Insertion of the Escherichia Coli Phosphofructokinase A (PFKA) Gene into a plasmid, pRSETA

We attempted to insert the Escherichia Coli (E. Coli) gene for Phosphofructokinase A (PFKA), an enzyme involved in glycolysis, into the plasmid pRSETA, place it under the control of the easily-inducible T7 RNA Polymerase, and his-tag the gene. We first designed appropriate primers and used PCR to amplify the PFKA gene with added restriction sites (HindIII and BamH1) at either end from several different DH5D K12 chromosomal DNA samples, some of which we had prepared ourselves. We then cut the gene, along with a sample of pRSETA, with the restriction enzymes HindIII and BamH1. Then we “gel purified” the resulting DNA, a mix of unwanted smaller fragments as well as the properly designed cut plasmid and gene. We then ligated the gene and the plasmid together, and chemically transformed the resulting ligation into DH5D competent cells. After screening numerous colonies for the presence of plasmid DNA using a miniprep and a restriction analysis using HindIII and BamH1, we had hoped to find evidence of at least one successful transformation of the pRSETA plasmid with the PFKA insert. However we have come up with no evidence of a succesful clone, and after redoing the procedure many times as well as eliminating many sources of error, we have decided to abandon the project with the conclusion that the cloning of this gene is very diffucult or impossible to carry out by our limited methods.