Difference between revisions of "Part:BBa K3972000"

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TtrS

E.coli codon-optimized TtrS(BBa_K3972000) and TtrR (BBa_K3972001) are two basic parts that are derived from the two-component system of the marine bacterium Shewanella Baltica. TtrS is the first component of this system and functions as the transmembrane sensor kinase (SK), which binds tetrathionate intracellular with high specificity. The second component, TtrR, is the DNA-binding response regulator (RR) that is activated by TtrS and subsequently binds to the pTtrB185-269 promoter(BBa_K2507019) as a phosphorylated dimer, which can induce gene expression (Figure 1) [1].

Figure 1. The two-component system TtrR TtrS.

Usage and Biology

Upon binding of the ligand tetrathionate, TtrS undergoes a conformational change, resulting in autophosphorylation of the transmembrane receptor protein. The phosphorylated TtrS activates the response regulator TtrR by transferring the phosphoryl group. The formed phosphorylated TtrR dimerizes and then acts as a transcription factor, which can bind to the pTtrB185-269 promoter with high selectivity. To characterize the activation and subsequent behavior of the TtrR/S system, a superfold GFP protein was expressed under the regulation of the PttrB185-269 promoter. The IPTG inducible promoter Ptac (BBa_K2558004), regulates TtrS, while TtrR is regulated by a leaky anhydrotetracycline inducible promoter pLtetO-1 (BBa_K3332034) [1].

Characterization

Expression
This part is optimized for the expression of TtrS in E.coli cells. As can be seen on the agar plate below (figure 2), the plasmid containing the TtrS protein was successfully transformed into E.coli BL21(DE3) cells. To successfully co-transform the TtrR and TtrS plasmids in BL21(DE3) cells, multiple attempts were needed to discover that SOC medium should be used to recover the cells after heat shock.

Figure 2. Agar plate with co-transfected TtrR TtrS in BL21(DE3) cells.

For protein expression, the cells were first harvested in a small culture and hereafter, in a large culture (figure 3 &4). The conditions used during these culturing experiments were based on literature. [1]

Figure 3. Small culture of TtrR TtrS in BL21(DE3).

Figure 4. Large culture of TtrR TtrS in BL21(DE3).

The expression of TtrS was tested using the two-component system TtrR/S. When TtrS is expressed and tetrathionate is present, TtrS will activate the production of TtrR, which subsequently expresses the superfold GFP gene. Furthermore, constitutively expressed mCherry will be used to normalize the sfGFP expression. To test the complete two-component system, multiple concentrations of tetrathionate inducer were used, combined with a constant concentration of doxycycline and IPTG. As can be seen in the graph below (figure 5), all samples display the same fluorescence intensity. The chosen doxycycline concentration was based on information from a research paper of Mazumder et al. [2]. The used IPTG concentration was based on consultation with our supervisors and a concentration was used, which is usually added in the lab at our university. Since the article of Daeffler et al. included a dose-response curve of tetrathionate, the used tetrathionate concentrations were based on this curve.

Figure 5. sfGFP measurement.

Since all samples displayed the same fluorescence intensity, the experiment was repeated with concentrations of IPTG, tetrathionate, and doxycycline shown in figure 6. The corresponding sfGFP graph can be seen in figure 7. As can be concluded from this graph, the presence of a higher concentration of doxycycline influences the fluorescence intensity.

File:T—TU-Eindhoven--second-TtrR-S-inducers.png

Figure 6. Concentrations of inducers.

Figure 7.sfGFP measurement.

To overcome this problem, two experiments were conducted, to understand which concentration of doxycycline would be optimal to use for the two-component system to function as a tetrathionate sensor. As can be seen in figure 8, doxycycline concentrations below 10 ng/mL show the same fluorescence intensity, while the doxycycline concentrations of 100 and 250 ng/mL give higher sfGFP expression levels. Since the tetrathionate concentrations were kept the same in all samples, it can be concluded that the optimal doxycycline concentration should be lower than 100 ng/mL. Figure 9 shows the doxycycline concentrations used in this experiment. As can be seen in figure 10, a concentration of 250 ng/mL doxycycline indeed overexpresses the sfGFP, even without the presence of tetrathionate. The presence of tetrathionate gives a 7-fold increase of signal with induction up to 10ng/mL. Between below 100 ng/mL samples, there is no difference between the GFP expression. After consulting with our supervisors, the optimal dox concentration for the sensor was 0 ng/mL. This corresponds with the article of Daeffler et al., since no inducer was used for the expression of TtrR. [1]

Figure 8. sfGFP measurement.

Figure 9. Concentrations of inducers.

File:T—TU-Eindhoven--fourth-TtrR-S.png

Figure 10. sfGFP measurement.

Using the optimal doxycycline concentration, the full dose-response curve with induction of tetrathionate was defined, using the concentrations of doxycycline, tetrathionate, and IPTG shown in figure 11. The full dose-response curve can be seen in figure 12.

File:T—TU-Eindhoven--S-Curve-inducers.png

Figure 11. Concentrations of inducers for the dose-response curve.

Figure 12. Dose-response curve of tetrathionate.

References

[1] Daeffler, K. N., Galley, J. D., Sheth, R. U., Ortiz-Velez, L. C., Bibb, C. O., Shroyer, N. F., Britton, R. A., & Tabor, J. J. (2017). Engineering bacterial thiosulfate and tetrathionate sensors for detecting gut inflammation. Molecular systems biology, 13(4), 923. https://doi.org/10.15252/msb.20167416 [2] Mostafizur Mazumder, Katherine E. Brechun, Yongjoo B. Kim, Stefan A. Hoffmann, Yih Yang Chen, Carrie-Lynn Keiski, Katja M. Arndt, David R. McMillen, G. Andrew Woolley (2015). An Escherichia coli system for evolving improved light-controlled DNA-binding proteins. Protein Engineering, Design, and Selection, Volume 28, Issue 9, Pages 293–302, https://doi.org/10.1093/protein/gzv033

==Sequence and Features=- Sequence and Features