Difference between revisions of "Part:BBa K2507012"

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==Characterization==
 
==Characterization==
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After validated this system in laboratory <i>Escherichia coli</i> Top10 and E.coli Nissle 1917, this system worked as a thiosulfate sensor. Link <i>thsR</i> with <i>sfgfp</i> (BBa_K2507008), chromoprotein genes (BBa_K2507009, BBa_K2507010, BBa_K2507011) or vioABDE(BBa_K2507012), this system can response to thiosulfate by produce sfGFP, chromoproteins (spisPink-pink chromoprotein, gfasPurple-purple chromoprotein, amilCP-blue chromoprotein) or dark-green small molecular(protoviolaceinic acid).
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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).
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[[File: SHSBNU 17 40a10.jpg|200px|thumb|left|alt text]]
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Figure 1. Schematic of ligand-induced signaling through ThsS/R and plasmid design of the sensor components. ThsS/R were tested under the situation BBa_K2507004 was in pSB4K5 backbone and BBa_K2507012 was in pSB1C3 backbone. We submitted the parts all to the iGEM registry in pSB1C3.
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[[File: SHSBNU 17 40a10.jpg|600px|thumb|center|Figure 1]]
  
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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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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_K2507012 into the pSB1C3 backbone. We submitted all of the parts to the iGEM registry in pSB1C3.
  
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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 40a22.jpg|200px|thumb|left|alt text]]
 
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Figure 2. Characterize thsS/R system by protoviolaceinic expression level. We add 1mM,0.1mM,0.01mM and NA Na2S2O3, it shows response while the leakage is heavey.
 
  
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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.  
+
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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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/>
+
 
 +
 
 +
<b>The conclusion is that while the system (ThsS/ThsR) works, the leaky expression is rather heavy.</b><br/>
 +
[[File: SHSBNU 17 40a22.jpg|600px|thumb|center|Figure 2]]
 +
Figure 2. Characterization of the ThsS/R system by observing the chromoprotein 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 <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 in aerobic and anaerobic condition seems
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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.
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图3
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Figure 3. We characterized ThsS/R system in E.coli Top10 and E.coli Nissle 1917 by sfGFP expression level measured by flow cytometry.
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图4
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Figure 4. We characterized ThsS/R system by flow cytometry.
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==Reference==
 
==Reference==

Latest revision as of 12:55, 1 November 2017


J23105-thsR-PphsA342-BBa_K274003

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
    Illegal NheI site found at 2312
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 5097
    Illegal AgeI site found at 5293
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 6522
    Illegal SapI.rc site found at 6597

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_K2507012 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 chromoprotein 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.

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