Experiment
Experiment (build) & Result (Test)
We first test the function of gut inflammation thiosulfate and tetrathionate sensors by sfGFP expression level in E. coli Top10 and E. coli Nissle 1917. And then we test what if we mix “excrement”(actually curry) with colorful bacteria(with chromoproteins). Then we replace sensors’ reporter by gfasPurple,asPink,amilCP. While thiosulfate+chromoprotein works well, tetrathionate sensor does’t response well. Then we change the reporter to a dark-green molecular-protoviolaceinic acid, it works well. We demonstrate that E. coli carrying our product can be a good reporter system to detect gut inflammation.
The function of ThsS/R & TtrS/R
ThsS/R
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. 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.
Because thiosulfate is an indicator of intestinal inflammation (Levitt et al, 1999; Jackson et al, 2012; Vitvitsky et al, 2015), this system can be used as a sensor for intestinal inflammation.
After validating the system in the laboratory strains Escherichia coli Top10 and E. coli Nissle 1917, we confirmed that the system indeed works as a thiosulfate sensor, as intended. By linking thsR with sfgfp (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).
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.
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.
The conclusion is that while the system (ThsS/ThsR) works, the leaky expression is rather heavy.
Figure 2. Characterization of the ThsS/R system by observing the sfGFP expression levels. We added 1mM, 0.1mM, 0.01mM and 0 Na2S2O3. The results demonstrate a response with rather heavy leaky expression without inducer.
Previously, Schmidl et al. have shown that thsR 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.
We realized that our thsR overexpression system is based on pSB4K5 which has several mutations in the pSC101 sequence, which means that pSB4K5 is actually a high-copy plasmid! http://parts.igem.org/Part:pSB4K5:Experience
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.
Next, we characterized the system under aerobic and anaerobic conditions. We measured sfGFP intensity by flow cytometry. (https://2017.igem.org/Team:SHSBNU_China/Protocol). The response curve of E. coli Top10 in aerobic and anaerobic condition seems almost the same while in E. coli Nissle 1917, gfp expression level is different in aerobic and anaerobic condition (Fighue5)
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.
Figure 4. We characterized the ThsS/R system by flow cytometry. The response curve seems different.
Figure5. We cultivated E. coli Nissle 1917 overnight under aerobic or anaerobic condition. Ths/R-sfGFP seems act better under anaerobic condition.
TtrS/R
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 gut inflammation.
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.
Figure 6. 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.
Figure7. 待补充
Figure8. We cultivated E. coli Nissle 1917 overnight under aerobic or anaerobic condition. Ths/R-sfGFP seems act better under anaerobic condition. While TtrS/R-sfGFP acted much worse than ThsS/R. So in TtrS/R system, we replaced gfp gene by vioABDE(BBa_...17). It worked better.
Choosing an Appropriate Chromoprotein
补充gfp需要氧气成熟的文献，一段文字
Human excrements have colors ranging from yellow to brown and even black. Thus, the bacteria in excrement must show a contrasting color in order to efficiently indicate the presence of gut inflammation.
At the beginning, we tested six different chromoproteins to find the most suitable candidate which will possibly be used as the reporter. We used pSEVA321(From Bluepha) as the backbone with a strong constitutive promoter. The following proteins were tested:
1. BBa_K1033910"fwYellow yellow chromoprotein"
2. BBa_K1033916 amajLime yellow-green chromoprotein"
3. BBa_K592010"amilGFP yellow chromoprotein"
4. BBa_K1033919 "gfasPurple purple chromoprotein"
5. BBa_K1033932 "spisPink pink chromoprotein"
6. BBa_K592009"amilCP blue chromoprotein"
Figure 9. Schematic diagram of chromoprotein devices.
After creating bacteria with different colors, we decided to conduct a color test with these bacteria to show which bacteria can best show the color.
First, we grew cultures of bacteria expressing each of the chromoprotein constructs we designed,
Figure10. Overnight-cultivated E. coli with different chromoproteins’ plasmids.
After centrifugation, we obtained the following bacterial pellets:
Figure11. Centrifuged Bacteria
Subsequently, we used curry to imitate the color of excrement, and mixed the bacteria with the resulting paste, obtaining the following result:
Figure12. Mix centrifuged bacteria with 1g curry and 500ul water.
The pink, blue, and purple chromoproteins showed the most obvious results.
Thus, we decided to use pink, blue, and purple chromoproteins as indicators.
Replace sfGFP by chromoproteins
After chosen three chromoproteins, we used Golden Gate method to change gfp gene by chromoprotein genes. We successfully constructed BBa_K2507009, K2507010, K2507011, K2507014, K2507015, K2507016. And Co-transformed with BBa_ K2507004/ BBa_K2507006 into E. coli Top10.
Figure 13 a. Schematic diagram of the ligand-induced signaling through ThsS/R and the plasmid-borne implementation of the sensor components. We combine BBa_ K2507004 with BBa_K2507009, BBa_K2507010, BBa_K2507011. b. Schematic diagram of ligand-induced signaling through TtrS/R and plasmid-borne implementation of the sensor components. We combine BBa_ K2507006 with BBa_K2507014, BBa_K2507015, BBa_K2507016.
We cultivated the bacteria in different concentration of thiosulfate or tetrathionate. After validating the system in the laboratory strains E. coli Top10, we confirmed that the system with chromoproteins indeed works as a thiosulfate sensor, as intended. By linking thsR with chromoprotein genes (BBa_K2507009, BBa_K2507010, BBa_K2507011) , this system can respond to thiosulfate by producing a signal visible to the naked eye.
Figure14. 待补充 ThsS/R-chromoproteins works! Whilc TtrS/R-chromoproteins doesn’t work.
Replace chromoproteins’ gene by vioAVBDE
Then we found than 2009 Cambrigde iGEM team researched several pigments. So we want to use violacien to be the reporter. After searching former iGEM part distribution kit in Beijing, finally we get the plasmid BBa_K274003 from Peking iGEM team a 2010 iGEM part distribution kit. BBa_K274003 coding vioABDE which coding enzymes produce a precursor of violacien- protoviolaceinic acid, which is dark-green.
Figure 15. The violacein biosynthetic pathway(From http://parts.igem.org/Part:BBa_K274002) .Genes for violacein biosynthesis are arranged in an operon consisting of vioA, vioB, vioC, vioDand vioE. VioA generates an IPA imine from L-tryptophan and VioB converts the IPA imine into a dimer. VioE then acts by transforming the dimer into protodexyviolaceinic acid (PVA), which can be spontaneously converted into a green pigment called deoxychromoviridans. VioD and VioC hydroxylate PVA to form violacein.
Figure16. 待补充
待补充
总结 Ths+色素蛋白ok，Ttr+protoviolaceinic acid 好用。
Medicine Quantity
Bacteria Quantity
On November 16, 2011, Pharma Zentrale company sent an application to FDA and states that each their capsule contains E.coli strain 1917 corresponding to 2.5-25*10^9 viable cells.
The total weight of one cell is 9.5e-13g.
Therefore, the weight of bacteria in one capsule is about 0.024g.
Color Testing
a. We cultured engineered bacteria overnight and then centrifuged them.
b. We added 1g curry, 500ul H2O and centrifuged bacteria.
c. We added 2g curry, 500ul H2O and centrifuged bacteria.
1.2. 50ml LB+ TtrS/R- vio system in E.coli Top10
3.4. 50ml LB+ ThsS/R- Pink system in E.coli Top10
Pink: spisPink vio:protoviolaceinic acid
取过夜的下列菌液100ul涂板
Therefore, there are 52*10^9 bacteria in 20ml ttr-vio bacteria liquid, and its weight is about 0.050g.
There are 76*10^9 bacteria in 20ml ths-pink bacteria liquid, and its weight is about 0.072g, and 50ml is about 0.180g
Dose
Therefore, it is effective to use 25*10^9 bacteria. We decided to make it 25*10^9 bacteria per capsule.
Replace sfGFP by chromoproteins
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.
Frederick C. Neihardt (1996), Escherichia coli and Salmonella: Cellular and Molecular Biology (1st volume), ASM Press. Available at: http://kirschner.med.harvard.edu/files/bionumbers/Composition%20of%20an%20average%20E.%20coli%20Br%20cell-Neudhart.pdf
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. Biochemistry 51: 6804 – 6815
Levitt MD, Furne J, Springfield J, Suarez F, DeMaster E (1999) Detoxification of hydrogen sulfide and methanethiol in the cecal mucosa. J Clin Invest 104: 1107 – 1114
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Vitvitsky V, Yadav PK, Kurthen A, Banerjee R (2015) Sulfide oxidation by a noncanonical pathway in red blood cells generates thiosulfate and polysulfides.
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