Reporter

Part:BBa_K1769003

Designed by: Ho-Tsang Tsai   Group: iGEM15_NYMU-Taipei   (2015-09-15)
Revision as of 18:16, 27 October 2020 by Lx0402 (Talk | contribs)

Salicylic acid sensor

This reporter is used to characterize the promoter J61051. GFP is produced when we add salicylic acid.

Improvement

The composite part BBa_K3524005 of rephos project was improved based on the composite part BBa_K1769003 of the NYMU-Taipei igem team. The BBa_K1769003 part is a salicylic acid biosensor and there is an inducible promoter with GFP response as reporter. The GFP is also used as a reporter in our part BBa_K3524005, when salicylic acid was added in the culture, we can detect the expression of EGFP. Besides, we did some modification: transcription factor NahR and Psal promoter are added in front of the EGFP gene. NahR can specifically bind to salicylic acid to activate the Psal promoter.

Figure 5

Besides, chassis engineering bacteria E. coli is replaced by Tatumella citrea, which can provide phosphorus for crops. The engineering Tatumella citrea can commensalism with crops when crops rhizospheres secrete salicylic acid.

Contribution

To build up a regulable genetic circuit, we chose the bio-sensor (nahR gene and Psal promoter) for the detection of salicylic acid we adopted is originally on the chromosome of Pseudomonas Stutzeri. With the appearance of salicylic acid, the protein translated form nahR binds with PsaI promoter and initiates the expression of the enhancer green fluorescent protein (EGFP).

Figure 1

Our team has verified this bio-sensor worked well. The future iGEM teams can exchange the EGFP to other functional proteins to make some significant utiles. For example, our team choose an essential gene GAPDH, belongs to Tatumella citrea, which can provide phosphorus for crops[1][2] to replace the EGFP . As a result, in the presence of salicylic acid, GADPH can be expressed, and T. citrea can survive. Since salicylic acid is a root exudate, this genetic circuit that T. citrea can commensalism with crops.

Engineering Success

Build: Results of plasmids construction:

Table 1
                        *n represents for pTrc99k-nahR plasmids.                   
                        *G represents for pMW119-Psa1-EGFP plasmids.

We then used the successfully constructed plasmids to examine the effectiveness of the bio-sensor system. Some possible factors that led to failure 1.Mistakes in homologous recombination Unable to recombine the right fragments into the vector. 2.Mistakes in PCR During the amplification, the fragments may not be correctly amplified by PCR, so some of the recovered fragments are wrong fragments, which will cause problems when used for vector construction Test: The pTrc99k-NahR and pMW119-Psa1-EGFP plasmids were co-transformed into Tatumella Citrea and screened by Kanamycin resistance and Ampicillin resistance LB solid plates. The co-transformed strains were inoculated into fresh LB medium, cultured at 37°C until OD600 was approximately equal to 0.6. Different concentrations of salicylic acid (dissolved in absolute ethanol) were added. After 24 hours, observations with fluorescence microscope were used to show the regulatory effect of salicylic acid on the PsaI promoter and it was indicated by fluorescence intensity. Data collected：

Figure 2
                                 Figure 2. Microscopic view of fluorescence shown by Tatumella Citrea


Learn: 1.With different salicylic acid concentration: We measured the fluorescence intensity to obtain a quantitative result. The data is plotted as the following: the measured fluorescence intensity is divided by the number of cells (OD600) to obtain a homogenized fluorescence intensity. The fluorescence intensity of all samples added with salicylic acid is subtracted from the fluorescence intensity of the background without salicylic acid. It can be seen that from 1 μM to 100 μM salicylic acid, the fluorescence intensity rises sharply. Under the condition of 1000 μM salicylic acid, the fluorescence intensity decreased, which is presumably due to the influence of salicylic acid solvent (ethanol) on cell growth, or other unknown regulatory effects of salicylic acid and NahR.

Figure 3
                              Figure 3. Relative fluorescence unit under different Salicylic acid concentration

2.With different hours: With the same concentration of salicylic acid, we also measured the change of fluorescence intensity over time. It can be seen from the figure 3. that within 3 to 22 hours after the addition of salicylic acid, the fluorescence intensity increases successively. But it mostly stops increasing at 24 hours.

Figure 4
                              Figure 4. Relative fluorescence unit with different time after addition of salicylic acid

The mechanism we created did work out well, and the expressions of EGFP protein was detected. This proves that the potential of the bio-sensor system we made is effective,. The two factors we examined are the most convenient, and available test we could conduct. For the concentration of salicylic acid, we found out that at 100 μM, it shows the highst fluorescence intensity. This means that the system works the best in that range of salicylic acid concentration. For the time after addition of salicylic acid, the highst fluorescence unit is shown at 22 hours, the trend is mainly increasing. But after 22 hours, it is decreasing. It is best to leave the mechanism for 15-22 hours after the addition of salicylic acid so it can show the most effective results. In conclusion, we have succeeded made a new Part: BBa_K3524005 that improves the function of the existing Part: BBa_E0040. At first, the sequence of our part BBa_K3524005 is different from the existing part BBa_E0040, and the blast result is as figure 5. Furthermore, we gave the GFP protein a new function that can be regulated by the presence of salicylic acid.

Figure 5
                                Figure 5. The blast result of the sequence of GFP in Part: BBa_E0040 and Part: BBa_K3524005.

Refernce: [1] Whitelaw, M.a. “Growth Promotion of Plants Inoculated with Phosphate-Solubilizing Fungi.” Advances in Agronomy, 1999, pp. 99–151., doi:10.1016/s0065-2113(08)60948-7. [2] Bar-Yosef, B., et al. “Pseudomonas Cepacia-Mediated Rock Phosphate Solubilization in Kaolinite and Montmorillonite Suspensions.” Soil Science Society of America Journal, vol. 63, no. 6, 1999, pp. 1703–1708., doi:10.2136/sssaj1999.6361703x.


Usage and Biology

The NYMU-Taipei igem team characterize this inducible as an inducible promoter with GFP response as reporter. The following data was obtained by microplate reader:

The inducible promoter was induced with different concentration(0, 1.50E-07, 6.10E-07, 2.40E-06, 9.80E-06, 3.90E-05, 1.60E-04, 6.20E-04, 1.00E-03) every 30 minutes. We started the induction when the OD600 was 0.6-0.7. The fluorescence response was measured with plate reader.

The inducible promoter was induced with different concentration(0, 1.50E-07, 6.10E-07, 2.40E-06, 9.80E-06, 3.90E-05, 1.60E-04, 6.20E-04, 1.00E-03) every 30 minutes. We started the induction when the OD600 was 0.6-0.7. The fluorescence response was measured with plate reader. The red line indicate the Hill equation(P = Pmax*[SA]^n/([SA]^n+Km^n) ) fit of our dose-response data with error bar showing the standard deviation and the R square value of this fit is 0.99531. "n" indicates the hill coefficient(unitless), "Pmax" indicates the maximal promoter activity, and "K" is the salicylic acid concentration while P equals 1/2*Pmax

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 786
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 77
    Illegal NgoMIV site found at 618
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1941


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