Question

8. Your lab assistant has tried to construct a genomic library of recombinant plasmids using only the EcoRI restriction enzyme, genomic DNA, a plasmid and DNA ligase. The plasmid used is a typical cloning plasmid with a multiple cloning site (MCS) in the middle of a Lacz gene. After transforming E. coli with the library, a few white colonies are observed but a most of the colonies are blue. Explain what happened and what you would tell your assistant to do in order to obtain more white colonies.

Answer 1

Okay, so he used a plasmid that contains an alpha peptide for galactosidase. This is about the blue-white screening. This peptide complements an incomplete galactosidase peptide due a deleted region in LacZ gene. If this alpha peptide gene is not disrupted, then the colinies will turn blue due to cleavage of a galactose compound. This blue coloration actually indicates he didn't succeed in inserting the genomic DNA into the plasmid, such genomic DNA would have disrupted the alpha peptide gene leading to a white coloration because of galactosidase inactivity.

To recover the white coloration in the future you need to disrupt such alpha gene in the plasmids with the use of restriction enzymes, the genomic DNA must get inserted into the plasmid. Th point to develop here is the genomic DNA insertion into the plasmid.