Knockout Of The Upp Gene In Escherichia Coli Dh5A And Its Application Research

Counterselectable markers are often instrumental for the construction of clean and unmarked mutations in bacteria. Under appropriate growth conditions, a counterselectable gene promotes the death of the microorganisms harboring it. Upp gene exists widely in prokaryotes and some primary eukaryotes cells. UPRT(Uracil Phosphoribosyltransferase), encoded by upp gene, can convert5-fluorouracil to5-fluoro-dUMP, which inhibits thymidylate synthase, causing cell death. So the upp gene can be used as a counterselectable marker. In this study, we construst DH5α-△upp mutant by Red homologous recombination and the upp counterselectable marker can be used in this upp deleted strain.We used two gene knockout methods. In the first scheme, the kanamycin-resistant cassette was PCR-generated by using primers with50-nt extensions that homologous to regions adjacent to the upp gene. After electroporated into competent DH5α strain cells containing pKD46, the kanamycin-resistant cassette took place of upp gene with the help of Red recombinase and construct the mutant strain called DH5a-△upp. In the second scheme, two pairs of primers, which was the homologous sequences of upstream and downstream regions of upp gene, was amplified from wild type DH5a strain by PCR. Then the fragment which was used in upp knockout was amplified by overlap extension PCR. PCR products containing two homologous sequences were introduced into competent cells as before and got the upp deleted mutant. Compared to the wild type, the upp deleted mutant grew a little slower, but increased resistance of5-fluorouracil significantly.We develpoed a one pladmid dual positive/negative selection system for the evolution of aminoacy-tRNA synthetases with DH5α-△upp. We demonstrate the utility of the system with para-iodophenylalanine synthetase. The study above lay the foundation of selecting other aninoacyl-tRNA synthetases.