Part:BBa_K3972000

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, which binds tetrathionate extracellularly with high specificity. The second component, TtrR, is the DNA-binding response regulator (RR) that is activated by TtrS and subsequently binds to the optimized pTtrB185-269 promoter (BBa_K2507019) as a phosphorylated dimer, which can induce gene expression (Figure 1) [1].

Figure 1. The two-component system TtrS/R.

Usage and Biology

The receptor protein TtrS can be activated in the presence of tetrathionate. After 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 phosphor group. The formed phosphorylated TtrR dimerizes and then acts as a transcription factor which can bind to the PttrB185-269 promoter. To characterize the activation 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
The DNA sequence for TtrS is optimized for the expression of TtrS in E.coli cells. As can be seen on the agar plates below (Figure 2), the plasmid containing the TtrS protein was successfully transformed into E.coli BL21 (DE3) cells and also co-transformed together with TtrR in E. Coli BL21 (DE3) cells. To successfully co-transform the TtrR and TtrS plasmids in BL21(DE3) cells, multiple attempts were required to discover that SOC medium should be used to recover the cells after heat shock, instead of LB medium.

Figure 2. Agar plates with (a) transfected TtrS in BL21 (DE3) cells and with (b) co-transfected TtrR TtrS in BL21(DE3) cells.

For protein expression, the cells were first multiplied in a small culture and thereafter, in a large culture in which they were induced with IPTG (Figure 3 & 4). The conditions used during these culturing experiments were based on literature [1].

Figure 3. Small cultures of TtrR TtrS in BL21(DE3). All four small cultures contain the TtrR/S sensing system.

Figure 4. Large culture of TtrR TtrS in BL21(DE3) induced with various inducer concentrations.

The expression of TtrS was tested using the entire two-component system TtrR/S. As described above, when TtrS is expressed and tetrathionate is present, TtrS will activate TtrR, which subsequently activates the expression of the superfold GFP gene. Furthermore, constitutively expressed mCherry will be used to normalize the sfGFP expression by dividing by the theoretical maximum of emission intensities of each sample. To characterize the complete two-component system, multiple concentrations of tetrathionate inducers were used, combined with a constant concentration of doxycycline and IPTG. As can be seen in the graph below (Figure 5), all samples displayed a similar fluorescence intensity. The tetrathionate concentrations used to generate a dose-response curve were based on the concentrations used in literature as described by Daeffler et al.[1]. Though the chosen doxycycline concentration was based on information from a research paper written by Mazumder et al. [2] and the used IPTG concentration was determined based on consultation with our supervisors, and on what concentration is conveniently used in our lab. Both these concentrations are higher than prescribed by the original paper [1].

Figure 5. Dose-response curve measured in BL21 (DE3) lysate, induced with 0.1 mM IPTG,250 ng/mL dox and various concentrations of tetrathionate. The GFP emission (at 512 nm) is normalized on the mCherry emission at 610 nm.

It is not clear what caused these results. Since the co-transformations worked and the ordered plasmids had not been changed, we suspected the inducers to cause these unexpected results. Therefore, eight different combinations of Ttr, doxycycline (dox), and IPTG were tested for induction, by measuring the sfGFP fluorescence. Therefore, the experiment was repeated with varying concentrations of IPTG, tetrathionate, and doxycycline and sfGFP intensities were measured (Figure 6).

From these experiments can be concluded that the sensor is working properly; however, the concentration of 250 ng/ml doxycycline causes overexpression of the TtrR proteins, resulting in high GFP expression.

Figure 6. a) Signal measured in BL21 (DE3) lysate, induced with 0.1 mM IPTG, with and without doxycycline (250 ng/ml) and with and without tetrathionate (1 mM). b) Signal measured in BL21 (DE3) lysate, induced with 0.01 mM IPTG, with and without doxycycline (250 ng/ml) and with and without tetrathionate (1 mM). The GFP emission (at 512 nm) is normalized on the mCherry emission at 610 nm.

To overcome this problem, two experiments were performed to determine the optimal concentration of doxycycline for minimal background fluorescence, and a maximal tetrathionate sensor performance of the two-component system (Figure 7). As can be concluded from these results, doxycycline concentrations of 100 and 250 ng/ml have higher sfGFP intensity compared to below 10 ng/mL while the tetrathionate concentrations were constant for all samples. According to the previous experiment, those higher sfGFP intensities result in overexpression of the sfGFP. Therefore, it could be concluded that the optimal doxycycline concentration should be below 10 ng/mL.

Figure 7. Signal measured in BL21 (DE3), induced with 0.1 mM IPTG, 1 mM tetrathionate and various concentrations of doxycycline. The GFP emission (at 512 nm) is normalized on the mCherry emission at 610 nm.

Based on these results, we choose to further test these doxycycline concentrations with and without tetrathionate to discover which of these concentrations have the largest difference between the background fluorescence and the sfGFP fluorescence from the induced cells (Figure 8). After discussions with our supervisors and based on these results and the literature, we decided to further test the sensor without dox [1]. Furthermore, these results show that a concentration of 250 ng/mL doxycycline indeed causes overexpression of sfGFP, even without the presence of tetrathionate.

Figure 8. Signal measured in BL21 (DE3), induced with 0.1 mM IPTG, with and without 1 mM tetrathionate and various concentrations of doxycycline. The GFP emission (at 512 nm) is normalized on the mCherry emission at 610 nm.

With the optimal inducer concentration of 0 ng/mL doxycycline, we performed one more characterization experiment with varying tetrathionate concentrations, which resulted in the dose-response curve (Figure 9).

Figure 9. Normalized GFP emission of the TtrS/R system induced with various concentrations of doxycycline, IPTG and tetrathionate. a) Dose-response curve measured in BL21 (DE3), induced with 0.1 mM IPTG, 0 ng/mL doxycycline and different concentrations tetrathionate. In addition a positive control (0.1 mM IPTG, 250 ng/mL doxycycline and 1 mM tetrathionate) and a negative control (no IPTG, doxycline and tetrathionate) were added. b) The dose-response from figure 9a plotted in a line-plot with logarithmic scale (EC50 = 49.1 +- 2.3 uM, n=1). The GFP emission (at 512 nm) is normalized on the mCherry emission at 610 nm.

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

Assembly Compatibility:

• 10
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  Illegal EcoRI site found at 1561
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  Illegal PstI site found at 1028
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  Illegal PstI site found at 1381
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• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1561
  Illegal NheI site found at 1309
  Illegal PstI site found at 1013
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  Illegal PstI site found at 1084
  Illegal PstI site found at 1381
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• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1561
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1561
  Illegal PstI site found at 1013
  Illegal PstI site found at 1028
  Illegal PstI site found at 1084
  Illegal PstI site found at 1381
  Illegal PstI site found at 1408
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1561
  Illegal PstI site found at 1013
  Illegal PstI site found at 1028
  Illegal PstI site found at 1084
  Illegal PstI site found at 1381
  Illegal PstI site found at 1408
  Illegal AgeI site found at 783
  
• 1000
  COMPATIBLE WITH RFC[1000]