Composite

Part:BBa_K2507013

Designed by: Huanding Ji   Group: iGEM17_SHSBNU_China   (2017-10-04)
Revision as of 13:00, 1 November 2017 by Weihang Guo (Talk | contribs) (→‎Characterization)


J23109-ttrR-PttrB185-sfGFP

Usage and Biology

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. TtrS is the membrane-bound sensor kinase (SK) which can sense tetrathionate outside the cell, and TtrR is the DNA-binding response regulator (RR). PttrB185-269 (BBa_K2507019) is a minimal TtrR-activated promoter which is activated when TtrR is phosphorylated by TtrS after TtrS senses tetrathionate.

Winter et al. have shown that reactive oxygen species (ROS) produced by the host during inflammation convert thiosulfate into tetrathionate, which this pathogen consumes to establish a beachhead for infection (Winter et al, 2010). Thus, tetrathionate may correlate with pro-inflammatory conditions and can therefore be used as a sensor for intestinal inflammation.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 11
    Illegal NheI site found at 34
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 318
  • 1000
    COMPATIBLE WITH RFC[1000]

Characterization

We first validated that this system can function as a tetrathionate sensor and reporter in the laboratory strains Escherichia coli Top10 and E. coli Nissle 1917.

Figure1

Figure 1.Schematic diagram of ligand-induced signaling through TtrS/R and plasmid-borne implementation of the sensor components. TtrS/R was tested with BBa_K2507006 integrated into the pSB4K5 backbone and BBa_K2507013 into the pSB1C3 backbone. We submitted all parts to the iGEM registry in pSB1C3.

Figure2

Figure 2.As a result, the Tetrathionate system does not work well.

Figure3

Figure3. We cultivated E. coli Nissle 1917 overnight under aerobic or anaerobic conditions. Ths/R-sfGFP seems to act better under anaerobic conditions. However, TtrS/R-sfGFP performed much worse than ThsS/R. We therefore replaced the gfp gene in the TtrS/R system, with the vioABDE operon (BBa_K2507017). It worked better. So in TtrS/R system, we replaced gfp gene by vioABDE(BBa_K2507017). It worked better.

Reference

Daeffler, K. N., Galley, J. D., Sheth, R. U., Ortiz‐Velez, L. C., Bibb, C. O., & Shroyer, N. F., et al. (2017). Engineering bacterial thiosulfate and tetrathionate sensors for detecting gut inflammation. Molecular Systems Biology, 13(4), 923.


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