Date of Graduation
Davis College of Agriculture, Natural Resources and Design
A method whereby DNA sequences of varying size may be accurately inserted into pre-determined locations within the Escherichia coli genome was developed. Using a binary vector system that takes advantage of either the E. coli RecET enzyme tandem or bacteriophage lambda's Redalphabeta recombinatory proteins, this method has the benefits of being both inducible and self-degrading, thereby offering a level of control to the experimenter as well as alleviating any problems that may be associated with leaving residual DNA in the bacterium. To demonstrate the usability of this system, two antibiotic resistance genes, as well as the pyrroloquinoline quinone (pqq) and gluconate dehydrogenase (gadh) operons were used as targeting molecules in three different strains of E. coli, with three distinct genomic targets. The location of the inserted sequences was confirmed through a primer-specific PCR detection assay. The combined expression of these operons demonstrates that E. coli can be made to produce 2-ketogluconate, an organic acid previously shown to dissolve insoluble rock phosphate, using glucose or gluconate as the sole source of carbon. Diversification of the substrates required for the production of 2-ketogluconate allows for the possibility of agricultural applications, while the recombinogenic targeting system offers a variety of possible uses in the modification of DNA molecules within the E. coli bacterium.
Coss, Dennis, "Insertion of genes and operons into the Escherichia coli genome through targeted recombination" (2004). Graduate Theses, Dissertations, and Problem Reports. 4130.