What is a pUC vector?
- Vector obtained by modifying pBR 322 vector
- Smaller in size 2686bp with high copy number
- ‘p’=plasmid “UC”: University of California
- Joachim
Messing and co-workers (1983)
Regions:
1.
Ori: E.coli origin
2
selectable markers: ampicillin
(amp r)& lacZ’ gene
This region has genes
providing resistance against ampicillin,
LacZ’
codes for β
Galactosidase enzyme
3.
Restriction sites: lacZ’ gene with multiple cloning sites for 13 Restriction
enzymes
Selection
of recombinant colonies using Blue White colony screening
After
transformation, 3 types of colonies
Grow
in a medium containing Agar+Ampicillin + Xgal+IPTG
IPTG
is an inducer of Beta galactosidase enzyme. It binds to the repressor activates
beta galactosidase transcription
1)
Non-transformed: without vector: Cannot
grow in Amp containing medium
2) Transformed:
a) Transformed with non recombinant: Can
grow in Amp medium, Active LacZ gene so can
convert X gal into blue product
b)Transformed with recombinant vector : can grow in Amp medium.
Lac Z gene is inactive by insertional
inactivation. It is the inactivation of a gene upon insertion of our gene of
interest. It cannot
convert X gal to Blue product
Recombinant
Colonies: White as LacZ gene is inactive by insertional inactivation
Non
Recombinant: Blue Colonies LacZ gene is active, the galactosidase
formed will convert Xgal into blue
product
This recombinant selection procedure
is called Blue White Colony Screening.
Advantages of pUC vector
- High copy number 500-600 copies per cell
- Easy one step selection of recombinant colonies
- Many restriction sites in MCS (Multiple Cloning Sites)
Disadvantage
- Insert size is 15 Kb
Ideal Characteristics of Gene Cloning Vector
