Illinois State University, Chemistry, Normal, IL 61790
Life is complex and in order to understand life processes, a reductionist approach is necessary. Instead of focusing on the product, analysis may facilitate modification or interruption of the underlying process which may yield another result. Choosing the modification of the process, allows for systematic variation for analysis of the product. Creation of a genetic library is the first step of directed evolution which is feasible using Taq Polymerase for mismatch PCR, a mutagenic technique. No special protocols or procedures are required. A 96-well iLap plate can be used to screen large numbers of small samples of cells expressing 6x His-tagged mutants of glycerol 3-phosphate cytidylyltransferase. An accurate and sensitive radiological assay allows small concentrations of enzyme to be tested for activity. Once enough protein is purified, enzyme kinetics can be used to determine effects of mutations on catalysis. JoAnn K. Bialobrzewski was supported by NSF grant no. CHE-0629174
[Abstract (DOC)]