Difference between revisions of "Part:BBa K2507002"

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===Usage and Biology===
 
  
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===Contribution===
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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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[[File:T—TU_Eindhoven--S-Curve-TtrR-S.png|600px|]]
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''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 (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--third-TtrR-S.png|400px|]]
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''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.''
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2507002 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2507002 SequenceAndFeatures</partinfo>
  
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==Reference==
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[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.
  
 
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<h2 id="results">Results</h2>
 
 
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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.