Skip to Main Content
 

Global Search Box

 
 
 
 

Files

File List


Ford with signature.pdf (2.11 MB)

ETD Abstract Container

Abstract Header

Knockout of the lacZ gene in Enterobacter sp. YSU

Ford, Kelsey L
http://orcid.org/0000-0003-0903-6010
http://rave.ohiolink.edu/etdc/view?acc_num=ysu1534337870735813

Abstract Details

2018, Master of Science in Biological Sciences, Youngstown State University, Department of Biological Sciences and Chemistry.
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. Enterobacter 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, lacZ. Thus, the gene of interest controls the expression of lacZ. 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 lacZ as a reporter gene to study metal resistance genes in Enterobacter sp. YSU. Before lacZ can be used as a reporter, it must be removed from the genome of Enterobacterr 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 lacZ gene. The DNA was ligated to produce a new recombinant plasmid lacking lacZ or it was ligated with a kanR/sacB DNA fragment to produce a new recombinant plasmid with lacZ 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 lacZ gene in Enterobacter sp. YSU will be a two-step process. First, the recombinant with the kanR/sacB replacement will be electroporated into Enterobacter sp. YSU. Homologous recombination into the chromosome of Enterobacter sp. YSU will allow it to grow on kanamycin plates. Second, the lacZ deletion plasmid will be electroporated into the new strain. Homologous recombination will remove the sacB gene allowing for growth on plates containing 5% sucrose and result in the complete removal of the lacZ gene. Bioinformatics analysis of the Enterobacter sp. YSU lacZ gene and LacZ protein showed that it is highly similar to thelacZ gene and LacZ protein from E. coli.
Jonathan Caguiat, Ph.D. (Advisor)
David Asch, Ph.D. (Committee Member)
Xiangjia Min, Ph.D. (Committee Member)
43 p.
Biology; Genetics; Microbiology; Molecular Biology
Beta-galactosidase; lacZ; Enterobacter; lactose operon; knockout; Escherichia coli

Recommended Citations

Citations

  • Ford, K. L. (2018). Knockout of the lacZ gene in Enterobacter sp. YSU [Master's thesis, Youngstown State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1534337870735813

    APA Style (7th edition)

  • Ford, Kelsey. Knockout of the lacZ gene in Enterobacter sp. YSU. 2018. Youngstown State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ysu1534337870735813.

    MLA Style (8th edition)

  • Ford, Kelsey. "Knockout of the lacZ gene in Enterobacter sp. YSU." Master's thesis, Youngstown State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1534337870735813

    Chicago Manual of Style (17th edition)

ysu1534337870735813
1,026
© 2018, all rights reserved.
This open access ETD is published by Youngstown State University and OhioLINK.