Knockout of the <i>lacZ gene in Enterobacter </i> sp. YSU

Knockout of the <i>lacZ gene in Enterobacter </i> sp. YSU

Bibliographic Details
Main Author: Ford, Kelsey L.
Language:English
Published: Youngstown State University / OhioLINK 2018
Subjects:
Biology
Genetics
Microbiology
Molecular Biology
Beta-galactosidase
lacZ
Enterobacter
lactose operon
knockout
Escherichia coli
Online Access:http://rave.ohiolink.edu/etdc/view?acc_num=ysu1534337870735813
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spelling ndltd-OhioLink-oai-etd.ohiolink.edu-ysu15343378707358132021-08-03T07:08:05Z Knockout of the <i>lacZ gene in Enterobacter </i> sp. YSU Ford, Kelsey L. Biology Genetics Microbiology Molecular Biology Beta-galactosidase lacZ Enterobacter lactose operon knockout Escherichia coli The lactose operon is responsible for the import and metabolism of lactose. β- galactosidase is a lactose operon enzyme that hydrolyzes lactose into glucose and galactose. <i>Enterobacter</i> sp. YSU, which was isolated from a heavy metal contaminated site in Oak Ridge, Tennessee, contains a lactose operon. Many studies used β- galactosidase as a reporter gene to study the expression levels of other genes. This was accomplished by replacing the gene of interest with the gene for β-galactosidase, <i>lacZ</i>. Thus, the gene of interest controls the expression of <i>lacZ</i>. Then, LacZ activity is detected using a color indicator such as 5-bromo-4-chloro-3-indolyl- β-D-galactopyranoside (X- gal) or o-nitrophenyl-β-D-galactopyranoside (ONPG) which turn blue or yellow, respectively, when hydrolyzed by ß-galactosidase. The overall goal is to use <i>lacZ</i> as a reporter gene to study metal resistance genes in <i>Enterobacter</i> sp. YSU. Before <i>lacZ</i> can be used as a reporter, it must be removed from the genome of <i>Enterobacter</i>r sp. YSU to eliminate background activity. A large section of the lac operon was cloned into a suicide plasmid and sequenced. Then, PCR was used to amplify the whole plasmid lacking the <i>lacZ</i> gene. The DNA was ligated to produce a new recombinant plasmid lacking <i>lacZ</i> or it was ligated with a kanR/sacB DNA fragment to produce a new recombinant plasmid with <i>lacZ</i> replaced by the kanR/sacB genes. KanR is a selectable marker for kanamycin resistance and sacB is a counter-selectable marker. Cells containing the sacB gene die when spread on agar plates containing 5% sucrose. Deletion of the <i>lacZ</i> gene in <i>Enterobacter</i> sp. YSU will be a two-step process. First, the recombinant with the kanR/sacB replacement will be electroporated into <i>Enterobacter</i> sp. YSU. Homologous recombination into the chromosome of <i>Enterobacter</i> sp. YSU will allow it to grow on kanamycin plates. Second, the <i>lacZ</i> deletion plasmid will be electroporated into the new strain. Homologous recombination will remove the <i>sacB</i> gene allowing for growth on plates containing 5% sucrose and result in the complete removal of the <i>lacZ</i> gene. Bioinformatics analysis of the <i>Enterobacter</i> sp. YSU <i>lacZ</i> gene and LacZ protein showed that it is highly similar to the<i>lacZ</i> gene and LacZ protein from E. coli. 2018-08-23 English text Youngstown State University / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=ysu1534337870735813 http://rave.ohiolink.edu/etdc/view?acc_num=ysu1534337870735813 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
Beta-galactosidase
lacZ
Enterobacter
lactose operon
knockout
Escherichia coli
spellingShingle Biology
Genetics
Microbiology
Molecular Biology
Beta-galactosidase
lacZ
Enterobacter
lactose operon
knockout
Escherichia coli
Ford, Kelsey L.
Knockout of the <i>lacZ gene in Enterobacter </i> sp. YSU
author Ford, Kelsey L.
author_facet Ford, Kelsey L.
author_sort Ford, Kelsey L.
title Knockout of the <i>lacZ gene in Enterobacter </i> sp. YSU
title_short Knockout of the <i>lacZ gene in Enterobacter </i> sp. YSU
title_full Knockout of the <i>lacZ gene in Enterobacter </i> sp. YSU
title_fullStr Knockout of the <i>lacZ gene in Enterobacter </i> sp. YSU
title_full_unstemmed Knockout of the <i>lacZ gene in Enterobacter </i> sp. YSU
title_sort knockout of the <i>lacz gene in enterobacter </i> sp. ysu
publisher Youngstown State University / OhioLINK
publishDate 2018
url http://rave.ohiolink.edu/etdc/view?acc_num=ysu1534337870735813
work_keys_str_mv AT fordkelseyl knockoutoftheilaczgeneinenterobacterispysu
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