Difference between revisions of "Part:BBa K2507002"
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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. | 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: | + | [[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.'' | ||
| − | [[File: | + | [[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.'' | ''Figure 3. Induction of the TtrS/R system with different concentrations of doxycycline, with and without tetrathionate.'' | ||
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.
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.
Figure 2. Induction of the TtrS/R system with different concentrations of doxycycline.
Figure 3. Induction of the TtrS/R system with different concentrations of doxycycline, with and without tetrathionate.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1309
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 783
- 1000COMPATIBLE 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.