bacteriophage M13& lambda by Praveen jntuacep

1. BACTERIOPHAGE
BY
K.PRAVEEN KUMAR REDDY
13191A2322
3RD
BIOTECHNOLOGY
JNTUACEP

2. 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

3. 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

4. TYPES:
•PLASMID AS VECTOR.
•BACTERIOPHAGE AS
VECTOR.
•COSMID AS VECTOR.
•PHAGEMID AS VECTOR.

5. THEY CAN UNDERGO TWO
LIFE CYCLE
•LYTIC CYCLE.
•LYSOGENIC CYCLE

6. 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.

7. Bacteriophage vector
Tow 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

8. • 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

9. DNAProtein coat
cos cosNonessential region
Long (left)
arm
short (right)
arm
Exogenous DNA
(~20-23 kb)
H2-1λ phage
H2 Bacteriophage vector

10. 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

11. λ phage
H2 Bacteriophage vector

12. 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

13. λ replacement
vector
2. 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

14. Plaques: the clear areas within the lawn
where lysis and re-infection have prevented
the cells from growing.
Recombinant λ DNA may be purified from
phage particles from plaques or from liquid
culture.
H2 Bacteriophage vector

15. Genes or foreign sequences may be
incorporated essentially
permanently into the genome of
E.coli by integration of a λ vector
containing the sequence of interest.
λ lysogens
in cloning techniques
H2 Bacteriophage vector

16. • 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 λ,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 phage
H2 Bacteriophage vector

17. M13 phage vectors
1. 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

18. Blue-white selection

19. • 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

20. THANK
YOU