Description:
Flavoenzyme methylenetetrahydrofolate reductase (MTHFR) is a critical component of folate metabolism in humans and Escherichia coli. Deleterious mutations to the MTHFR gene can cause mild and severe chronic ailments. To study the consequences of specific mutations, we can construct DNA vectors encoding the mutant protein of interest. Since the catalytic domain of MTHFR is highly conserved between E. coli and humans, E. coli is an ideal model organism for this sort of study. Here we have begun to produce a collection of new plasmid vectors encoding eMTHFR containing substitution mutations at sites hypothesized to be important in MTHFR’s enzymatic activity. Our methods are based on Stratagene’s protocol for their QuikChange II site‐directed mutagenesis kit. To date we have successfully produced and isolated plasmid vector pJUS‐1D, encoding eMTHFR substitution mutant Glu28Asp. Sequencing results have confirmed the plasmid’s desired identity. We are now in the process of producing additional vectors encoding eMTHFR mutants Ser26Thr and Ser26Ala.