BACTERIOPHAGE VECTORS

BACTERIOPHAGE

Virus that infect bacteria is known as bacteriophage.

It was discovered by Frederick W.Twort and Felix d’ Herelle .

D’ Herelle coined the term bacteriophage meaning ‘bacterial eater’ to describe the agent’s bacteriocidal activity.

Phages are very simple in structure , consisting merely of a DNA or occasionally RNA molecule carrying a number of genes , surrounded by a protective  coat or capsid made up of protein molecules.

They can undergo two life cycle

Lytic cycle – in this phages multiplies inside the host bacterium and new viral particle comes out by lysing or rupturing the host bacterial cell wall.

Lysogenic cycle – in this phages donot undergo multiplication or induce lysis but the viral DNA  get integrated into bacterial DNA without causing lysis.

Bacteriophage as a vector

Bacteriophage is used as vector because it can accept very large pieces of foreign DNA.

Genetic engineers have constructed numerous derivatives of phage vectors that contain only one or two sites for a variety of restriction enzymes.

Phage that have a stuffer fragment are called substitution vectors because they are designed to have a piece removed and substituted with something else.

Examples are lambda phage , M13 vector , T4 ,T7 phage etc.

M13 PHAGE

Bacteriophage M13 was isolated from wastewater in Munich . Hence named as M13 phage.

It is a filamentous phage which has 6407 nucleotides.

It possess single stranded circular DNA.

It was sequenced by Sanger in 1982.

CONSTRUCTION OF M13 AS PHAGE VECTOR

The first step in construction of an M13 cloning vector was to introduce the lacZ gene into the intergenic sequence.

This give rise to M13mp1 , which forms blue plaques on X –gal agar.

M13mp1 does not possess any unique restriction sites in the lacZ’ gene.

M13mp2 VECTOR

It contain the hexanucleotide GGATTC near the start of the gene.

A single nucleotide change would make this GGATTC , which is an EcoR1 site.

This alteration was carried out using in vitro mutagenesis resulting in M13mp2.

M13mp7 VECTOR

A polylinker , which consists of a series of restriction sites and has EcoR1 sticky ends.

This polylinker was inserted into  EcoR1 site of M13mp2 , to give M13mp7 a more complex vector with four possible cloning sites (EcoR1 , BamH1 , Sal1 and Pst1).

The polylinker is designed so that it does not totally disrupt the lacZ gene . Although it is altered , b – galactosidase enzyme is still produced.

SELECTION OF RECOMBINANTS

The vector is then inserted into a competent host cell viable for transformation , which are then grown in the presence of X-gal.

Cells transformed with vectors containing recombinants will produce white colonies ; non – recombinant plasmids grow into blue colonies.

LAMBDA PHAGE VECTORS

The lambda phage particle contains two major structural components called a head and tail.

The lambda genome is a double stranded DNA molecule of 48,503 bp that is packaged within the phage head . The tail proteins are required for phage attachment to E.coli outer membrane and injection of phage DNA into the host cell.

At each 5’ End of lambda genome there is a single stranded region of 12 bases in length. These single stranded regions are complementary to each other and are able to pair with one another and are therefore referred to as cohesive ends sites . After injection of DNA into the cell , the cohesive ends pair together by hydrogen bonds to form circular DNA molecule.

Advantages of lambda vector

The size of cloned DNA is relatively large ( 9 -25 kb).

The multiplication of cloned DNA is very easy by infecting a suitable host with bacteriophages containing recombinant DNA .

There is direct selection of clones containing recombinant DNA during in vitro packaging.

Storage of clones is easy.

Lambda gt10 and lambda gt11

These are inserton vectors . These are modified lambda phages designed to clone cDNA fragments .

Lambda gt10 is a 43kb double stranded DNA for cloning fragments that are only 7 kb in length . The insertion of DNA inactivates the cl (repressor) gene, generating cl- bacteriophage . Non recombinant lambda gt10 is cl+ and forms cloudy plaques on appropriate E.coli host , whereas recombinant cl- lambda gt10 forms clear plaques . Thus recombinant lambda gt10 plaques can be easily selected.

Lambda gt11 is a 43.7kb double stranded phage for cloning DNA segments that are less than 6 kb in length . Recombinant lambda gt11 can be screened using either nucleic acid or antibody probes . The recombinant lambda gt11 becomes gal- whereas non recombinant lambda gt11 remains gal+ so that an appropriate E.coli host with recombinant phage will form white or clear colonies . Non recombinant phage gal+ form blue colonies thus permitting screening in the presence of IPTG and X-GAL.

LAMBDA  REPLACEMENT  VECTORS

A replacement vector has two recognition sites for the restriction endonuclease used for cloning . These sites flank a segment of DNA that is replaced by the DNA to be cloned.

Often the replaceable fragment or stuffer fragment in cloning carries additional restriction sites that can be used to cut it into small pieces , so that its own re-insertion during cloning is very unlikely.

Replacement vectors are generally designed to carry larger pieces of DNA than insertion vectors.

An example of replacement vectors is ;

LAMBDA EMBL4 can carry upto 20kb of inserted DNA by replacing a segment flanked by pairs of EcoR1 , BamH1 and Sal1 . Any of these three restriction endonucleases can be used to remove the stuffer fragment , so DNA fragments with a variety of sticky ends can be cloned .

EMBL 3 &  EMBL4 have polylinkers with reverse orders of restriction sites with respect to each other.

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