Toxin-encoding plasmids

Navarro-Garcia F, Canizalez-Roman A, Luna J, Sears C, Nataro JP Plasmid-encoded toxin of enteroaggregative Escherichia coli is internalized by epithelial cells. Infect Immun 2001 69 1053-1060. 

Fig. 1. Outline of the strategy to construct GST-fused expression plasmids by the in vitro recombination-assisted method. Am, Gm, and Cm are abbreviations for ampicillin-, gentamicin-, and chloramphenicol-resistance, respectively. The figure also indicates the ccdB gene encoding a toxin targeting the co//essential DNA gyrase and the phage X recombination sites (attB, attP, attL, and attR).

Stinear TP, Mve-Obian A, Small PLC, Frigui W, Pryor MJ, Brosch R, Jenkin GA, Johnson PDR, Davies JK, Lee RE, Adusumilli S, Gamier T, Haydock SF, Leadlay PF, Cole AT. Giant plasmid-encoded polyketide synthases produce the macrolide toxin of Mycobacterium ulcerans. Proc. Natl. Acad. Sci. U.S.A. 2004 101 1345-1349. 

One or more plasmids are usually found in clinical isolates of S. aureus. There are at least three families of S. aureus plasmids (Novick 1990), which are classified into fifteen incompatibility groups (Novick 1987). Most plasmids that have been desaibed encode antimicrobial resistance determinants, and some have also been attributed other clinically significant properties, such as toxin production (e.g. entero-toxins SED and SEJ, and exfoliative toxin ETB), whereas some staphylococcal plasmids are phenotypically cryptic. 

Plasmids contain the DNA encoding the recombinant toxin under control of the T7 promoter (14), and contain an ampicillin or chloramphenicol resistance gene. The latter is preferred for the preparation of proteins for clinical use, since patients allergic to penicillin might react to recombinant proteins purified from bacteria grown in the presence of ampicillin. Plasmids are stored at a concentration of 1 mg/mL in 0.01 M Tris-HCl, pH 8.0, and 1 mM EDTA at 4°C. 

This chapter will not focus on the recombinant DNA techniques used to construct plasmids encoding recombinant toxins, since such methods are not unique to recombinant toxins. The method described for plasmid expression in E. coli BL21/A.DE3 cells is applicable for plasmids containing the T7 promoter (14) and the ampicillin resistance gene. 

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