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Development of an IPTG inducible expression vector adapted for Bacteroides fragilis.

Plasmid (2012-04-11)
Anita C Parker, C Jeffrey Smith
ABSTRACT

The genus Bacteroides are gram-negative, obligate anaerobes indigenous to the gastrointestinal tract of humans and animals. The Bacteroides and other members of the Bacteroidetes phylum have diverged from the Proteobacteria. These organisms evolved a unique promoter structure for the initiation of transcription, hence common genetic tools are of limited use in the Bacteroides. An expression vector that can control gene expression in the Bacteroides was constructed by engineering the lacO₁,₃ repressor binding sites into the promoter of the cfxA β-lactamase gene. The gene for the LacI repressor was placed under control of the Bacteroides tetQ gene promoter for constitutive expression and inserted into the vector. Studies utilizing the xylosidase reporter gene, Xa, showed that the gene was induced by Isopropyl β-d-1-thiogalactopyransoide (IPTG) in a time and concentration dependent manner from 10 to 250 μM over a 10-240 min time frame. The utility of the vector was demonstrated by insertion of the Bacteroides fragilis trxA gene into the plasmid. TrxA synthesis was monitored by Western hybridization and the results indicated that it was regulated by the presence of IPTG in the media. This is the first transcriptional regulatory system developed for the Bacteroides that has incorporated components from the Proteobacteria and demonstrates the feasibility of modifying existing genetic tools for use in these organisms.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
IPTG, ≥99% (TLC), ≤0.1% Dioxane
Sigma-Aldrich
Isopropyl β-D-1-thiogalactopyranoside, ≥99% (TLC)
Sigma-Aldrich
Isopropyl β-D-thiogalactopyranoside solution, ReadyMade IPTG solution for Blue-white screening