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Sunday, May 16, 2021

Blue-White Screening Mechanism - CSIR/ ICMR/ DBT (Life Sciences)

Blue-White Screening Mechanism

  • It is a rapid and efficient technique for the identification and recombinant bacteria.
  • It relies on the activity of β- galactosidase ( β- gal) an enzyme that occurs in E. coli which cleaves lactose into glucose and galactose.
Schematic representation of typical plasmid vector that can be used for blue-white screening.

Fig: Schematic representation of typical plasmid vector that can be used for blue-white screening.

LacZ gene & their Disruption:

  • It also called the heart of the Blue/white selection and is allows the selection of insert DNA fragment into the vector.

  • It has been engineered to contain multiple cloning sites (MCS), which is an oligonucleotide sequence with a series of different restriction endonuclease recognition sites arranged in tandem in the same reading frame as the lacZ gene itself.

  • The lacZ gene codes for the β- gal enzyme, which metabolizes the β- galactosidase bond into lactose. It will also cleave the galactosidase bond in an artificial substrate called X-gal (5-Bromo-4-chloro- 3-indoyl-beta-D-galactopyranoside), which can be added to bacterial growth media and has a blue color when cleaves by intact enzymes.

  • Most plasmid vectors carry a short segment of the lacZ gene that contains coding information for the first 146-amino acids of β- galactosidase. The host E.coli strains used are competent cells containing lacZΔM15 deletion mutation. 

  • When the plasmid vector is taken up by such cells, due to the α- complementation process, a functional β- galactosidase enzyme is produced. 

  • If the fragment of DNA is cloned (inserted) into multiple cloning sites (MCS), the lacZ gene will be disrupted, inactivating it, and the resulting β- galactosidase will no longer be able to cleave X-gal, resulting in white bacterial colonies rather than blue colonies.

How does Blue/white screening work:

For screening the clones containing recombinant DNA, a chromogenic substrate, X-gal is added to the agar plate. If β- galactosidase is produced, X-gal (substrate) is produced, X-gal is hydrolyzed to form 5-Bromo-4-chloro-indoxyl, which spontaneously dimerizes to produce an insoluble blue pigment called 5,5'-dibromo-4,4'-dichloro indigo. The colonies formed by non-recombinant cells, therefore appear blue in color while the recombinant ones appear white. The desired recombinant colonies can be easily picked and cultured.

Isopropyl β-D-1- thiogalactopyranoside (IPTG) is used along with X-gal for blue-white screening. IPTG (inducer) is a non-metabolizable analog of galactose that includes the expression of the lacZ gene.

A schematic representation of a typical blue-white screening procedure

Fig: A schematic representation of a typical blue-white screening procedure

Limitations of the Blue/white screening:

  • The lacZ gene in the vector may sometimes be non-functional and may not produce β- galactosidase. The resulting colony will not be recombinant but will appear white. 
  • Even if a small sequence of foreign DNA may be inserted into MCS and change the frame of the lacZ gene. This results in false-positive white colonies.
  • Small inserts within the reading frame of lacZ may produce ambiguous light blue colonies as β- galactosidase is only partially inactivated.

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