Part:BBa_K895007:Experience

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User Reviews

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••••• iGEM Dundee 2012

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

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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 Serrata. 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.

<p>E. coli MG1655 was transformed with BBa_K895007 grown aerobically overnight in LB medium. The culture was then used to inoculate 30 ml 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 aassay 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

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