Artificial evolution of aconitase via heat-shock induced oxidative stress

Oxidative stress, where oxygen has an unpaired electron, has been shown to damage cellular components. These electrons injure local cellular machinery and have the potential to interrupt major protein pathways. Aconitase is a key polypeptide with multiple niches in the cell, and has been shown to be a target for free radical impairment. We utilize artificial evolution using heat shock to cause major oxidative damage. With up to 99% fatality and repeated exposure we have an effective way to select against aconitase mutants via respiratory deficient yeast on glycerol, a non-fermentable growth medium. In this experiment, we use the previously described artificial evolution coupled with error-prone PCR to select for heat-resistant aconitase mutants. The results are in the form of purified DNA from different clones. These will give future insight on the important enzymatic domains of aconitase.