Bacteriophage lambda is a double-stranded DNA virus that can be engineered as an insertion vector for cloning foreign DNA fragments. Lambda has a 48.5 kb genome that is linear with single-stranded overhangs. Insertion vectors like GT10 and GT11 contain a unique restriction site, like EcoRI, within a gene where foreign DNA can be inserted. This inactivates genes required for lysogeny, allowing selection of recombinant phage by plaquing on lysogenic E. coli strains. Larger DNA fragments can be cloned using substitution vectors.

1. INSERTION VECTORS

2. • Bacteriophage
• Double stranded DNA virus
• that can be engineered to serve as a receptor
for foreign DNA fragments in recombinant
DNA experiments

3. • Bacteriophages are viruses that infect
bacteria. Bacteriophages can have a protein
"tail" attached to the capsid (protein coat that
envelopes the genetic material), which is used
to infect the host bacteria.
• The genetic material can be ssRNA, dsRNA,
ssDNA, or double-stranded DNA between 5
and 500 kilo base pairs long with either
circular or linear arrangement

4. Essential features
• The DNA of phage λ is a linear duplex
molecule of about 48.5 kb
• At each end are short single-stranded 5′
projections of 12 nucleotides,
• are complementary in sequence
• the DNA becomes a circular structure when
injected into host cell,
• λ DNA naturally has cohesive termini, which
associate to form the cos site.

5. Double-stranded, linear DNA vector –
suitable for library construction

6. • Lambda is the most common phage. The lambda
genome is 48.5Kb in length and contains about 46
genes.
• It has an EcoR1 site inserted within the c1
gene(lambda repressor) and so could accept a DNA
frag of 7.6kb before becoming 2 large to be
packaged into virus particle.
• There are two main types of lambda vectors;
insertion vectors and replacement vectors

7. λ insertion vector
• In this vector part or all of the optional DNA has been
removed and a unique restriction site introduced at same
position within the trimmed down genome.
• This vector is used for cDNA cloning and expression.
• example- GT10, GT11, Zap

9. • 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)
• Since insertion vectors are large enough to be
packaged into viable viruses, it is useful to have a
• selection system that would allow us to identify
recombinant phage containing foreign DNA
inserts from non-recombinants containing only
vector sequences

10. • Lambda gt10 and gt11 are examples of
insertion vectors.
• Gt10 has a unique EcoR1 site, within the c1
repressor gene and can accept inserts up to
7.6kbp in size.
This is a bit larger than inserts of wild type
phage would be able to accept because there is
a small deletion elsewhere in the genome of
gt10

11. • For Lambda gt 10
• For lambda insertion vectors, this selection system is
based on the choice between lytic and lysogenic life
cycles.
• the phage genome choses between these alternatives
based on the competition between positive and
negative transcription factors expressed during the
immediate early phase of the life cycle.
• The negative transcription regulator is the lambda
repressor
• The lambda repressor acts to shut off lambda
transcription.
• Insertion of foreign DNA into the cI coding sequence
therefore inactivates this negative regulator and forces
all recombinant phage to replicate via the lytic cycle.
• Non-recombinants can follow either the lytic or the
lysogenic pathway

12. • E. coli containing a lambda provirus (a lambda
lysogen) are immune to subsequent phage infection
and so can grow in the presence of the virus.
• This results in a 'cloudy plaque' morphology (cloudy
appearance is due to the presence of lysogenic
bacteria that continue to grow within the plaque).
• Recombinant phage carrying a foreign DNA insert
are unable to lysogenize (no negative regulator) and
therefore have a 'clear plaque' morphology (no
lysogenic hosts growing within the plaque).

13. • For Lambda gt 11
• Lambda gt11 contains a lacZ gene with a unique
EcoR1 site
• is an expression vector where DNA is expressed as
β-galactosidase fusion protein
• Removal of the lacZ gene results in no beta-
galactosidase being produced.
• Recombinant phage will form white colonies and
non-recombinant phage will form blue colonies in
the presence of X-gal and IPTG( which is an inducer)
• recombinant λgt11 can be screened using antibody
probes

14. • a selection system that allows only recombinant phage to
grow can be used.
• Is the identification of E coli Hfl strains (Hfl = High frequency
lysogeny)
• which affect the lambda lytic-lysogenic decision.
• Instead of 99.9% of all phage infections following the lytic life cycle
and 0.1% following the lysogenic pathway, infection of an Hfl strain
results in 99.9% of infections following the lysogenic pathway and
only 0.1% following the lytic pathway.
• Infection of an Hfl host with a mixture of recombinant and non-
recombinant insertion phage (where insertion inactivates cI - the
lambda repressor) results in progeny viruses that are exclusively
recombinants. All non-recombinant phage go lysogenic and
produce no viral progeny.

15. • The limitation to using lambda insertion vectors is
their small insert size capacity. The analysis of
transducing phage suggested that as much as 40%
of the wild-type genome is dispensible for lytic
growth. However viral genomes only 60% of the
wild-type length aren't packaged into viable phage
particles. Therefore, in order to utilize the full
carrying capacity of the lambda vector,
• substitution phage vectors were developed

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