FOR THOSE WHO TRIED THE 'Lac operon stuff' questions...

Lactose operon home-made questions :
1) WT strain on MM with lactose as sole source of carbon‡ white colonies
On MM with lactose and X-gal and IPTG will make blue colonies (how blue kind of depends, but blue indeed).
A lacO[c] mutant on MM+lactose grows into white colonies.
A lacO[c] mutant on MM+sucrose+IPTG grows into white colonies.
A lacO[c] mutant on MM+glucose+IPTG+X-gal grows into blue-ish colonies.
A lacI[S] mutant plated on MM+lactose as a sole source of carbon will make no colonies at all. Phenotypically it will be a Lac- mutant (auxotrophic).

Note that I have not explained the logic here…but you should for full marks.
Example : A lacO[c] mutant on MM+lactose grows into white colonies…because it will be able to express high levels of B-Gal. The fact that there is a lacO[c] mutation is irrelevant in this case because the system is derepressed anyway thanks to the lactose. The low concentration of glucose allows the CAP/cAMP to bind its target site on the DNA and activate/enhance expression of the lacZ. No blue colour is produced because there’s no X-gal.



2) Strain 1 : this strain is Lac[c] as it producez B-Gal (blue colour in the presence of X-gal) even without inducer. It could be a lacO[c] or a lacI- mutation.
Strain 2 : it is Lac- (does not show B-Gal activity). However, the B-Gal protein, or at least one of its epitopes, is detected, and the permease is too. Therefore, it is probably a point mutation in the lacZ gene.
Strain 3 : makes no B-Gal. Could be lacI[s], promoter mutation, or an early nonsense mutation in the lacZ gene.
Strain 4 : small colonies on lactose only may mean that it can never fully induce the lacZ gene, so it takes it a long time to metabolize lactose. It may be a crp mutant.

For strain 1 : if it’s lacI- it should be complemented in trans by lacI+, but not if it’s lacO[c].

For strain 3 : genetically it would be hard to figure out if it’s an early nonsense mutation or a promoter mutation. However, we can distinguish between these two possibilities and lacI[s] by testing whether strain 3 can be complemented in trans by a WT lac operon. If it’s a lacI[s] mutant, then it should not, while if it’s a promoter or lacZ mutant, it should.

For strain 4 we could confirm that it’s a crp mutant by growing the strain in the presence and absence of glucose and measuring the B-gal activity on each medium : if it’s really a crp mutant, the presence or absence of glucose should be irrelevant (have no effect on B-gal expression).

3) I am very sorry about question 3—the formatting disappeared…


THE WINTEROSE OPERON :
1) The winterose metabolizing system is inducible and shows transcriptional regulation. It could be either a positively or a negatively regulated system. The 2 interesting mutants (mut 1 and mut 5) could be mutants in the winterose digesting enzyme(s).
b) mut1 and mut5 complement each other…so they are very likely mutations in 2 different genes. It may be that the proteins encoded by these 2 genes need to interact in order to digest the winterose sugar, and when both of them are mutated they can still interact, but when only one is, then they can’t (remember heterodimer suppression in 334?).
There may be other possible answers-and it’s OK to get creative, as long as you don’t run into a contradiction.

2) We have dealt with mutants 1 and 5.
Mutant 2 : could be a mutant in a ‘CAP-type’ protein (a factor that enhances expression) or in this ‘CAP-type protein’ binding site
Mutant 3 : same as mutant 2 (so pick the binding site for one and the protein for the other).
Mutant 4 : if it’s a negatively regulated system, could be a mutation in the repressor that does not bind the inducer anymore (superrepressor)
Mutant 6 : could be a mutation in the binding site of the repressor, if it’s a negatively regulated system (kind of like lacOc).