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| | <p>SHSBNU_China has sent the parts from China via Genscript, by DHL, and the tracking number is 2016823992.</p> | | <p>SHSBNU_China has sent the parts from China via Genscript, by DHL, and the tracking number is 2016823992.</p> |
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| − | <p style = "font-family:arial;color:#444444;font-size:32px; text-align: center"> BBa_K2507004 J23104-thsS </p> | + | |
| − | <p style = "font-family:arial;color:#444444;font-size:32px; text-align: center"> BBa_K2507008 J23105-thsR-PphsA-sfGFP</p>
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| − | <p style = "font-family:arial;color:#000000;font-size:20px; text-align: left"> Usage and Biology</p>
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| − | <p>ThsS (BBa_K2507000) and ThsR (BBa_K2507001), both codon-optimized for <i>E. coli,</i> are two basic parts which belong to the two-component system from the marine bacterium <i>Shewanella halifaxensis.</i> ThsS is the membrane-bound sensor kinase (SK) which can sense thiosulfate outside the cell, and ThsR is the DNA-binding response regulator(RR). PphsA(BBa_K2507018) is a ThsR-activated promoter which is turned on when ThsR is phosphorylated by ThsS after ThsS senses thiosulfate.</p>
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| − | <p>Because thiosulfate is an indicator of gut inflammation (Levitt et al, 1999; Jackson et al, 2012; Vitvitsky et al, 2015), this system can be used as a sensor for gut inflammation. </p>
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| − | <p style = "font-family:arial;color:#000000;font-size:20px; text-align: left"> Characterization</p>
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| − | <p>After validating the system in the laboratory strains <i>Escherichia coli</i> Top10 and <i>E. coli</i> Nissle 1917, we confirmed that the system indeed works as a thiosulfate sensor, as intended. By linking <i>thsR</i> with <i>sfgfp</i> (BBa_K2507008), 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, either under normal or UV light, such as sfGFP, chromoproteins (spisPink-pink chromoprotein, gfasPurple-purple chromoprotein, amilCP-blue chromoprotein) or a dark-green small-molecule pigment (protoviolaceinic acid).</p>
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| − | <img src="https://static.igem.org/mediawiki/2017/4/4f/SHSBNU_17_40a01.jpg"/>
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| − | <p>Figure 1. Schematic diagram of the ligand-induced signaling through ThsS/R and the plasmid-borne implementation of the sensor components. ThsS/R was tested by introducing BBa_K2507004 into the pSB4K5 backbone and BBa_K2507008 into the pSB1C3 backbone. We submitted all of the parts to the iGEM registry in pSB1C3.</p>
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| − | <p>We first tested whether the system works as intended. Characterization experiments were performed aerobically. Bacteria were cultured overnight in a 96-deep-well-plate, with 1ml LB media + antibiotics + different concentrations of inducer (thiosulfate) in each well. </p>
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| − | <p><strong>The conclusion is that while the system (ThsS/ThsR) works, the leaky expression is rather heavy.</strong></p>
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| − | <img src="https://static.igem.org/mediawiki/2017/4/4a/SHSBNU_17_40a02.jpg"/>
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| − | <p>Figure 2. Characterize thsS/R system by sfGFP expression level. We add 1mM,0.1mM,0.01mM and NA Na2S2O3, it shows response while the leakage is heavey.</p>
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| − | <p>Previously, Schmidl et al. have shown that <i>thsR</i> overexpression in the absence of the cognate SK and input can strongly activate the output promoter (Schmidl et al, 2014), possibly due to RR phosphorylation by alternative sources (small molecules, non-cognate SKs), or low-affinity binding by non-phosphorylated RRs. </p>
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| − | <p>We realized that our thsR overexpression system is based on pSB4K5 which has several mutations in the pSC101 sequence, which means that pSB4K5 <strong>is actually a high-copy plasmid! </strong><a href="http://parts.igem.org/Part:pSB4K5:Experience">http://parts.igem.org/Part:pSB4K5:Experience</a></p>
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| − | <p>Due to the limited time, we were not able to change the backbone to another low-copy plasmid, but we will certainly do it after the 2017 iGEM Jamboree.</p>
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| − | <p>Next, we characterized the system under aerobic and anaerobic conditions. We measured sfGFP intensity by flow cytometry. (<a href="https://2017.igem.org/Team:SHSBNU_China/Protocol"> https://2017.igem.org/Team:SHSBNU_China/Protocol</a>). The response curves under aerobic and anaerobic conditions seemed almost indistinguishable. </p>
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| − | <img src="https://static.igem.org/mediawiki/2017/2/29/SHSBNU_17_40a03.jpg"/>
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| − | <p>Figure 3. We characterized the ThsS/R system in E. coli Top10 and E. coli Nissle 1917 by measuring the sfGFP expression levels via flow cytometry. </p>
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| − | <img src="https://static.igem.org/mediawiki/2017/f/fb/SHSBNU_17_40a04.jpg"/>
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| − | <p>Figure 4. We characterized the ThsS/R system by flow cytometry.</p>
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| − | <p style = "font-family:arial;color:#000000;font-size:20px; text-align: left"> Reference</p>
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| − | <p><span>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. </span><i>Molecular Systems Biology, 13</i><span>(4), 923.</span></p>
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| − | <p><span>Jackson MR, Melideo SL, Jorns MS (2012) Human sulfide: quinone oxidoreductase catalyzes the first step in hydrogen sulfide metabolism and produces a sulfane sulfur metabolite. </span><i>Biochemistry</i><span>51: 6804 – 6815 </span></p>
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| − | <p><span>Levitt MD, Furne J, Springfield J, Suarez F, DeMaster E (1999) Detoxification of hydrogen sulfide and methanethiol in the cecal mucosa.</span><i>J Clin Invest</i><span>104: 1107 – 1114 </span></p>
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| − | <p><span>Schmidl SR, Sheth RU, Wu A, Tabor JJ (2014) Refactoring and optimization of light-switchable Escherichia coli two-component systems. </span><i>ACS Synth Biol</i><span>3: 820 – 831</span></p>
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| − | <p><span>Vitvitsky V, Yadav PK, Kurthen A, Banerjee R (2015) Sulfide oxidation by a noncanonical pathway in red blood cells generates thiosulfate and polysulfides.</span><i>J Biol Chem</i><span></span>290: 8310 – 8320 </p>
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| − | <p style = "font-family:arial;color:#444444;font-size:32px; text-align: center"> BBa_K2507004 J23104-thsS </p>
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| − | <p style = "font-family:arial;color:#444444;font-size:32px; text-align: center"> BBa_K2507008 J23105-thsR-PphsA-sfGFP</p>
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| − | <p style = "font-family:arial;color:#000000;font-size:20px; text-align: left"> Usage and Biology</p>
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| − | <p><i>E. coli</i>-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 <i>Shewanella baltica.</i> 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.</p>
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| − | <p>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 gut inflammation.</p>
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| − | <p style = "font-family:arial;color:#000000;font-size:20px; text-align: left"> Characterization</p>
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| − | <p>We first validated that this system can function as a tetrathionate sensor and reporter in the laboratory strains <i>Escherichia coli</i> Top10 and <i>E. coli</i> Nissle 1917.</p>
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| − | <img src="https://static.igem.org/mediawiki/2017/1/1b/SHSBNU_17_40a05.jpg"/>
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| − | <p>Figure 1. Schematic of ligand-induced signaling through TtrS/R and plasmid design of the sensor components. TtrS/R were tested under the situation BBa_K2507006 was in pSB4K5 backbone and BBa_K2507013 was in pSB1C3 backbone. We submitted the parts all to the iGEM registry in pSB1C3.</p>
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| − | <img src="https://static.igem.org/mediawiki/2017/c/c4/SHSBNU_17_40a06.jpg"/>
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| − | <p><i>Figure2</i></p>
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| | <p style = "font-family:arial;color:#000000;font-size:20px; text-align: left"> Reference</p> | | <p style = "font-family:arial;color:#000000;font-size:20px; text-align: left"> Reference</p> |
| | <p><span>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.</span><i>Molecular Systems Biology,</i><span>13(4), 923.</span></p> | | <p><span>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.</span><i>Molecular Systems Biology,</i><span>13(4), 923.</span></p> |
