Development of a recombineering system in Enterobacter sp. YSU

Abstract

Recombineering, also known as recombination-mediated genetic engineering, is a molecular genetics technique that utilizes homologous recombination to modify the genome of prokaryotes and eukaryotes in vivo. One recombination system is the lambda Red recombination system that is controlled by the lambda bacteriophage, which contains the red genes that encode the Exo, Beta, and Gam proteins. The Exo, Beta, and Gam proteins are involved in the process of double strand break repair and are responsible for homologous recombination. This method of recombination has replaced the more conventional and time-consuming genome modification technique using restriction endonuclease enzymes and DNA ligase. I hypothesize that the lambda Red recombination system will be successful in Enterobacter sp. YSU because it was developed in Escherichia coli and has been proven to be successful in Enterobacter cloacae, Enterobacter aerogenes and Saccaromyces cerevisiae. pKD46 plasmid was PCR amplified to remove the ampicillin resistance gene because Enterobacter sp. YSU is already resistant to ampicillin which is the selectable marker for pKD46. A chloramphenicol and kanamycin resistance gene was PCR amplified and mixed with the pKD46 PCR product without the ampR gene, treated with T4 Polynucleotide Kinase, and ligated to construct two new plasmids, pKD46-cm and pKD46-kan. pKD46-cm and pKD46-kan were used for recombination with pBR322 plasmid to replace the ampR gene with chloramphenicol or kanamycin resistance. AmpR was successfully replaced with kanamycin resistance using pKD46-cm and with chloramphenicol resistance using pKD46-kan in E. coli. Recombination in Enterobacter sp. YSU via transformation was attempted using pKD46-kan to replace cmR with kanamycin resistance because it was successful in E. coli. However, it was not successful because cmR from pACYCY184 does not appear to be expressed well in YSU. This recombination system can be useful in helping understand gene function by creating gene replacements, deletions, insertions,