromosomal state.
• Most plasmids are not required for the survival of
the host cell.
• Plasmids in E. coli
– F Factor (Fertility Factor)
– R Plasmids (Resistance Plasmids)
– Col Plasmids (synthesize compounds that kill
sensitive cells)
Features of many modern Plasmids
•Small size or low molecular weight plasmid
•Origin of replication
•Multiple cloning site (MCS)
•Selectable marker genes
•Some are expression vectors and have sequences that allow
RNA polymerase to transcribe genes
•DNA sequencing primers
Essential Features of a Cloning
Vector
• Origin of replication
– essential for selfreplication in host
cells
• Dominant selectable
marker gene
– usually confers drug
resistance
• One or more unique
restriction sites
A Polycloning Site is a Cluster
of Unique Restriction Sites
pBR322
Essential Features
pBR322
Essential Features
• pBR322 is early example of a widely used, purpose-built
cloning vector
• pBR322 contains the ApR and TcR genes of RSF2124
and pSC101, respectively
• Replication elements of pMB1, a Col E1-like plasmid
• completely sequenced with 4361 bp
• it is totally characterized in terms of its restriction sites
such that the exact length of every fragment can be
calculated. These fragments can serve as DNA markers
for sizing any other DNA fragment in the range of several
base pairs up to the entire length of the plasmid
• There are over 40 enzymes with unique cleavage
sites on the pBR322 genome (Fig. 4.8).
• The target sites of 11 of these enzymes lie within the
tetracycline resistant (TcR) gene, and there are sites
for a further two (ClaI and HindIII) within the promoter
of that gene.
• There are unique sites for six enzymes within the
ampicillin resistant (ApR) gene.
• Thus, cloning in pBR322 with the aid of any one of
those 19 enzymes will result in insertional
inactivation of either the ApR or the TcR markers.
• However, cloning in the other unique sites does not
permit the easy selection of recombinants, because
neither of the antibiotic resistance determinants is
inactivated.
Selectable Markers
• The commonest selectable markers are ones
encoding resistance to antibiotics such as ampicillin
(ApR), chloramphenicol (CmR), tetracycline (TcR),
streptomycin (SmR), and kanamycin (KmR).
• Another type of positive selection is reversal of
auxotrophy.
• For example, if the hisB+ gene is cloned in a vector
then it is easy to select recombinants by
transforming a hisB auxotroph and growing it in a
medium lacking histidine.
pBR322
Essential Features
• Plasmid pBR322 has been a widely used cloning vehicle.
• In addition, it has been widely used as a model system for
the study of prokaryotic transcription and translation, as
well as investigation of the effects of topological changes
on DNA conformation.
• The popularity of pBR322 is a direct result of the availability
of an extensive body of information on its structure and
function.
• A large number of improved vectors have been derived
from pBR322 eg. pBR325, pUC vectors
• pBR325 encodes chloramphenicol resistance in addition to
ampicillin and tetracycline resistance and has a unique EcoRI
site in the CmR gene
pUC vectors
• use of polylinkers or multiple cloning sites (MCSs)
• An MCS is a short DNA sequence, carrying sites for many
different restriction endonucleases.
• An MCS increases the number of potential cloning strategies
available by extending the range of enzymes that can be
used to generate a restriction fragment suitable for cloning
• The pUC vectors also incorporate a DNA sequence that
permits rapid visual detection of an insert.
• The MCS is inserted into the lacZ′ sequence, which encodes
the promoter and the α-peptide of β-galactosidase.
• The insertion of the MCS into the lacZ′ fragment does not
affect the ability of the α-peptide to mediate
complementation, but cloning DNA fragments into the MCS
does.
The Blue-White Color Test
• Therefore, recombinants can
be detected by blue/white
screening on growth medium
containing Xgal
• The E. coli lacZ gene
encodes -galactosidase.
-galactosidase converts the
colorless substrate Xgal into
a blue product.
• Cells with -galactosidase
activity produce blue colonies
when grown on Xgal; cells
lacking -galactosidase
activity produce white
colonies.
Reporter Genes
• Reporter genes are ones whose phenotype can be discerned
by visual examination of colonies growing on a plate and/or
ones that can be used to measure levels of gene expression.
• In terms of analysis of recombinants, the most widely used
reporter gene is the lacZ gene encoding b-galactosidase. As
noted in Box 3.2, the presence or absence of b-galactosidase
activity is easily detected by growing cells on medium
containing the chromogenic substrate Xgal.
• Another gene whose activity can be detected in a similar way
is the gusA gene encoding b-glucuronidase. This enzyme
cleaves 4-methylumbelliferyl-b-D-glucuronide (MUG) to
release a blue pigment.