The carB gene codes for the production of the carbamoyl phosphate synthetase I enzyme, which converts ATP, carbonate, and ammonia to carbamoyl phosphate. Carbamoyl phosphate then reacts with ornithine to produce citrulline. If the activity of carbamoyl phosphate synthetase I is decreased by mutations, then carbamoyl phosphate will not be produced, therefore preventing the production of arginine, and making cells with the mutated ΔcarB plasmid arginine auxotrophs. The purpose of this experiment is to create mutant enzymes that no longer function. By mutating the carB gene in a way that disrupts enzyme activity, for instance by changing a negatively charged glutamate residue within the binding site of the enzyme, the change will block enzyme activity and prohibit cell growth on limited media. Bacterial growth was observed only on rich media for three of four samples produced by random mutagenesis, indicating the presence of a mutation producing a nonfunctional enzyme. The amount of inorganic phosphate produced by each of the three mutated samples in comparison to the native enzyme was quantified through an inorganic phosphate assay, showing that less inorganic phosphate was produced from the mutated samples than in the native enzyme. The reduced production of inorganic phosphate by the mutated enzymes confirms the presence of a mutation that reduces the activity of the enzyme.<br/>

Analysis of mutations in <i>E. Coli</i> carbamoyl phosphate synthetase I

253rd National Meeting (2017)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.

Analysis of mutations in E. Coli carbamoyl phosphate synthetase I