BACTERIOPHAGE

BYK.PRAVEEN KUMAR REDDY

13191A23223RD BIOTECHNOLOGY

JNTUACEP

BACTERIOPHAGE• VIRUS THAT INFECT BACTERIA IS KNOWN AS

BACTERIOPHAGE• IT WAS DISCOVERED BY FREDRICK.W.T.WORT IN

GREAT BRITIAN (1915) AND FELIX D’HERELLE IN FRANCE(1917).

• D’HERELLE COINED THE TERM BACTERIOPHAGE MEANING ‘BACTERIAL EATER’ TO DESCRIBE THE AGENT’S BACTERIOCIDIAL ACTIVITY.

• PHAGES ARE VERY SIMPLE IN STRUCTURE,CONSISTING MERELY OF A DNA OR OCASSINALLY RNA MOLECULE ARRYING NUMBER OF GENES COATED MADE UP OF PROTEIN MOLECULES.

INTRODUCTION

•A CLONING VECTOR IS DNA MOLECULE IN WHICH FOREIGN DNA CAN BE INSERTED ARE INTEGRATED AND WHICH IS FURTHER CAPABLE OF REPLICATING WITH IN HOST CELL PRODUCE MULTIPLE CLONES OF RECOMBINANT DNA.

•EXAMPLES: PLASMIDS, PHAGE OR VIRUS

TYPES:•PLASMID AS VECTOR.

•BACTERIOPHAGE AS VECTOR.

•COSMID AS VECTOR.•PHAGEMID AS VECTOR.

THEY CAN UNDERGO TWO LIFE CYCLE

•LYTIC CYCLE.•LYSOGENIC CYCLE

WHY BACTERIOPHAGE AS A VECTOR?

• 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 SUFFER FRAGMENT ARE CALLED “SUBSTITUTION VECTORS “ BECAUSE THEY ARE DESIGNED TO HAVE PIECE REMOVED AND SUBSTITUTED WITH SOMETHING ELSE.

• EXAMPLES: ARE LAMBDA PHAGE ,M13 PHAGE,T4,T7 PHAGE,P1PHAGE ETC.

Bacteriophage vectorTow examples:H2-1 λ phage bacteriophageλ λ replacement vector H2-2 M13 phage M13 phage vector Cloning in M13 Hybrid plasmid-M13 vectors

Cloning vectors

• viruses that can infect bacteria. •48.5 kb in length•Linear or circular genome (cos ends)

λ phage

Lytic phase (Replicate and release)

Lysogenic phase (integrate into host genome)

H2 Bacteriophage vector

DNAProtein coat

cos cosNonessential region

Long (left)arm

short (right)arm

Exogenous DNA(~20-23 kb)

H2-1λ phageH2 Bacteriophage vector

5‘-CGGGGCGGCGACCTCG-3’3’-GCCCCGCCGCTGGAGC-5’

Cleavage Ligation(during packaging) (after infection)

GGGCGGGCGACCTCG-3’5’-CG + GC-5’3’-GCCCCGCCGCTGGA

The phage λ cos ends

Circular form

Linear form

H2 Bacteriophage vector

λ phage

H2 Bacteriophage vector

λ replacement vector• Replace the nonessential

region of the phage genome with exogenous DNA (~ 20 kb)

• high transformation efficiency (1000-time higher than plasmid)

H2 Bacteriophage vector

λ replacement vector2. Packing with a

mixture of the phage coat proteins and phage DNA-processing enzymes

1. Ligation

3. Infection and formation of plaques

H2 Bacteriophage vector

Plaques: the clear areas within the lawn where lysis and re-infection have prevented the cells from growing.

Recombinant DNA may be purified from phage particles from plaques or from liquid culture.

H2 Bacteriophage vector

Genes or foreign sequences may be incorporated essentially permanently into the genome of E.coli by integration of a vector containing the sequence of interest.

lysogens in cloning techniques

H2 Bacteriophage vector

• A filamentous phage• Phage particles contain a 6.7kb circular single strand of DNA.•After infection of a sensitive E.coli host,the complementary strand is synthesized,and the DNA replicated as a double-stranded circle,the replicative form(RF) with about 100 copies per cell. •Contrasting to phage ,the cell are not lysed by M13,but continue to grow slowly,and single-stranded forms are continuously packaged and released from the cells as new phage particles.

H2-2 M13 phageH2 Bacteriophage vector

M13 phage vectors1. Replication form (RF, dsDNA) of M13

phage can be purified and manipulated like a plamid.

2. Phage particles (ssDNA): DNA can be isolated in a single-stranded form

• DNA sequencing (Topic J2)• Site-directed mutagenesis (Topic J5)

Cloning (RF, like plasmid) transfection (recombinant DNA) growth (plating on a cell lawn) plaques formation (slow growth)

H2 Bacteriophage vector

Blue-white selection

• Small plasmid vectors being developed to incorporate M13 functionality• Contain both the plasmid and M13 origin of replication• Normally propagate as true plasmids• Can be induced to form single-stranded phage particles by infection of the host cell with a helper phage.

Hybrid plasmid-M13 vectors

H2 Bacteriophage vector

THANK

YOU