Question

A reporter gene--that is, a gene whose activity is relatively easy to detect--is commonly used to study promoter activity under a variety of investigative circumstances. Recombinant DNA technology is used to attach such a reporter gene to the promoter of interest, after which the recombinant molecule is inserted by transformation or transfection into the cell or host organism of choice. Green fluorescent protein (GFP) from the jellyfish Aequorea victoria fluoresces green when exposed to blue light. Assume that you wished to study the regulatory apparatus of the lac operon and you developed a plasmid (p) that contained all the elements of the lac operon except that you replaced the lac structural genes with the GFP gene (your reporter gene). You used this plasmid to transform E. coli.

Answer 1

lacI^S - This is a super-repressor which have a mutation in lactose binding site such that the presence of lactose can no longer bind to the repressor and operon is in constitutive repressed condition.

lacO^{C –} In this mutation, the binding site of lac repressor is mutated in such a way that it can no longer bind to the operator site. Operon which has LacO^C is constitutively active When the operon has LacI^s and LacO^C, the operon is in constitutively expressed condition as LacI^s cannot bind to the operator site.

Glucose inhibits the activity of adenyl cyclase. This enzyme converts ATP into cAMP. cAMP binds to CAP and forms a complex which binds to the promoter and helps RNA polymerase to transcribe the operon.

So in the presence of Glucose Lac operon is in repressed condition.

LacO works in cis-manner- It can only regulate the expression of operon where it is present.

LacI works in trans-manner. It means that LacI present in the merodiploid condition can bind to the operator of the other operator and prevents its expression.

Medium Condition Lactose Glucose Green B-galactosidase? Colonies? Genotype rotzt/ptotGFP T + + + + rotz pitoʻGFP I + + rotzt/pOʻGFP I + + + III I Toʻzt/pfo GFP I + + 1