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Development of a Recombineering System in Enterobacter sp. YSU

Curtis, Christine
http://orcid.org/0000-0001-7092-6253
http://rave.ohiolink.edu/etdc/view?acc_num=ysu1452363978

Abstract Details

2015, Master of Science in Biological Sciences, Youngstown State University, Department of Biological Sciences and Chemistry.
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, gene/protein tagging, and gene cloning.
Jonathan Caguiat, Ph.D. (Advisor)
David Asch, Ph.D. (Committee Member)
Xiangjia Min, Ph.D. (Committee Member)
50 p.
Biology; Genetics; Microbiology; Molecular Biology
recombineering; lambda Red recombination; Red genes; genetic engineering; pKD46; recombination system; genetic modifications

Recommended Citations

Citations

  • Curtis, C. (2015). Development of a Recombineering System in Enterobacter sp. YSU [Master's thesis, Youngstown State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1452363978

    APA Style (7th edition)

  • Curtis, Christine. Development of a Recombineering System in Enterobacter sp. YSU. 2015. Youngstown State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ysu1452363978.

    MLA Style (8th edition)

  • Curtis, Christine. "Development of a Recombineering System in Enterobacter sp. YSU." Master's thesis, Youngstown State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1452363978

    Chicago Manual of Style (17th edition)

ysu1452363978
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This open access ETD is published by Youngstown State University and OhioLINK.