Question

10. Mutations in the lacI gene (lacI-) constitutively transcribe the lac operon. Mutations in the operator region can also occur, also resulting in the constitutive expression of the lac operon. Using the “reagents” described below, design an experiment to test if a mutant that constitutively expresses the lac operon is mutated in the lacI gene or in the operator.
X-gal – a colorless chemical that is a substrate for B-galactosidase, when cleaved it produces a blue dye that “stains” any cells expressing B-galactosidase.
An F’ strain of E. coli that contains an F plasmid with a functional lacI gene
An F’ strain that contains an F plasmid with a functional operator site
The F- mutant strain that constitutively expresses the lac operon

You can make whatever media you want but must specify what media you use in each step.

Answer 1

You need to perform two crosses :

1. Mutant × F' containing wild-type lacI gene
2. Mutant × F' containing wild-type operator

After each cross select Mutants containing F' plasmid by antibiotic selection method in plate 1 F'1 (contains plasmid having wild-type lacI gene) and in plate 2 F'2 (contains plasmid having wild-type operator).

There should be two types of plate 1 and 2. Contains lactose and do not contain lactose. (So basically four plates).

Media composition in these four plates :

1) basal medium + glucose (grow F'1)

2) basal medium + lactose (grow F'1)

3) basal medium + Glucose (grow F'2)

4) basal medium + lactose (grow F'2)

(All four media will contain X-gal and sole carbon source as mentioned above)

Interpretation of Results :

If you see all blue colonies in each four plates then there would be mutation in operator region. Because Mutation on operator is dominant and cannot be complemented by inserting wild-type operator.

If there is blue colonies only in plates 2, 3 and 4 and not in 1 and 2 then there would be mutation in lacI gene in Mutant strain. Because Mutation in lacI gene is recessive and can be complemented by inserting wild-type allele of lacI gene. After complementation, the expression of lac Operon will be indusible, so lac Operon will only be expressed when lactose is present.