BS 406 Genetic Engineering

DR.GAYATRI DAVE
Cloning vectors
• Cloning vectors are DNA molecules that are used to
"transport" cloned sequences between biological hosts and
the test tube.
• Most vectors are genetically engineered.
• A vector is used to amplify a single molecule of DNA into many
copes. Cloning vectors share four common properties:
• 1. Ability to replicate.
• 2. Contain a genetic marker for selection.
• 3. Unique restriction sites to facilitate cloning of insert DNA.
• 4. Minimum amount of nonessential DNA to optimize cloning
Cloning vectors for E.coli
M13 BASED PHAGE VECTORS
 M13mp1

+ LacZ’
 M13mp2

LacZ’
EcoR1 site
+
 Site directed mutagenesis
Aspartic acid
GGATTC

GAATTC
Aspargine

Altered beta galactosidase enzyme but still functional...

 M13mp7

LacZ’
Symmetric
+
MCS
EcoR1 site
LacZ’ disrupted Clear Plaque

LacZ’ reformed Blue Plaque

Features of phage In
Phage Inside
lambda host
Head

Terminally
Figure:1
redundant
Structure

Linier/circular, concatemeric, terminally redundant

DNA, packaged through headful mechanism
Phage lambda as vector
• Points to ponder… • The lambda virus particle
 Manipulation of Restriction contains a linear DNA of
Sites 48,502 bp with a single-
 Size Limitation for stranded 5' extension of 12
bases at both ends; these
Packaging extensions are
 Transfection of complementary to each
Recombinant Molecules other. These ends are
 Two-strain packaging called cohesive ends or
 Single-strain packaging cos. During infection, the
right 5' extension (cosR),
followed by the entire
genome, enters the host
cell. Both the cos ends are
ligated by host DNA ligase
 • Size-limitation • The second problem was the
requirement of a minimum
and maximum genome length
Solution: (38 and 53 kbp, respectively)
Genetic studies of specialized for the efficient packaging
transducing bacteriophages and for the production of
showed, however, that the viable phage particles (8,
central one-third of the genome, 111). The viability of the
i.e., the region between the J bacteriophage decreases
and N genes, is not essential for when its genome length is
lytic growth. The presence of a greater than 105% or less
nonessential middle fragment of than 78% of that of wild-type
the phage genome was also lambda.
revealed during construction of
viable deletion mutants
λ phage based vectors

15 kb fragment
Use of ‘natural’ selection
Insertion vectors
Insertion vectors
• Insertion vectors are the simplest form of lambda cloning
vectors.
• The vector itself can be grown (therefore must contain at least
75% of the wild-type genome length).
• Foreign DNA fragments are inserted into a unique restriction
site in the vector genome. Packaging requirements thus limit
insert fragment size to 0 - 10 KB - due to the limitations on
viral genome size (75% to 105% of the wild-type length = 50
KB)
Lambdagt10
•Size based selection
•Clear and turbid plaque
morphology
ZAP
Selection methods
• EZAPII (Figure 6.12c), with which
insertion of up to 10 kb DNA into
any of 6 restriction sites within a
polylinker inactivates the lacZ′
gene carried by the vector.
• Recombinants give clear rather
than blue plaques on X-gal agar.
Replacement vector
• A replacement vector has two recognition sites for the
restriction endonuclease used for cloning. Can carry up tp 20
kb
• These sites flank a segment of DNA that is replaced by the
DNA to be cloned
Spi-phenotype
• Wild-type λ cannot grow on E. coli strains lysogenic
for phage P2; in other words, the λ phage is Spi+
(sensitive to P2 inhibition).
• It has been shown that the products of λ genes red
and gam, which lie in the region 64–69% on the
physical map, are responsible for the inhibition of
growth in a P2 lysogen
• Hence vectors have been derived in which the stuffer
fragment includes the region 64–69%, so that
recombinants in which this has bee replaced by
foreign DNA are phenotypically Spi− and can be
positively selected by plating on a P2 lysogen
Replacement vectors
Summary
• lambda gt11 –– has complete lacZ gene cloned in it
• – blue plaques (with X-gal, IPTG)
• – if insert cloned into unique EcoRI site
• – get insertion inactivation – colourless plaques
• – also is expression vector
• – if cDNA cloned in frame with lacZ gene
• – get expression of fusion protein in lambda-infected cells
• – screen for proper cDNA clone
• – using antisera for desired protein
• potential problems: – insert must be in frame
• – antisera must recognise protein produced in E. coli
• – proper folding, glycosylation may be problem
• – fusion protein must not be lethal
• – kills cells before progeny phage produced
• Lambda ZAP – (Stratagene) – (Fig. 3.13)
• – has Bluescript plasmid cloned in it
• – cDNAs cloned in Bluescript multiple cloning site
• – get insertion inactivation of lacZ ' gene
• (in lacZΔM15 host)
– get fusion protein with lacZ ' gene
• – multiple cloning site
• – allows cloning in 3 reading frames
• – better chance of expression
• note – not all sites can be used
• – some duplicated in lambda vector
• – lacIq – overexpressed lac operon repressor
• left arm__SalI BamHI EcoRI_stuffer_EcoRI BamHI SalI__right arm
• – different order in lambda EMBL 4 (Fig. 3.11)
• – EcoRI on outside, SalI on inside
• cloning – double digest vector with BamHI & EcoRI
• – get arms with BamHI sites, stuffer with EcoRI ends
• selection – vector allows Spi– selection
• – Spi– phenotype – not sensitive to P2 inhibition
• – lambda + and lambda EMBL with stuffer – have red & gam genes
• – will not grow in cells lysogenized by phage P2
• – if stuffer replaced with cloned DNA
• – red & gam genes lost
• – phage will grow in P2 lysogen strain
• – must also have recA+ host cell & chi sites in vector
• – for packaging
Plasmid based cloning
vectors
• The simplest cloning vectors, and the ones
most widely used in gene cloning, are
those based on small bacterial plasmids.
• E.g PBR322
• The name “pBR322” conforms with the
standard rules for vector nomenclature:
• l “p” indicates that this is indeed a
plasmid.
• “BR” identifies the laboratory in which the vector was
originally constructed
• (BR stands for Bolivar and Rodriguez, the two researchers who
developed pBR322).
• “322” distinguishes this plasmid from others developed in the
same laboratory
• (there are also plasmids called pBR325, pBR327, pBR328, etc.)
 PBR322

Size-less than
10 kb

High copy Antibiotic resistant

number gene as selectable
marker
Cosmid
Cosmid Cloning Vectors
• Fragments from 30 to 46 kb can be accommodated by a
cosmid vector.
• Cosmids combine essential elements of a plasmid and
Lambda systems.
• Cosmids are extracted from bacteria and mixed with
restriction endonucleases.
• Cleaved cosmids are mixed with foreign DNA that has
been cleaved with the same endonuclease.
• Recombinant cosmids are packaged into lambda
caspids
• Recombinant cosmid is injected into the bacterial cell Shown above is a 50,000 base-pair long
where the rcosmid arranges into a circle and replicates DNA molecule bound with six EcoRI
molecules, and imaged using the atomic
as a plasmid. It can be maintained and recovered just as force microscope. This image clearly
indicates the six EcoRI "sites" and allows an
plasmids. accurate restriction enzyme map of the
cosmid to be generated.
http://homer.ornl.gov/cbps/afmimaging.htm
Cosmid
BAC an alternative
of Cosmid
Introduction
• This BAC system (bacterial artificial chromosome) is based on
the single-copy sex factor F of E. coli. This vector (Fig. 5.5)
includes the λ cos N and P1 loxP sites, two cloning sites
(HindIII and BamHI) and several G+C restriction enzyme sites
(e.g. SfiI, NotI, etc.) for potential excision of the inserts.
• BACs are capable of maintaining human and plant genomic
fragments of greater than 300 kb for over 100 generations
with a high degree of stability (Woo et al. 1994) and have
been used to construct genome libraries with an average
insert size of 125 kb
BAC map
Vectors for Eukaryotes
 Yeast derived vectors

Can not
Survive
independently
Yeast based vectors
• Four types of plasmid vector have been developed:
• yeast episomal plasmids (YEps),
• yeast replicating plasmids (YRps), yeast
• centromere plasmids (YCps) and
• yeast artificial chromosomes (YACs).
• All of them have features in common.
• First, they all contain unique target sites for a number of
restriction endonucleases.
• Secondly, they can all replicate in E. coli, often at high copy
• number.
• Finally, they all employ markers that
• can be selected readily in yeast
Shuttle vector
YAC
• a yeast artificial chromosome (YAC) contains
• a yeast origin of replication a centromere,
• a telomere at each end
• a large inserted DNA sequence of up to about 500 kb
• Prior to insertion of the foreign DNA, the essential
components of the YAC are maintained in bacterial cells as
circular plasmids.
Vecto
r for
other
highe
r
eukar
yotes
P element for Drosophilla
For mammals
Choice of Vector
Salient features of Host strain
• E.coli and Saccharomyces strain
• Common features for any host strain
• Nomenclature:
• Genes are given three-letter, lowercase, italicized names that are
often mnemonics suggesting the function of the gene.
• If the same function is affected by several genes, the different
genes are distinguished with uppercase italic letters, for example
recA,recB, recC, and recD all affect recombination. By convention,
E. coli genotypes list only genes that are defective, but the
superscript symbols “–” and “+” are occasionally used
redundantly for clarity or to emphasize a wild-type locus.
• Phenotypes are capitalized and the letters are followed by either
superscript “ +” or “ –,” or sometimes “ r”for resistant or “ s ” for
sensitive
• Although convention dictates that phenotypes are not specified in
the genotype designation, they are sometimes included, when not
easily inferred. For example, rpsL(Strr) indicates that a mutation
in the gene for ribosomal protein small subunit S12 confers
resistance to streptomycin.
• Specific mutations are given allele numbers that are usually italic
arabic numerals such as hsdR17. If the exact locus is not known,
then the capital letter is replacedby a hyphen, as in arg-3.
• An amber mutation is denoted by am following the gene
designation and
• a temperature-sensitive mutation that renders the gene inactive
at high temperature, is denoted by ts.
• A constitutive mutation is denoted by superscript q; thus lacIq
indicates constitutive expression of the gene for the lac repressor.
Method for Gene transfer in
prokaryote and eukaryote
and method of selection
Transformation
Selection of Transformants
Continued
The other type of insertional
inactivation
Transfection
Selection method
Gene transfer in eukaryotes
GENE LIBRARY
Overview of Cloning
Problem??

WE WANT TO ISOLATE A SINGLE

GENE FROM HUMAN CHROMOSOME
Genomic DNA librarys
• We wish to clone a single-copy gene from the human genome.
How might this be achieved?
• We could simply digest total human DNA with a restriction
endonuclease, such as EcoRI, insert the fragment into a suitable
phage-λ vector and then attempt to isolate the desired clone.
How many recombinants would we have to screen in order to
isolate the right one?
The concept
• Assuming EcoRI gives, on average, fragments of about 4 kb,
and given that the size of the human haploid genome is 2.8 ×
106 kb, we can see that over 7 × 105 independent
recombinants must be prepared and screened in order to
have a reasonable chance of including the desired sequence.
In other words we have to obtain a very large number of
recombinants, which together contain a complete collection
of all of the DNA sequences in the entire human genome, a
human genomic library.
Step 1
• There are two problems with the above approach.
• First, the gene may be cut internally one or more times by
EcoRI so that it is not obtained as a single fragment. This is
likely if the gene is large.
• Also, it may be desirable to obtain extensive regions flanking
• Problem:
• Fragments averaging about 4 kb are likely to be
inconveniently short.
• Alternatively, the gene may be contained on an EcoRI
fragment that is larger than the vector can accept
• Solution
• Random Cloning in lamda vector
Chromosome walking
• How many clones are required?
• Let n be the size of the genome relative to a single cloned
fragment. Thus, for the human genome (2.8 × 106 kb) and an
average cloned fragment size of 20 kb, n = 1.4 × 105. The
number of independent recombinants required in the library
must be greater than n, because sampling variation will lead
to the inclusion of some sequences several times and the
exclusion of other sequences in a library of just n
recombinants.
Method for Selection of clone
• The Problem in selection
Two Basic Strategy to obtain
clone
• Direct selection for the desired gene (Figure 8.2a), which
means that the cloning experiment is designed in such a way
that the only clones that are obtained are clones of the
required gene. Almost invariably, selection occurs at the
plating-out stage.
• Identification of the clone from a gene library (Figure 8.2b),
which entails an initial “shotgun” cloning experiment, to
produce a clone library representing all or most of the genes
present in the cell, followed by analysis of the individual
clones to identify the correct one.
Direct Selection
Limitation
• Direct cloning has limited application, only useful for
screening of Antibiotic resistant gene.
• For other purpose the mutant host strain is required for the
screening through Direct Selection.This method is known as
Marker rescue
• E.g. trp gene for tryptophan synthatase
• Limitation of Marker rescue
Marker Rescue
Concept of C-DNA Library
Method for Identification of
clone from Library
Colony hybridization
Plaque hybridization
Probe and its labeling
Labeling Methods
Probe and its labeling
Nonradioactive labeling of
probe
Southern Hybridization
Immunoscreening
Unit-4

APPLICATION AND LIMITATION OF

GENETIC ENGINNERING
Contents
• Applications- Production of Proteins from cloned genes
• Application of Genetic Engineering in Medicine
• Application of Genetic Engineering in Agriculture engineering
• Application of genetic engineering in forensic and archeology
Limitations:
Problem caused by host strain
Problem resulting form the sequence of foreign gene
Genetic engineering ethics
Medicine
• Through Cloning
• Synthesis of insulin
• Growth Hormone
• Recombinant vaccine
• Through Animal Pharming
Animal Pharming
Application in agriculture
• BT-cotton
Herbicide resistance crop
Terminator technology
Application in forensic
science
Sex determination
Archeogenetics
Random Mutagenesis
Gene shuffeling
Production of chimeric
proteins
• In protein production there are two aspects that require
optimisation:
• (1) the biology of the system and
• (2) the production process , Suitable engineering aspects
Native

Fusion
Importance of correct
reading frame
Expression system-
Eukaryotes
Protein engineering concepts
• Proteins that have been engineered by the incorporation of
mutational changes have become known as muteins
• Two Apparoches
• Rational Design
• Directed evolution
Rational Design
Error Prone PCR
DNA shuffling