Operon Essay

727 Words2 Pages

The lac operon is a transcriptional control of lactose metabolism in bacteria. The operon contains three transcriptional genes, lac Z, lac Y and lac A, which encodes for β-galactosidase, permease and transacetylase respectively. Lac P and lac O copes for the lac promoter and the lac operator, essential to the functioning of this operon. β-galactosidase converts lactose to allolatose, while permease allow lactose to be transported into the cell. Transacetylase does not have a role in lactose usage. In the absence of lactose, there is no allolactose, converted from lactose by β-galactosidase, to the active regulatory repressor, and thus the repressor binds to the operator and transcription is inhibited, as the RNA polymerase bound to the promoter is blocked. In the presence of lactose, allolactose binds to the repressor, rendering it inactive and unable to bind to the operator, allowing the transcription of the three structural genes.
In this experiment, uvrA phr E. coli cells, repair-deficient mutant strain, were first exposed to one or two seconds or UV radiation. Since this strain lack both nucleotide excision repair and phtoreactivation to repair resulting pyrimidine dimers, mutations resulting from error-prone repair may occur in the lac operon. By plating out the mutated E. coli on MacConkey/lactose medium, we can determine whether the cells are lac operon mutants by assessing the colour of the colonies. MacConkey agar, acts as a pH indicator and stains bacteria that ferment lactose red (acidic pH). Bacteria that cannot use lactose will use the agar’s other constituent, peptone, instead, producing ammonia that gives a basic pH, and thus the colonies will appear pale.
Thus, in our screening process, red streaked colonies are a...

... middle of paper ...

...lactose.
By comparing the data from both the β-galactosidase activity assay experiment and the complementation experiment, we can determine the likely gene encoded in the lac operon, which had been mutated by UV exposure, and thus prevent lactose usage, in each of the two mutants.
Thus, at the end of this project, we would have created and successfully isolated E.coli mutants that had a mutation in the lac operon, as a result of UV exposure, as well as identify the genotype of those two lac operon mutants.
1.1 AIM:
1. To isolate E. coli lac operon mutants via mutant screening using MacConkey/lactose and confirmation by growth in MacConkey/maltose plates
2. To determine the genotypes of two different E. coli lac operon mutants via β-galactosidase activity assay by spectrophotometry and the results of complementation test, with the introduction of various plasmids.

Open Document