Question

You hypothesize that a transcriptional regulator may control expression of specific genes in LB medium, and want to find out what these genes are. You have made a set of strains (i.e., library) in which a promoterless lacZ was inserted at varied chromosomal locations. Design an experiment to identify these genes using agar plates with LB and X-gal. Describe the steps in this procedure.

Answer 1

if the promoterless lacZ is inserted close to any gene(s) or operon, then the laz gene will also be transcribed when the operon gets expressed. The polycystronic RNA will eventually produce a beta-galactosidase protein(product of lacZ gene) which will cleave X-Gal( structural analog to lactose), which will produce blue chromogenic substance.

prepare LB agar. Allow it to cool, add X-gal before it solidifies, pour to plates.

create grids and patch plate each strain on it. Just pick the strain that looks blue, amplify the sequence upstream of the lacZ and sequence it.

Step-by-step explanation:

Just pick the strain that looks blue, amplify the sequence upstream of the lacZ, and sequence it.