A two-step chromosomal lacZ-fusion method in Salmonella enterica serovar Typhimurium
Abstract
I describe a new method for creating chromosomal lacZ fusions to any non-essential gene in S. typhimurium. This new method utilizes uses a combination of linear transformations, facilitated by λ-RED recombination and gene duplications facilitated by P22 transductions. A partial lac operon containing lacZ (nt 1-500), a kanamycin resistance cassette, and lacA (nt 579-614) was used to disrupt a gene of interest, facilitated by RED recombinase. P22 phage grown on a strain of S. typhimurium containing a MudA transposon, which includes an intact lac operon fused to a gene located upstream of the gene of interest was used to transduce, the disrupted gene, resulting in duplication of the disrupted gene and complementation of the lac operon. This duplication fuses an intact lac operon to the promoter of interest, and once carried into the wild-type background, maintains the wild-type gene of interest to monitor any self-regulatory activity. This new method was tested by fusing the lacZ gene to the promoter of the putative osmotic stress ABC glycine-betaine/choline-O-sulfate transporter OsmU. The expression of OsmU was monitored during osmotic shock and in the presence of the osmoprotectants, glycine-betaine, and choline-O-sulfate. Using this method we have determined that the osmU operon is induced approximately 1.3-3 fold during stationary phase.
Degree
M.S.
Advisors
Csonka, Purdue University.
Subject Area
Genetics|Microbiology
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