Bacteriophage by reshma

Seminar
On
BACTERIOPHAGE VECTOR
Submitted
By RESHMA SHEIKH
M.Sc. III Semester (Botany)
GUIDED BY : HOD PRINCIPAL
Miss Pushplata Kanwar. Miss R. Kuldeep. Dr. Komal
Sarava
Mr. Amarnath Nishad.

* Bacteriophages are the viruses that infect bacterial cells after injecting
their genetic material.
(DNA or RNA) and kill them.
* The viral DNA or RNA replicates and expressed inside the bacterial cells,
and produce a number of phage particles released after bursting the
bacterial cell.
* This is called lytic cycle of bacteriophage.
* Bacteriophage are used as a phage vector in gene cloning.
* Phage are affect on Virus.
VIRUS – NUECLEO PROTEIN = NUCLIC ACID + PROTEIN
* This is a infection agent.
VECTOR – “Vector is a career. Vector is a type of DNA, in which
there is a desired or (DNA) insert present, which reappears by
entering the host cell.”
VECTOR + DNA = Recombinant DNA

PHAGE VECTORS
* Commonly E.coli Phage – Ex. l Phage and
M13 phage are use as phage vector.
* It can recombinant to large size of DNA.
* Large DNA insert cloning can be easy.
* Maximum 24 DNA inserted can be cloned in
lphage.
* Its selection and screening are easy.
 Some phage vectors:

1. l PHAGE
 This is a DNA virus.
 ori present for Replication .
 l -genome size is 48.5kb (kilobase pairs).
 Phage l contains a proteinaceous head and a
long tail attached to the head.
 There is a gene for the required enzymes for
DNA replication.

 There is gene for the Lysis and Lytic cycle.
 It has a single stranded cohesive protruding, which is
of 12 base sequence that is fixed.
 The 12 nucletide long projections
(5’GGGCGGCGACCT-3’) show cohesiveness and
form the Cos site (cohesive site).
 Phage genome has a larger non-essential region
which is not involved in cell lysis.
 Replication cycle of phage l is accomplished into two
pathways:
 Lytic

FIG. Relation of phage in E.Coli Cell.
A- lytic cycle; 1. Roling circle replication; 2. Production of concatemers;
3. Cleavage of cos site; 4. Transcriptionand translation; 5. Packaging;
6. Lysogenic cycle.

Types of l phage vector:-
 Insertion vector
 Replacement vector
Insertion vector :-
 Insertion vectors have unique cleavage site into which relatively small piece
of foreign DNA is inserted.
 Foreign DNA fragment does not affect the function of phage.
 The non essential part in the insertion vector is completely deleted.
 The upper and lower limits of size of DNA that may be packed into phage
into phage particles is between 35-53 Kb.
 The minimum size of vector must be above 35 Kb. It means the maximum
size of foreign DNA to be inserted is about 18 Kb.
 In this type of vector it should have at least one restriction site.
 Ex: l gt10, l gt11, l ZAPII


lgt10 :-
 Phage l is modified to construct lgt10 that clone
cDNA fragment.
 The lgt10 is a dsDNA of 43 kb which may clone
7kb long foreign DNA fragment.
 After inserting DNA, cl+(repressor) gene is
inactivated; therefore, cl- recombinant
bacteriophage is formed.
 The recombinant cl- lgt10 after infecting E. coil
forms clear plaques which can easily be screened
from cloudy plaques formed by non-recombinant
lgt10 cl+ vector.

FIG. Insertion cloning by using phage lgt10
vector

Replacement vector :-
 The replacement vectors have cleavage sites present on
either side of a length of non-essential DNA of phage.
 As a result of cleavage left and right arms are formed each
arm has a terminal cos site and longer a stuffer region, the
non-essential region, which can be substituted by foreign
DNA fragment.
 It has been found that about 25-30 Kb of genome codes for
essential products for lytic cycle.
 The remaining 20-25 Kb of genome could be replaced with
the foreign DNA fragments of known essential products.
 In this type of vector it should have at least 2 restriction site.
 Ex:- lWES, ls, lEMBL4, charons.

Charons:-
 In 1977, Blattner and cp-workers developed the
charon series insertion vectors.
 Charon 7 consists of imm434 and seves as an
insertion vector for both HindIII and EcoRI.
Whereas charon 12 contains lacZ gene. Insertion
of DNA into to single EcoRI will inactivate the lacZ
gene of charon 12.
 The active enzyme results in formation of blue
colonies or plaque, whereas after inactivation of
lacZ colourless colonies are formed.

FIG. Cloning of a replacement vector, A-
optimization of the suitable sized ligation products
for efficient packaging a phage.

M13 Phage Vector :-
 M13 is a filamentous phage.
 It is a single stranded DNA, that is called sense.
 It enters in the bacteria which has sex pili.
 F+ or HRr infect the E-coli bacteria.
 M13 phase has single stranded liner genome, after entering in host cell it changes
into double stranded circular replicative intermediated.
 M13 phage can deliver only 6.4 kb genome to host cell by F pili. Therefore;

 M13 genome’s double stranded forms are used to get recombinant
molecules, because single stranded DNA is not cleavage through type-II
restriction end nuclease. but to get the DNA purest’s single stranded copy it
uses M13’s single stranded DNA.
 M13 genome are used to create M13 mp series’ vector.
Ex:- M13mp8, M13mp9,etc
 M13mp series’ first vector is M13 mpI, this are build by inserting lacZ α gene
into non coding region of M13 genome, that are present beside the
Origin(ori).
 There is no unique restriction site in lacZ α sequence, but the Hex
nucleotide sequence are replaced by GGATTC through mutagenesis that are
present in starting of lacZ α gene to build GAATTC EcoRI site.
 By this way M12mp2 vector is build from M13mp1 that are used in a
selection of blue/white plaque present in x-gal.
 The extra unique restriction site are created by connecting synthetic
polylinker sequence in the lacZ gene.
 M13MP1 are created by connecting polylinker sequence in M13mp1’s
EcoRI site, in that EcoRI, BamH1, Salt and Pst1 cloning site are present.
 Latest M13 vector M13 mp8 has more complex polylinker.

FIG. M13 Phage Vectors :
(A) M13mp1
(B) M13mp2
(C) M13mp8
Properties of M13 vector-
* Very large DNA insert can be clone.
* Double stranded DNA insert are uses
more widely by single stranded copy.
* The infected bacteria cell are not destroy
Because of this vectors, maintenance is easy.
* They are creating plaque in the vector bacterial
lawn, so selection of recombinant DNA will be easy.

P1 Phage Vector :-
 Phage vector is used for cloning large DNA segment (about 1000 kb)
producing 105 clones per µg of DNA insert.
 On the basis of cloning efficiency and capability, it is put between YAC and
cosmids.
 Ex:- pNS%82tet14ad10(pAd10) and pAd10sacBII.

Conclusion :
 The use of Bacteriophage in cloning as a
vector.
 Large DNA fragment can be cloned by this.
 This is standard cloning vector.
 After replication isolation and screening is
easy.
REFERENCE :
(i) A text Book of Biotechnology - R. C.
Dubey.

Bacteriophage by reshma

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