Question

You are asked to develop a demonstration to show how the lac operon works. You decide to use X-gal and IPTG to determine if the enzyme ?-galactosidase is active. X-gal is a lactose analog that turns blue when metabolized by ?-galactosidase, but it does not induce the lac operon. IPTG is an inducer of the lac operon, but is not metabolized by ?-galactosidase.

a. (2pts) Which of the following would you expect to bind to ?-galactosidase. Circle all that apply.

Lactose (or allolactose)                                Xgal                                 IPTG

b. (2pts) Which of the following would you expect to bind to the lac repressor? Circle all that apply.

Lactose (or allolactose)                                Xgal                                 IPTG

After mutagenesis, you find three mutants with the phenotypes shown in the table below (last row). Note that all of these mutants are the result of a single loss-of-function mutation.

c. (2pts) A loss-of-function mutation in which component(s) (I, Pi, CAP binding site, Plac, O, lacz) could produce the constitutive phenotypes seen in these mutants?

d. (2pts) To investigate constitutive mutant 8, you create a merozygote by introducing an F? carrying the following: lac I and its promoter (Pi), lacz, lacY and lacA, and their promoter (Plac) into mutant 8 cells. This does not rescue the mutant phenotype in mutant 8; that is, the cells are still constituitive. From these results, what can you conclude about which component (there is only one) is mutant in strain 8?    

Cell Type Media + glucose No lactose X White colonies White coloniesWhite colonies + glucose No glucose No glucose + lactose X-gal Dark blue colonies Dark blue colonies -ga +lactose + X-gal No la tose + X-gal No lactose + X-gal Wild type Mutants 8-10 White colonies White colonies Dark blue colonies

Answer 1

a. Lactose (allo lactose ) and X-gal- as Beta-galactosidase has a high specificity for Galactose, it hydrolyses X-gal to insoluble indole which dimerizes to give an insoluble blue color.  β-galactosidase is the gene product of Lac Z, it cleaves lactose to glucose and galactose.

b. Lactose and IPTG- In the presence of Lactose, allolactose binds to the Lac repressor, causing allosteric change in its shape. In this state it is unable to bind to the cognate operator. This is refereed to as induction as it induces rather than represses the metabolism of the metabolic genes. IPTG is Isopropyl -β- D-1 thiogalactopyranoside is a commonly used allo-lactose mimic, used to indice the transcription of genes.

c. I, Pi, O

d. We know that mutation in mutant 8 cell is in the O region of the operon.Mutation in I or Pi would be complemented "in trans" as having a single functioning repressor protein would regulate expression from the genomic lac operon and the inserted lac operon sequences.If the O region in the genomic lac operon is mutated such that it cannot bind to the repressor protein then the added DNA cannot stop the trancription of the genes adjacent to the mutated O region.