Difference between revisions of "Part:BBa K2507008"

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We first tested whether the system work. Characterization experiments were performed aerobically. Bacteria were cultured overnight in a 96-deep well plate, 1ml LB media +antibiotics+different concentration of inducer(thiosulfate).  
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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.  
  
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<b>The conclusion is the system(ThsS/ThsR) works, while the leakage is very heavy.</b><br/>
 
  
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[[File: SHSBNU 17 40a02.jpg|200px|thumb|left|alt text]]
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<b>The conclusion is that while the system (ThsS/ThsR) works, the leaky expression is rather heavy.</b><br/>
  
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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.
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[[File: SHSBNU 17 40a02.jpg|600px|thumb|center|Figure 2]]
  
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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.  
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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.
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We thought that our thsR overexpression is originate from pSB4K5 which have several mutation at pSC101 sequence. It means pSB4K5 <b>is actually a high-copy plasmid! </b><br/>
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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.  
 +
 
 +
We realized that our thsR overexpression system is based on pSB4K5 which has several mutations in the pSC101 sequence, which means that pSB4K5 <b>is actually a high-copy plasmid! </b><br/>
 
  https://parts.igem.org/Part:pSB4K5:Experience
 
  https://parts.igem.org/Part:pSB4K5:Experience
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Due to the limited time, we didn’t have time to change the backbone to another low copy number plasmid, while we would try after iGEM Jamboree 2017.
 
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Then, We characterize the system at aerobic and anaerobic condition. We measured sfGFP intensity by flow cytometry.(Protocol的链接).The response curve of <i>E.coli</i> Top10 in aerobic and anaerobic condition seems almost the same while in <i>E.coli</i> Nissle 1917, gfp expression level is different in aerobic and anaerobic condition (Figure5)
 
  
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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. (http://2017.igem.org/Team:SHSBNU_China/Protocol). The response curves of <i>E. coli</i> Top10 under aerobic and anaerobic conditions seemed almost indistinguishable, while in <i>E. coli</i> Nissle 1917, the GFP expression level was different under aerobic and anaerobic conditions (Figure 5).
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[[File: SHSBNU 17 40a03.jpg|600px|thumb|center|Figure 3]]
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Figure 3. We characterized the ThsS/R system in <i>E. coli</i> Top10 and <i>E. coli</i> Nissle 1917 by measuring the sfGFP expression levels via flow cytometry.
  
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[[File: SHSBNU 17 40a03.jpg|200px|thumb|left|alt text]]
 
  
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Figure 3. We characterized ThsS/R system in <i>E.coli</i> Top10 and <i>E.coli</i> Nissle 1917 by sfGFP expression level measured by flow cytometry.
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[[File: SHSBNU 17 40a04.png|600px|thumb|center|Figure 4]]
  
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[[File: SHSBNU 17 40a04.png|200px|thumb|left|alt text]]
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Figure 4. We characterized the ThsS/R system by flow cytometry. The response curve seems different.
  
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Figure 4. We characterized ThsS/R system by flow cytometry. The response curve seems different
 
  
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[[File: SHSBNU 17 40a12.png|200px|thumb|left|change this]]
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[[File: The_last_one.png|600px|thumb|center|Figure 5]]
  
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Figure5. We cultivated E.coli Nissle 1917 overnight under aerobic or anaerobic condition. Ths/R-sfGFP seems act better under anaerobic condition.
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Figure5. We cultivated <i>E. coli</i> Nissle 1917 overnight under aerobic or anaerobic condition. Ths/R-sfGFP seems act better under anaerobic condition.
  
 
==Reference==
 
==Reference==

Latest revision as of 13:01, 1 November 2017


J23105-thsR-PphsA342-sfGFP

Usage and Biology

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.


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
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1313

Characterization

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

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

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! 

https://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. (http://2017.igem.org/Team:SHSBNU_China/Protocol). The response curves of E. coli Top10 under aerobic and anaerobic conditions seemed almost indistinguishable, while in E. coli Nissle 1917, the GFP expression level was different under aerobic and anaerobic conditions (Figure 5).


Figure 3

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

Figure 4. We characterized the ThsS/R system by flow cytometry. The response curve seems different.


Figure 5

Figure5. We cultivated E. coli Nissle 1917 overnight under aerobic or anaerobic condition. Ths/R-sfGFP seems act better under anaerobic condition.

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.

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

Schmidl SR, Sheth RU, Wu A, Tabor JJ (2014) Refactoring and optimization of light-switchable Escherichia coli two-component systems. ACS Synth Biol 3: 820 – 831

Vitvitsky V, Yadav PK, Kurthen A, Banerjee R (2015) Sulfide oxidation by a noncanonical pathway in red blood cells generates thiosulfate and polysulfides. J Biol Chem 290: 8310 – 8320