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Applications of BBa_K895007

User review of SHSBNU_China

Before our marine Shewanella baltica TtrS/R (BBa_K2507013, BBa_K2507014, BBa_K2507015, BBa_K2507016, BBa_K2507017)system, the only known genetically encoded tetrathionate sensor is the Salmonella typhimurium TtrS/R two-component system (TCS) (Hensel et al, 1999; Price-Carter et al, 2001). However, they found S.typhimurium’s promoter PttrB is repressed by oxygen and nitrate via the global regulator Nitrate Reductase Regulator (FNR). While the oxygen levels in the intestinal are poorly understood by human beings Kristina et al think the oxygen level maybe relatively high near the epithelial mucosal boundary because of near to the blood. Meanwhile, nitrate levels in intestinal have been shown to be improved during inflammation (Winter et al, 2013). In iGEM 2012, Dundee used Salmonella TtrS/R system BBa_K895007 and the system works well.

The sensor we use S.baltica TtrS/R which is only weakly repressed by oxygen and not repressed by nitrate in E. coli. And our S.baltica TtrS/R system can work well using protoviolaceinic acid as the reporterBBa_K2507017,which display dark-green color. And our system could also work poorly using sfGFP as the reporter.

Figure 1

Figure 1 TtrS/R system and ThsS/R system ThsS (BBa_K2507000) and ThsR (BBa_K2507001), both codon-optimized for E. coli, are two basic parts which belong to the two-component system from the marine bacterium Shewanella halifaxensis. By linking thsR with chromoprotein genes (BBa_K2507009, BBa_K2507010, BBa_K2507011) or the violacein producing operon vioABDE (BBa_K2507012), this system can respond to thiosulfate by producing a signal visible to the naked eye, such as chromoproteins (spisPink-pink chromoprotein, gfasPurple-purple chromoprotein, amilCP-blue chromoprotein) or a dark-green small-molecule pigment (protoviolaceinic acid). E. coli-codon-optimized TtrS(BBa_K2507002) and TtrR (BBa_K2507003) are two basic parts which are derived from the two-component system of the marine bacterium Shewanella baltica.

By linking ttrR with chromoprotein genes (BBa_K2507009, BBa_K2507010, BBa_K2507011) or the violacein producing operon vioABDE (BBa_K2507012), this system can respond to thiosulfate by producing a signal visible to the naked eye such as chromoproteins (spisPink-pink chromoprotein, gfasPurple-purple chromoprotein, amilCP-blue chromoprotein) or a dark-green small-molecule pigment (protoviolaceinic acid).

Figure 2

Figure 2. Characterization of the ThsS/R and TtrS/R system by observing the chromprotein expression levels. We added 1mM, 0.1mM, 0.01mM and 0 Na2S2O3 to ThsS/R system and added 2.5mM, 1mM, 0.1mM and 0 Na2S4O6·2H2O to ThsS/R system. The results demonstrate there is an obvious response in the ThsS/R system. In a. gfasPurple system, the response curve is obvious, and b. spisPink & c. amilCP with rather heavy leaky expression without inducer. d.e.f. However, in TtrS/R system there is no clear result.

Figure 3

Figure 3. Characterization of the ThsS/R and TtrS/R system by observing the protoviocaceinic acid( dark-green pigment). We added 1mM, 0.1mM, 0.01mM and 0 Na2S2O3 to ThsS/R system and added 2.5mM, 1mM, 0.1mM and 0 Na2S4O6·2H2O to ThsS/R system. The results demonstrate there is an obvious response in the ThsS/R system with rather heavy leaky expression without inducer. And the Ths/R+protoviolaceinic acid works rather well!

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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.

The Western immunoblot experiment (Figure 1) was corroborated by miniature-scale whole cell fluorscence assays (Figure 2). MG1655 harbouring BBa_K895007 was grown in LB medium supplented with tetrathionate or thiosulfate. The cultures were 200 ul in volume and contained within the wells of a 96-well fluorescence plate. The plate was incubated at 37 C in a Biotek incubator/plate reader and cell growth and GFP fluorescence recorded at 20 minute intervals. The data clearly show that GFP production is induced by tetrathionate in the growth medium (Figure 2).

Figure 2: Induction of GFP production from BBa_K895007 in a miniaturised whole cell assay. The E. coli wild-type strain MG1655 was transformed with BBa_K895007. The strain was then grown micro-aerobically (i.e. with shaking just before data point collection) in 200 ul cultures in a 96-well fluorescence plate. Cultures (in triplicate) either contained LB only ('no addition') or were supplemented with 0.1% (w/v), 0.2% (w/v) or 0.4%(w/v) potassium tetrathionate were indicated. Another control, supplemented with 0.4% (w/v) potassium thiosulfate, was also included. The Optical Density at 600 nm (as a measure of cell density) was recorded at 20 minute intervals together with GFP fluorescence (Ex. 485 nm / Em. 528 nm, slit widths 20 nm). The background fluorescence from non-inoculated LB medium was subtracted from the data before the average fluorescence was divided by the OD-600 to give a ready indication of 'relative fluorescence per cell' in each experiment.

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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