Synthetic construction of vectors Introduction, enzymes in genetic engineering, linkers, adaptors... Construction of pBR322 pBR322 derivatives (pUC series) Modular vectors (pZ series, BioBricks and pSEVA) Yeast artificial chromosomes Pseudomonas putida superbug

1. Synthetic construction of
vectors
Darshan

2. Contents
• Introduction
• Components of vectors
• Tools in synthetic construction
• pBR322 construction
• pBR322 derivatives
• Modular vectors
• Superbug
• Cosmids
• YAC

3. Introduction
• Naturally occurring plasmids-
phenotypes….
• ColE1 and RSF1010
• Limitations: no. of selection markers and
recognition sites for restriction
enzymes
• pBR322

4. Components of vectors
• Ori
Recognized by the cellular replication
machinery
Define the number of copies of a given
plasmid in the cell

5. • Selection marker
 Any gene allowing a selective advantage to
the positive transformants
 Antibiotic resistance
 Auxotrophy, etc..
• Multiple cloning site (MCS)
 To facilitate cloning of desired DNA,
containing several sites recognized by
different restriction enzymes

6. • Promoters
To drive transcription of vector’s
transgene as well as other genes that
encode certain phenotypic
characteristics
• Ribosome binding sites
• Termination sequence

7. Ideal vector
• Small size (low mol wt.)
• Having multiple selection markers
• Having unique sites for a large number
of restriction enzymes, preferably
genes with readily scorable phenotypes
• Copy no- cloning
• Promoters- expression

8. Tools in synthetic construction
• Enzymes
Restriction enzymes (typeII being used
the most)
Polymerases
Ligases

9. • Linkers
Blunt ligation
Contain sites for one or more REs
Can be ligated to both ends of DNA and
then digested

11. • Adaptors
Preformed cohesive ends

12. pBR322 construction
• Why construction?
• Natural plasmids have limited genetic
markers for selecting transformants
• Contains the ApR and TcR genes of
RSF1010 and pSC101, respectively
combined with replication elements of
pMB1

13. R7268
ApR
R1 drd 19
ApR CmR
SmR SuR
KmR
pMB1
pMB3
ApR
ColE1
pSF2124
ApR
pSC101
TcR
pMB9
TcR
pBR312
ApR TcR
pMB8
pBR313
ApR TcR
pBR322
ApR TcR
1
5
2
8
7
4
3
6
6

14. pBR322 derivatives
• To fulfill special purpose cloning needs
• Early works- insertion of additional
unique restriction sites and selectable
markers
• Eg. CmR in pBR325 with unique EcoRI
site

15. pUC plasmids
• Incorporate a DNA sequence that
permits rapid visualization of DNA
insert
• MCS in lacZ (encodes promoter and α-
peptide to mediate complementation)
• pUC7 is descended from pBR322

17. Modular vectors

18. Superbug

19. Cosmids
• Plasmids that can
be packaged into
bacteriophage λ
particles

20. YAC construction