INSERTION VECTORS

• Bacteriophage

• Double stranded DNA virus

• that can be engineered to serve as a receptor for foreign DNA fragments in recombinant DNA experiments

• 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

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.

Bacteriophage lambda (l) infects E. coli

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

• 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

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

• 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

• 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

• 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

• 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).

• 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

• 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.

• 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

Group1

• Anesu Chikaka

• Tatenda Gunda

• Ngonidzashe Dedza

• Liberty Machonisa

• Tanyaradzwa Ngara