Difference between revisions of "Part:BBa K895007:Experience"

Revision as of 17:57, 1 October 2012

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Applications of BBa_K895007

User Reviews

UNIQd4521b3f177c52c2-partinfo-00000000-QINU

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iGEM Dundee 2012

This part was seen work in practice. The device produces GFP in response to extracellular tetrathionate (Figures 1,2). The device is not activated by thiosulfate, which is the natural product of tetrathionate reduction (Figures 1,2).

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Results

Characterisation by Dundee iGEM Team 2012

Tetrathionate reductase is an enzyme found in most Salmonella strains and some other opportunistic pathogens such as Serratia. The expression of the tetrathionate reductase genes (ttrBCA) in Salmonella is regulated by a two-component system. This two-component system senses extracellular tetrathionate via a membrane-bound histidine kinase (TtrS), which in turn serves to promote phosphorylation of the DNA-binding TttR response regulator (Hensel et al. 1999). It is possible that this two-component system could be used to regulate any reporter protein or other enzyme system.

Tetrathionate has recently been shown to be a compound generated during the inflammation process during infection of the gut epithelium (Winter et al. 2010). For the natural Salmonella organism, this means it can use tetrathionate as a terminal electron acceptor during infection to out-compete other microbes as well as, possibly, to survive attack by macrophages (Winter et al. 2010). This suggests that the presence of tetrathionate in the gut could signal inflammation and infection are occuring and a synthetic device could be designed to read-out this signal.

To test this BBa_K895003 was designed and GFP was placed under its control to give BBa_K895007. Next, an E. coli chassis (strain MG1655) was transformed with BBa_K895007 and grown aerobically overnight in LB medium. The culture was then used to inoculate 30 ml sealed, anaerobic cultures in LB only, LB + 0.4% (w/v) tetrathionate or LB + 0.4% (w/v) thiosulfate. Following 16 hours growth aliquots were taken and analysed by SDS-PAGE and Western immunoblotting using an anti-GFP monoclonal.

This small scale assay reveals that GFP was only expressed from the plasmid in the presence of tetrathionate. The device therefore works as a putative inflammation biosensor.

Figure 1: Production of GFP from BBa_K895007 is tetrathionate dependent. The RED ARROW points to the lane producing GFP.

References

Hensel,M., Hinsley,A.P., Nikolaus,T., Sawers,G. and Berks,B.C. (1999) The genetic basis of tetrathionate respiration in Salmonella typhimurium. Mol. Microbiol. 32:275-287.

Winter,S.E., Thiennimitr,P., Winter,M.G., Butler,B.P., Huseby,D.L., Crawford,R.W., Russell,J.M., Bevins,C.L., Adams,L.G., Tsolis,R.M., Roth,J.R. and Bäumler,A.J. (2010) Gut inflammation provides a respiratory electron acceptor for Salmonella. Nature 467:426-429.

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