Development of a Recombineering System in <i> Enterobacter</i> sp. YSU

Bibliographic Details
Main Author:	Curtis, Christine
Language:	English
Published:	Youngstown State University / OhioLINK 2015
Subjects:	Biology Genetics Microbiology Molecular Biology recombineering lambda Red recombination Red genes genetic engineering pKD46 recombination system genetic modifications
Online Access:	http://rave.ohiolink.edu/etdc/view?acc_num=ysu1452363978

id	ndltd-OhioLink-oai-etd.ohiolink.edu-ysu1452363978
record_format	oai_dc
spelling	ndltd-OhioLink-oai-etd.ohiolink.edu-ysu14523639782021-08-03T06:34:50Z Development of a Recombineering System in <i> Enterobacter</i> sp. YSU Curtis, Christine Biology Genetics Microbiology Molecular Biology recombineering lambda Red recombination Red genes genetic engineering pKD46 recombination system genetic modifications 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 <i>in vivo</i>. One recombination system is the lambda Red recombination system that is controlled by the lambda bacteriophage, which contains the <i>red</i> 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 <i>Enterobacter</i> sp. YSU because it was developed in <i>Escherichia coli</i> and has been proven to be successful in <i>Enterobacter cloacae, Enterobacter aerogenes</i> and <i>Saccaromyces cerevisiae</i>. pKD46 plasmid was PCR amplified to remove the ampicillin resistance gene because <i>Enterobacter</i> 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 <i>ampR</i> 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 <i>ampR</i> gene with chloramphenicol or kanamycin resistance. <i>AmpR</i> was successfully replaced with kanamycin resistance using pKD46-cm and with chloramphenicol resistance using pKD46-kan in <i>E. coli</i>. Recombination in <i>Enterobacter</i> sp. YSU via transformation was attempted using pKD46-kan to replace <i>cmR</i> with kanamycin resistance because it was successful in <i>E. coli</i>. However, it was not successful because <i>cmR</i> 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. 2015 English text Youngstown State University / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=ysu1452363978 http://rave.ohiolink.edu/etdc/view?acc_num=ysu1452363978 unrestricted This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws.
collection	NDLTD
language	English
sources	NDLTD
topic	Biology Genetics Microbiology Molecular Biology recombineering lambda Red recombination Red genes genetic engineering pKD46 recombination system genetic modifications
spellingShingle	Biology Genetics Microbiology Molecular Biology recombineering lambda Red recombination Red genes genetic engineering pKD46 recombination system genetic modifications Curtis, Christine Development of a Recombineering System in <i> Enterobacter</i> sp. YSU
author	Curtis, Christine
author_facet	Curtis, Christine
author_sort	Curtis, Christine
title	Development of a Recombineering System in <i> Enterobacter</i> sp. YSU
title_short	Development of a Recombineering System in <i> Enterobacter</i> sp. YSU
title_full	Development of a Recombineering System in <i> Enterobacter</i> sp. YSU
title_fullStr	Development of a Recombineering System in <i> Enterobacter</i> sp. YSU
title_full_unstemmed	Development of a Recombineering System in <i> Enterobacter</i> sp. YSU
title_sort	development of a recombineering system in <i> enterobacter</i> sp. ysu
publisher	Youngstown State University / OhioLINK
publishDate	2015
url	http://rave.ohiolink.edu/etdc/view?acc_num=ysu1452363978
work_keys_str_mv	AT curtischristine developmentofarecombineeringsysteminienterobacterispysu
_version_	1719439899823702016

Development of a Recombineering System in <i> Enterobacter</i> sp. YSU

Similar Items