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pUC Vector: Features, Selectable markers, Blue White Screening for Selection of Recombinants using pUC Vector

 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)
    pUC Vector: Features, Selectable markers,

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

Blue White Screening for Selection of Recombinants using pUC Vector

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
Learn More: Insertional-inactivation-in-pBR322
Ideal Characteristics of Gene Cloning Vector

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