The document discusses the plasmid vector pBR322, which was constructed in 1977 and is one of the most commonly used cloning vectors. It describes the origins and components of pBR322, including two antibiotic resistance genes, the origin of replication, and restriction enzyme cleavage sites. The document also summarizes the construction of several derivatives of pBR322, including pBR327, pUC18, and pBR118/119, and notes their applications and advantages over the original pBR322 vector.

1. DERIVATIVES OF pBR322
By
Ankur Sharma

2. Vector pBR322
 This artificial vector was
constructed by Bolivar and
Rodriguez in 1977 .
 It is one of the most popular
and commercially used early
purpose built plasmid based
vector.

3.  Its size is 4361bp(Watson 1988)
 Contain two antibiotic resistance genes: –
a) Ampicillin resistance gene (ApR)
b) Tetracycline resistance gene (TcR)
 These genes were taken from plasmid RSF2124 and pSC101
respectively.
 Origin of replication from plasmid pMB1(ColE1 like plasmid)
Basics

5.  Its total genome has been sequenced in 1979.
 It contain unique restriction sites for >40 different restriction
endonucleases.
 Out of these, 11 are present within tetracycline resistant
gene(TcR ) two for ClaI and HindIII within the promoter of that
gene.
 Ampicillin resistant ApR gene contain unique cleavage sites for
6 restriction enzymes
 If any of these 19 R.E. is used for inserting the desired DNA
fragment then it will result in insertional inactivation of that
particular gene(either Ap or Tc)
Cont….

7.  Use of any RE out of 19 different RE will result in insertional
inactivation of that particular antibiotic resistant gene and it
will no longer remain functional.
 For eg. If it is inserted in TcR
gene then the cells containing it
will be resistant to ampicillin but sensitive to tetracycline.
 Transformed cells are selected on the basis of their ApR
and
then replica plated onto a Tetracyclin containing media.
Selection of transformed cells and the cells
containing rDNA :-

8.  Cells containing DNA insert in their TcR gene are sensitive to
it so unable to grow in its presence and only those cells
containing recircularized vector will survive on this medium
and the cells containing r-DNA can be recovered from master
plate.
Importance :-
 Model system for study of prokaryotic transcription and
translation.
 Investigation of the effects of topological changes on DNA
conformation.
Cont….

9. L’[;
pBR322 construction
Plasmid R7268 (R1) was
isolated
Variant of this plasmid
R1drd19 isolated
Tansposon Tn3 was
transported to pMB1 to
form pMB3

10. Due to EcoR1 rearrangement formation of pMB8
(carries colicin immunity)
combination of EcoR1 fragments from pSC101
with pMB8 (opened at its unique EcoR1 site) to
generate pMB9
*pMB9 code for both tetracycline resistance
and colicin immunity
Cont….

11. Transfer of Tn3 of R1drd19 to ColE1 to form pSF2124
Transport of Tn3 from pSF2124 to pMB9 to form
pBR312
EcoR1 rearrangement results in formation of pBR
313
Isolation of two fragments and their ligation to
form pBR322
Cont….

12. k
Generation of plasmid pBR322 from pBR313

13.  The phenotype of plasmid pBR313 is ApRTcR Colimm
 It contain three restriction sites of R.E. Pstl , one of them is
located within ampicillin resistant gene.
 In order to eliminate the two other restriction sites and to
maintain the ApR in functional state two new plasmid had to
be constructed. These are-
1. pBR318
2. pBR320
Cont….

14. Construction of pBR318:-
pBR313 was digested with Pstl
E.Coli strain RRl was transformed with these
three pBR313 Pstl fragments and selected for
tetracycline resistance
Two small fragments lost.
pBR318 (tetracycline-resistant but
sensitive to ampicilline)

15. Construction of pBR320 :-
pBR313 was digested with Eco Rll
Bacteria were transformed with unligated Eco Rll
fragment of pBR313
Ampicillin resistant cells were selected and
screened for tetracycline and colicin sensitivity
Plasmid pBR320(containing R.S. for Pstl within
amp. Resistant gene.)

16. Formation of pBR322 from pBR318 and pBR320 :-
pBR318
digested with Pstl and
Hpal simultaneously
Two fragments –
1.95Da and 2.2×106Da
Tetracycline resistance
gene and a part of amp.
Resistance gene.
pBR320
Digested with Pstl
and Hincll
3 fragments
largest
Origin of replication
and remaining part of
amp. Resistance gene.

17. Transformation of mixture in
E.Coli strain RRl
pBR322
Cont….

18. Construction of pBR327 from pBR322 :-

19. pBR322 contain 6 restriction sites for EcoRll and a long non
essential region flanked by two EcoRll cleavage sites at position
1442 and 2502 (between origin of replication and tetracycline
resistance gene)
partial digestion with EcoRll
The two EcoRll 1442(CCTGG) and 2502(CCAGG) differ so
resulting non complementary ends had to be trimmed with
nuclease S1
Gel electrophoresis
Desired 3.3 Kb DNA fragment isolated, circularized with T4
DNA ligase
transformation
Cont….

20. Isolation of pBR327
Advantages :-
 Small size(1089bp less than pBR322)
 It is a EK2 vector and lacks mobilization function.
Cont….

21. pUC18/19 vector :-
 Size – 2.69 Kb
 They are identical but
differ in orientation of
MCS (opposite)
pUC18/19 contain :
1. pMB1 replicon rep
for replication
Source – pBR322

22. 2. Bla gene code for beta lactamase
Resistance to ampicillin
High copy no. due to lack of rop gene and single
point mutation in this gene.
encode promoter and α- peptide of β-
galactosidase
3. lacZ’ sequence
Cont….

23. i4. MCS(Multiple Cloning Site) or Poly linker sequence
Short DNA sequence (2.8Kb in pUC19) carrying R.S. for
different R.E.
 Use of MCS increase the no. of potential cloning
strategies available by extending the range of enzymes.
 The usual site for insertion is between initiator ATG
codon and codon 7
Cont….

24. s

25. Selection of recombinant cells
 For selection of recombinant cells blue-white screening is
used.
 Colonies are cultured on medium containing X-gal (5-Bromo-
4-chloro-3-indolyl-β-D-galactoside) and IPTG is used as inducer.

27. pUC 118/119
 Phagmid based ds DNA vectors
which are derivatives of pUC
18/19.
 Size – 3162bp
 Constructed by insertion of
intergenic region of M13 phage
DNA into pUC18/19.
 Infection by helper phage
M13K07 induce the production of
vector as ssDNA.

28. Function :-
 Cloning, sequencing, oligo-nucleotide directed mutagenesis,
strand specific probe synthesis.
 Host - E.coli JM103, JM105, JM110.

29. se
Some other important vectors :-
pAT153 :-
• It is a dsDNA plasmid based vector
• Size – 3658bp
• Constructed by deletion of two Hae ll
fragments (positions 1644-2349)
• Immobilization due to deletion of bom
site.
• High copy number – 2-4 fold greater
than pBR322 and pBR327.

30. Bluescript M13 :-
• Size – 2.96 kb
• Origin of replication from
bacteriophage M13 which is
inserted in opposite direction.
• Derived from pUC plasmid
having promoters for
• bacterophage T3 and T7
• Use in generation of ssDNA(in
vivo) or RNA(in vitro).

31. pGEM-3/4 :-
• Contain SP6 and T7
promoter flanking the
cloning site.
• In pGEM-4 the orientation
of MCS is opposite to that of
pGEM-3

32. pGEM3Z :-
• pGEM3Z and pGEM4Z
carry lac Z gene Hinc ll
fragment encoding α-
peptide and the same MCS
that of pGEM3 and 4.
• The promoter for T7 and
SP6 RNA Poly. Are present
at opposite ends of MCS in
the vector.

33. `
References :-
I. Principles of Gene Manipulation and Genomics – S.B.
Primrose and R.M. Twyman (seventh edition)
II. From Genes to Clones – Ernst-L Winnacker
III. A Practical Guide To Molecular Cloning – Bernard Perbal
(second edition)