Difference between revisions of "Part:BBa K2507002"
(→‎Contribution)
 
(7 intermediate revisions by 2 users not shown)
Line 8: Line 8:
  
 
<p>
 
<p>
−
TtrS/R are two basic parts which belong to the two-component systems. This system is from marine Shewanella species. After validate this system in laboratory Escherichia coli, this system can function as a tetrathionate sensor and reporter. Because of tetrathionate is an indicator of gut inflammation, this system can be used as a sensor of gut inflammation. Connect TtrR with sfGFP or chromoprotein， this system can response to tetrathionate by produce protein.
+
<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).  
−
 
+
 
<p/>
 
<p/>
  
 
</html>
 
</html>
 
<br/><br/>
 
<br/><br/>
−
<!-- Add more about the biology of this part here
 
−
===Usage and Biology===
 
  
−
<!-- -->
+
===Contribution===
 +
The [https://2021.igem.org/Team:TU-Eindhoven/Contribution iGEM team TU-Eindhoven 2021] further characterized the TtrS/R system by establishing a dose-response curve. Our project also consists of the two-component TtrS/R system with a different DNA sequence than noted above, however, the same amino acid sequence was used. Our TtrS/R system was transformed into <em> E. coli </em> BL21(DE3) cells for GFP and mCherry measurements. Subsequently, a small culture and a large culture were made. The GFP and mCherry concentrations were measured with a spectrophotometer and a full dose-response curve was constituted (Figure 1). On part page [https://parts.igem.org/Part:BBa_K3972000 BBa_K3972000] more details can be found on how this dose-response curve was established.
 +
 
 +
[[File:T—TU_Eindhoven--S-Curve-TtrR-S.png|600px|]]
 +
 
 +
''Figure 1. Dose-response curve TtrS/R system, under control of tetrathionate.''
 +
 
 +
The team of iGEM SHSBNU 2017 was not able to create such a dose-response curve. After analyzing our own results and the results of Daeffler et al.[1], we think it might be a result of overexpression of TtrR that will result in GFP expression, even though tetrathionate is not present. Corresponding to this behavior, the sensor will lose its sensitivity to tetrathionate (Figure 2 and Figure 3), as can also be seen in the results of iGEM SHSBNU. In contrast to the iGEM team SHSBNU 2017, we made use of the pLtetO-1 promoter, which is a leaky promoter. By not inducing this promoter, we are able to receive a full dose-response curve, making it sufficiently adjustable for our concept to work.
 +
 
 +
[[File:T—TU-Eindhoven--third-TtrR-S.png|400px|]]
 +
 
 +
''Figure 2. Induction of the TtrS/R system with different concentrations of doxycycline.''
 +
 
 +
[[File:T—TU-Eindhoven--fourth-TtrR-S-inducer.png|400px|]]
 +
 
 +
''Figure 3. Induction of the TtrS/R system with different concentrations of doxycycline, with and without tetrathionate.''
 +
 
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2507002 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2507002 SequenceAndFeatures</partinfo>
  
 +
==Reference==
 +
[1] 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 intestinal inflammation. Molecular Systems Biology, 13(4), 923.
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
Line 29: Line 44:
 
<html>
 
<html>
 
<br/>
 
<br/>
−
<h2 id="results">Results</h2>
 
 
<p>  </p>
 
<p>  </p>

Latest revision as of 13:01, 19 October 2021

TtrS

Background

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).



Contribution

The iGEM team TU-Eindhoven 2021 further characterized the TtrS/R system by establishing a dose-response curve. Our project also consists of the two-component TtrS/R system with a different DNA sequence than noted above, however, the same amino acid sequence was used. Our TtrS/R system was transformed into E. coli BL21(DE3) cells for GFP and mCherry measurements. Subsequently, a small culture and a large culture were made. The GFP and mCherry concentrations were measured with a spectrophotometer and a full dose-response curve was constituted (Figure 1). On part page BBa_K3972000 more details can be found on how this dose-response curve was established.

T—TU Eindhoven--S-Curve-TtrR-S.png

Figure 1. Dose-response curve TtrS/R system, under control of tetrathionate.

The team of iGEM SHSBNU 2017 was not able to create such a dose-response curve. After analyzing our own results and the results of Daeffler et al.[1], we think it might be a result of overexpression of TtrR that will result in GFP expression, even though tetrathionate is not present. Corresponding to this behavior, the sensor will lose its sensitivity to tetrathionate (Figure 2 and Figure 3), as can also be seen in the results of iGEM SHSBNU. In contrast to the iGEM team SHSBNU 2017, we made use of the pLtetO-1 promoter, which is a leaky promoter. By not inducing this promoter, we are able to receive a full dose-response curve, making it sufficiently adjustable for our concept to work.

T—TU-Eindhoven--third-TtrR-S.png

Figure 2. Induction of the TtrS/R system with different concentrations of doxycycline.

T—TU-Eindhoven--fourth-TtrR-S-inducer.png

Figure 3. Induction of the TtrS/R system with different concentrations of doxycycline, with and without tetrathionate.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1309
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 783
  • 1000
    COMPATIBLE WITH RFC[1000]

Reference

[1] 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 intestinal inflammation. Molecular Systems Biology, 13(4), 923.