about Natural and Artifical vectors

1. PLASMID AS VECTORS
IN
GENETIC ENGINEERING
JINAL A. MEHTA
M.Sc. SEM-I

2. CHARACTERISTICS OF PLASMID
VECTOR :
1. Small Size
2. Autonomous Replication
3. Presence Of Selectable Marker Genes
4. Presence Of Unique Restriction Enzyme Site
5. Nonconjugated & nonmobilizable
6. Replicas Under Relaxed Control

3. I. NATURAL PLASMID VECTORS FOR
E.coli :
i. pSC101
ii. pSF2124(RSF2124)
iii. Col E1 Plasmid
II. ARTIFICAL CONSTRUCTED PLASMID
VECTORS FOR E.coli :
i. pBR322
ii. pUC series
iii. pGMR

4. I. pSC101
• First used for in vitro coloning of eukaryotic DNA.
• 9kbs size.
• Low copy number(1-2 copies).
• Has ADVANTAGE of a single Eco RI site at which DNA
be inserted.
• Has selectable marker for tetracycline resistance.
• Derived from the conjugative plasmid R6-5.
DISADVANTAGE :
• Large size
• Stringent replicative control
• Low copy number
• Low insert capability

5. II. pSF2124(RSF2124)
 Produced by transfer of the ampicillin resistance
gene.
 Has ability for colicin biosynthesis as well as
ampicillin resistance.
 Has high copy number.
 Mobilizable plasmid
 Has single site fro Bam H1 & EcoR1
 Not currently used as vector as it does not provide
easy selection by insertional inactivation

6. III. Col E1 Plasmid
• Small circular colicingenic plasmid.
• Size is 6,466bp
• Codes for 57kDa protein toxin that can
kill other E.coli cells by depolarizing the
bacterial membrane.
• Give 1000 – 3000 copies.

7. imm Immunity protein
kill Lysis protein
inc RNA I incompatibility determinant
rop/rom Protein , That regulates priming and
copy number
mob Proteins for mobilization during
conjugation
cer Maintain plasmid as monomers
exc Exclusion protein
cea Colicin toxin
GENE FUNCTION

9. I. pBR322
• pBR322 is plasmid and was the first widely-used E.coli
cloning vector .
• Created in 1997 , it was named after its Mexican creators
, p standing for plasmid & BR for Bolivar and Rodriguez .
• pBR322 is 4,361 bp in length and contains a replicon
region (source plasmid pMB1).
• The ampR gene , encoding the ampicillin resistance
protein (source plasmid RSF2124).
• The tetR gene , encoding the tetracycline resistance protein
(source plasmid pSC101).

10. R7268
ampr
R1 drd19
ampr cmr
smr sur
pMB1
pMB3
ampr
pBR312
ampr tetr
pSF2124
ampr
ColE1
pSC101
tetr
pBR322
ampr tetr
pBR313
ampr tetr
pMB9
tetr
pMB8
1
2
3
4
5
6
7
8
GENEOLOGY OF
PLASMID
pBR322

11. • Has unique restriction site for
more than 40 restriction enzyme.
• 11 of these 40 sites lie within the
tetR gene.
• Has 2 sites for restriction enzyme
HindIII and Clal within the
promoter of the tetR gene.
• Six key restriction sites inside the
ampR gene.
• The origin of replication or ori
site in this plasmid is pMB1(a close
relative of ColE1).
• The ori endoes two RNAs (RNAI
and RNAII) and one protein (called
Rom or Rop ).

13. DERIVATIVES OF pBR322
 pBR324
 pBR325
 pBR327
 pBR328
 pBR329
 pAT153

15. II. pUC series
 pUC series are derived
from pBR322 .
 This work done by messing
and co-workers in the
university of
California(1982).
 The p in its name stands
for plasmid and UC
represents the university in
which it was created.
 It is a circular double
stranded DNA and has
2686 bp.

17.  It has one ampR gene (ampicillin resistance gene).
 Has an N-terminal fragment of -galactosidase (lac Z)
gene of E.coli.
 The multiple cloning site (MCS) region is split into the lac
Z gene (Codons 6-5 of lac Z are replaced by MCS), Where
various restriction site for many restriction endonucleases
are present .
 The ori site or replicon , rep is derived from pMB1 vector.
 pUC vector is small but has a high copy number. The high
copy number of pUC plasmid is a result of the lack of the
rop gene and a single point mutation in rep of pMB1.
 The lac z gene codes for -galactosidase

18. pGEMR
• The pGEM-3Z vector is intended for use as a standard cloning vector
, as well as for the high efficient synthesis of RNA in vitro.
• The vector carries the lacZ -peptide and the multiple cloning
region arrangement from pUC18 allowing selection of recombinants
by blue/white screening.
• In addition , the vector contains both the SP6 and T7 RNA
polymerase promoters flanking the multiple cloning region.
• The pGEM-3Z and pGEM-4Z vectors are essentially identical except
for the orientation of the SP6 and T7 promoters.

20. • The vector is very similar to a pUC vector and is almost exactly the same
size.
• It contains the following functional modules
i. Ampᴿ
ii. Col E1 origin
iii. Lac Zα sequence
iv. MCS within the Lac Zα
v. Promoter for T7 phage RNA polymerase (located at one end of Lac
Zα)
vi. Promoter for SP6 phage RNA polymerase (located at another end of
Lac Zα)
• The T7 and SP6 promoters are used by T7 & SP6 RNA polymerases,
respectively, in separate in vitro conditions to produce RNA transcripts of
two strands of the DNA insert cloned within the MCS.
• T7 & SP6 RNA polymerases are very active enzymes and are able to
synthesis 1-2mg of RNA per minute.
• The RNA copies so obtained can be used as hybridization probes or used for
studying RNA processing or for protein synthesis.