Subcloning Methanococcus thermolithotrophicus FK506 Binding Protein Using Polymerase Chain Reaction
McGhee, Claire E.
MetadataShow full item record
Bicelles doped with cholesterol sulfate have shown capabilities of greater thermostability then preexisting bicelles; however, they have not yet been properly characterized. To find the limits of these bicelles, a medium-small thermostable protein called to Methanococcus thermolithotrophicus FK506 Binding Protein (MTFK) has been selected go be spectroscopically analyzed in a cholesterol sulfate doped bicelle solution. MTFK is a PPIase with chaperone-like abilities. As such it has the capability of catalyzing protein folding and preventing the aggregation of said proteins. Despite its possible applications in regulating proteins during experiments with temperature variation, the MTFK gene does not exist commonly on the market. Thus using PCR to produce the gene for MTFK from eight oligodeoxyribonucleotides, codon-optimized for expression in E. coli, is a viable option to make this gene more accessible. PCA-PCR was unsuccessful at producing the MTFK gene; however, oligodeoxyribonucleotides were effectively pieced together by PCRing two to four primer sequences at once, instead of all eight oligodeoxyribonucleotides. Due to the successive PCR runs mutation of the gene was a prominent occurrence. But the mutated MTFK gene underwent a planned mutation to produce the proper MTFK gene.