Difference between revisions of "Part:BBa K2507002"
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===Contribution===
 
===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.  
 
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.  
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''Figure 1. Dose-response curve TtrS/R system, under control of tetrathionate.''
 
''Figure 1. Dose-response curve TtrS/R system, under control of tetrathionate.''
  
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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 as can be seen in the results of iGEM SHSBNU (Figure 2). 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.
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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.
  
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[[File:T—TU_Eindhoven--S-Curve-TtrR-S.png|600px|]]
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[[File:T—TU-Eindhoven--third-TtrR-S.png|400px|]]
  
 
''Figure 2. Induction of the TtrS/R system with different concentrations of doxycycline.''
 
''Figure 2. Induction of the TtrS/R system with different concentrations of doxycycline.''
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[[File:T—TU-Eindhoven--fourth-TtrR-S-inducer.png|400px|]]
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''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>

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.