The Construction of a Reporter Plasmid for the Detection of virB Promoter Activation Through the Use of a β-galactosidase Assay
Baran, Brenda L.
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Shigellosis, endemic throughout the world predominantly in third world countries, causes severe dysentery and 1.1 million deaths yearly. Shigella flexneri is one of four species of Shigella responsible for Shigellosis. The key virulence genes are encoded and organized within operons within a 31 kb region of a 230 kb virulence plasmid. These genes are positively regulated by the virB gene. The VirF protein is a transcriptional activator of the virB gene. VirF directly interacts with DNA regions upstream of the virB promoter (Tobe, T., Yoshikawa, M., Mizuno, T., and Sasakawa, C. (1993) Transcriptional Control ofthe Invasion Regulatory Gene virB of Shigella flexneri: Activation by VirF and Repression by H-NS. JBacteriol 175, 6142-6149). A reporter plasmid was to be constructed and act as a detection system for VirF and contain the LacZ gene which encodes for β-galactosidase. β-galactosidase would be activated by VirF, by incorporation of a virB promoter, producing a chromogenic reaction by the use of a β-galactosidase assay. LacZ was obtained from plasmid pTZl 8U after its multiple cloning site, MCS, was replaced with a synthetic sequence, putting the plasmid back into frame, as to not interfere with the need of unique restriction sites. The replacement of the MCS did not interfere with LacZ transcription; β-galactosidase activity was demonstrated. LacZ was ligated into a plasmid pACYC184, compatible with the virF expression plasmid. virB was not successfully ligated. Ultimately the reporter plasmid could be used in high-throughput β-galactosidase assay drug screening (Thibodeau, A. S., Fang, R., and Joung, J. K., (2004) High-throughput β-galactosidase assay for bacterial cell-based reporter systems. Biotechniques 36,410-415.)