Question

Principle of blue- white screening method(why do the colonies appear blue or white? what do we try to recover?what are the roles of substrate (Xgal) and the gene expression inducer(IPTG)?)

this experiment was GENETIC DEFECT CORRECTION WITH BACTERIAL TRANSFORMATION

In this experiment we did that first of all,1 µl of plasmid DNA was added into the tube which contains competent cells and the tube was tapped gently to mix DNA and the competent bacteria.After that it was placed on ice for 30 minutes. Then, the tube with the competent bacteria and plasmid were transferred to heating block at 42 °C and the tube was leaved in there exactly 90 seconds. 0.25 ml of LB broth was added to the tube and incubated 20 minutes at 37°C . After that, with using aseptic techniques pipette, the entire contents of the 500 µl X-gal/IPTG vial was put onto the agar plate which is labelled and with using glass spreader, it was dispersed. 200µl of bacteria which has the plasmid was spreaded onto the agar plate with glass beads.Then, the plate was inverted and incubated 37°C overnight and one week later the results were observed and there was some blue color on the agar plate.

Answer 1

The E.coli enzyme B-galactosidase is a large polypeptide that is the product of lacZ gene. The active form of the enzyme is homotetramer. Certain deletion mutations in the lacZ gene result in the formation of protein that lack some N- terminal residues and prevent homotetramer formation. In such mutants , subunit assembly and enzyme assembly can be restored by presence of a small amino-terminal fragment of the lacZ product. Such lacZ mutants are said to subject to alpha complementation.

In beta-galactosidase gene complementation method, the mutant host cell synthesize Omega peptide part of beta galactosidase and thus cannot make any functional beta-galactosidase. The vector is engineered to contain afragment of the beta-galactosidase gene that synthesize alpha-peptide.

After transformation by the vector, intragenic complementation occurs resulting in active beta galactosidase which can be assayed by acting on a colorless substance , Xgal , a chromogenic substrate to make a blue product .

IPTG act as a inducer if lacZ gene.