scriptural control of lactose metabolism in bacteria. The operon contains three transcriptional genes, lac Z, lac Y, and lac A, which encode β-galactosidase, permease, and transacetylase, respectively. Lac P and lac O cope with the lac promoter and lac operator, which are essential for the functioning of this operon. β-galactosidase converts lactose to allolatese, while permease allows the transport of lactose into the cell. Transacetylase has no role in the utilization of lactose. In the absence of lactose, there is no allolactose, converted from lactose by β-galactosidase, in the active regulatory repressor, and therefore the repressor binds to the operator and transcription is inhibited, since promoter-bound RNA polymerase is blocked. In the presence of lactose, allolactose binds to the repressor, rendering it inactive and unable to bind to the operator, allowing transcription of the three structural genes. In this experiment, uvrA phr cells of E. coli, a repair-deficient mutant strain, were first exposed to one to two seconds or to UV radiation. Because this strain lacks both nucleotide excision repair and photoactivation to repair the resulting pyrimidine dimers, mutations resulting from error-prone repair can occur in the lac operon. Plating the E. coli mutated on MacConkey/lactose medium, we can determine whether the cells are mutants of the lac operon by evaluating the color of the colonies. MacConkey Agar, acts as a pH indicator and stains lactose fermenting bacteria red (acidic pH). Bacteria that cannot use lactose will instead use the other constituent of agar, peptone, producing ammonia which provides a basic pH and therefore colonies will appear pale. Therefore, in our screening process, the red-streaked colonies are......middle of the sheet......lactose. By comparing the data from both the β-galactosidase activity assay experiment and the complementation experiment, we can determine the probable gene encoded in the lac operon, which was mutated by UV exposure, and thus prevent the use of lactose, in each of the two mutants. Therefore, at the end of this project, we would have successfully created and 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 these two lac operon mutants . 1.1 PURPOSE:1. Isolate E. coli lac operon mutants by mutant screening using MacConkey/lactose and confirmation by growth in MacConkey/maltose2 plates. To determine the genotypes of two different E. coli lac operon mutants by testing β-galactosidase activity by spectrophotometry and complementation test results, with the introduction of various plasmids.

Topic > scriptural control of lactose metabolism in bacteria. The operon contains three transcriptional genes, lac Z, lac Y, and lac A, which encode β-galactosidase, permease, and transacetylase, respectively. Lac P and lac O cope with the lac promoter and lac operator, which are essential for the functioning of this operon. β-galactosidase converts lactose to allolatese, while permease allows the transport of lactose into the cell. Transacetylase has no role in the utilization of lactose. In the absence of lactose, there is no allolactose, converted from lactose by β-galactosidase, in the active regulatory repressor, and therefore the repressor binds to the operator and transcription is inhibited, since promoter-bound RNA polymerase is blocked. In the presence of lactose, allolactose binds to the repressor, rendering it inactive and unable to bind to the operator, allowing transcription of the three structural genes. In this experiment, uvrA phr cells of E. coli, a repair-deficient mutant strain, were first exposed to one to two seconds or to UV radiation. Because this strain lacks both nucleotide excision repair and photoactivation to repair the resulting pyrimidine dimers, mutations resulting from error-prone repair can occur in the lac operon. Plating the E. coli mutated on MacConkey/lactose medium, we can determine whether the cells are mutants of the lac operon by evaluating the color of the colonies. MacConkey Agar, acts as a pH indicator and stains lactose fermenting bacteria red (acidic pH). Bacteria that cannot use lactose will instead use the other constituent of agar, peptone, producing ammonia which provides a basic pH and therefore colonies will appear pale. Therefore, in our screening process, the red-streaked colonies are......middle of the sheet......lactose. By comparing the data from both the β-galactosidase activity assay experiment and the complementation experiment, we can determine the probable gene encoded in the lac operon, which was mutated by UV exposure, and thus prevent the use of lactose, in each of the two mutants. Therefore, at the end of this project, we would have successfully created and 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 these two lac operon mutants . 1.1 PURPOSE:1. Isolate E. coli lac operon mutants by mutant screening using MacConkey/lactose and confirmation by growth in MacConkey/maltose2 plates. To determine the genotypes of two different E. coli lac operon mutants by testing β-galactosidase activity by spectrophotometry and complementation test results, with the introduction of various plasmids.