Biotechnology : A general account of Cloning Vectors

2. Cloning Vectors
 Vectors / Vehicle DNA
 DNA molecules that can carry a foreign DNA
fragment to be cloned into a recipient cell.
 Stable, self replicating DNA molecule to
which a foreign DNA fragment can be
attached for introduction into a cell.
 Should be small, ideally less than 10
kilobase (1 Kilobase – 1000 bp) [ bp = base
pairs]

3. Vector has
an origin of replication (ori) –
ensure the replication of vector
within the cell
selectable markers (Antibiotic
resistant genes ) – enable selection
of cell containing vector.
 one or more unique restriction sites
(Cloning sites) into which a DNA

4. Types of Cloning Vectors
1. Plasmids
 Extra chromosomal, small, circular, self
replicating DNA in the cytoplasm of Bacteria.
 Has a few (<30) genes – useful but not essential-
these genes give some additional properties like
 Antibiotic & heavy metal resistance
 Nitrogen fixation (e.g. Rhizobium
leguminosarum)
 Pollutant degradation
 Toxic production etc.

5. Types of Plasmids
 Single copy/ low copy/ stringent – one plasmid in a
cell.
 Multicopy / high copy number/ relaxed – 100 or
more copies.
 Conjugative / self transmissible – carry transfer
genes (tra genes) – enable them to transfer from
one bacterium to another.
 Non conjugative plasmids – do not carry transfer
genes – replicate autonomously but can not
transfer to another bacterium.

6.  Plasmids – used as vectors in Genetic
engineering
 Stanley Cohen et. al., (1973) first reported the
use of bacterial plasmids as molecular cloning
vectors.
 Modified to have selectable marker genes-
antibiotic resistance genes.

7.  Multiple cloning sites ( Poly linker region) –
has a number of unique target sites (unique
restriction sites) for restriction
endonucleases.
 Circular plasmid vector is cut with a
restriction enzyme linear plasmid.
 Foreign DNA (insert) cut with the same
enzyme can be joined to the vector by a
ligation reaction.

8. E.coli. Plasmid vector pBR 322
( P – Plasmid
B – Boliver
R – Rodriguez
322 – Strain number )
 Most commonly used multicopy cloning vector –
“work house” of a gene cloning laboratory.
 Small sized – 4363 bp – allows addition of a further
6 Kb fragment of DNA and facilitates entry into
cells.
 Has an origin of replication (of E. coli plasmid Col
E 1) – required to propagate the plasmid and

9.  Unique restriction sites for –
 Eco RI ( from E. coli)
 Bam HI ( from Bacillus amyloliquefaciens H)
 Sal I (from Streptomyces albus G)
 Pvu I ( from Proteus vulgaris)
 Pst I (from Providencia stuartii)
 Has two selectable marker genes that give
resistance to antibiotics tetracycline (tet R) and
amplicillin (amp R) – help for the selection and
identification of clones carrying the plasmid.

10. pBR 322

11. 2. Bacteriophage based vectors
 Filamentous bacteriophages with single stranded DNA
 M 13
 fd
 Bacteriophage with double stranded DNA
 Lambda phage ( phage)
 High effieciency in transferring DNA into bacterial cells
(E. coli.)
 Most commonly used
 Lambda phage genome – 48502 bp.
 Contain several non essential regions which can be
replaced with almost equal length of foreign DNA.

13. About 15 kb of foreign DNA can be
incorporated in a Lambda phage –
resulting r DNA can be packaged in
phage heads in vitro. Phage particles
can inject the r DNA into E.coli 
Replication  clones of rDNA.

14. 3. Cosmids
 Used as vectors in genetic engineering.
 Can be used to clone DNA inserts of up to 45 kb.
 Constructed using r DNA technology
 They are essentially plasmids that contain a
minimum of 250 bp of Lambda phage DNA which
includes –
 Cos site – sequence yielding cohesive ends.

15.  Sequences needed for binding of and cleavage by
terminase so that under appropriate conditions
they are packaged in vitro into empty Lambda
heads.
 Has origin of replication
 Unique restriction sites.
 Selectable markers from the plasmid – selection of
recombinant vector is based on the procedure
applicable to the plasmid making up the cosmid.
 E.g. C2RB , p JB 8

16. Cosmid vector C2RB
• Size - about 6.8 kb .
• Has the origin of replication of col EI
derived from pKC 7 plasmid.
• It has 2 cos sites derived from MUA -3
vector.
• Has selectable marker genes amp R and
Kan R
• Has unique restriction sites for Bam HI,
Eco RI, Cla I and Hind III .
• Can be used for cloning DNA fragments of

17. Cosmid vector pJB8
• Size – about 5.4 Kb
• Has an origin of replication of colEI
plasmid
• Has a single cos site derived from
charon 4A.
• Restriction sites for Bam HI, EcoRI,
ClaI and HindIII .
• Has selectable marker gene amp R

18. 4. Phagemid Vectors
 Have components of phage chromosomes and plasmids
 Origin of replication of a plasmid (Col E1 plasmid)
 Origin of replication of a filamentous phage (M13)
 Multiple cloning sites (a polylinker)
 A marker gene
Examples
pUC 118
pUC 119 p – Plasmid
UC – University of California where they are
constructed .

19. Source: www.creative-biogene.com
Phagemid vectors pUC 118
pUC 119

20. Uses of phagemid vectors pUC 118, pUC 119
1. Cloning vector for making double – stranded
copies of DNA insert
• Can replicate as double – stranded plasmids in
the absence of helper phage (Replication is
controlled by the plasmid ori – origin of
replication )
• The DNA to be cloned is inserted through the
multiple cloning sites and the phagemid is then
inserted into a bacterium (transformation).

21. 2. For making single stranded copies of
DNA insert
• Phagemids pUC 118 and pUC 119 can replicate as
single stranded DNAs which are packaged in M13
phage coats and extruded from the cell. (through
the cell membrane and cell wall without killing the
host cell) in presence of a helper phage (Replication
is controlled by the phage M 13 Ori. The phagemid
carrying bacteria are infected with a helper phage
M13 which promotes replication from the phage
origin in the phagemid). When freed from the
phage coat proteins, the single stranded copies of
the DNA insert can be used – (eg: for sequence
analysis).

22. Colour Test for selection of transformed
cells by using pUC vectors
 Non-transformed cells – Appear as blue colonies on
agar medium containing a colourless substrate X-
gal (5-bromo-4-chloro-3-indolyl- galactoside).
 The vector has E.coli lac Z gene  -galactosidase
cleave X-gal  blue coloured 5-bromo-4-chloro
indigo and galactose.
 Transformed cells – Appear as white colonies due to
functional inactivation of the 5’segment of the lac
Z-gene because of insertion of foreign DNA into the
polycloning region  lack of  - galactosidase
activity.

23. COLOUR TEST FOR SELECTION O F TRANSFORMED
CELLS BY USING pUC VECTORS
NON-TRANSFORMED CELLS – APPEAR AS BLUE COLONIES
TRANSFORMED CELLS – APPEAR AS WHITE COLONIES

24. 5. Ti Plasmid (Tumour inducing plasmid,
pTi)
 Ideal vectors for plant genetic engineering
 Plasmid in crown gall causing, soil bacterium,
Agrobacterium tumefaciens  Natural plant
genetic engineer –
 Contain genes for the transfer, stable
incorporation and expression of genetic
information in plants –
 A large double stranded circular DNA (200 Kb)

25.  Has a sequence for
 Replication
 Transfer of plasmid from one organism to
another
 Transferred DNA (T-DNA) induce crown gall
(tumour)
 Production of opines – unusual amino acid (a
derivative of Arginine)
 Virulence (virulent gene – Vir) – transfer of T
– DNA into host cell
 Oncogenicity  tumour growth

26. Ti Plasmid
Image: https://www.biologyexams4u.com/

27. 6. Shuttle Vectors
 Created by recombinant techniques
 Designed to replicate in cells of two different
species – contain two origins of replication, one
specific for each species. (two different
prokaryotic species/ one prokaryotic species
(E.coli) and another eukaryotic sps (yeast,
plant, animals).

28. Yeast Shuttle Vector
Image: https://www.biologyexams4u.com/2014

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