Part:BBa_K3995005

Ptat_AtzR

Ptat_AtzR

Usage and Biology

Ptat_AtzR is a coding sequence for expressing atzR. Cyanuric acid combination with AtzR can activate amilGFP expression to show the signs of fluorescence. The content of cyanuric acid is detected by detecting the fluorescence concentration. So Ptat_AtzR should be used with Pprovoin5_amilGFP.

Figure 1. Genetic design of our biosensor..

Construct design

The atzR is under Ptat promoter. The Ptat_atzR is inserted in the pUC57 mini vector to get plasmid A (Figure 2).

Figure 2. Ptat_AtzR box..

Figure 3. Schematic maps of Ptat_AtzR (sensor plasmid)..

BBa_K3995009

Usage and Biology

Tat promoter is a promoter from Pseudomonas aeruginosa PAO1. Shah and Naseby constructed plasmids carrying lux genes which are under the control of constitutive promoters and tested the strength of five different constitutive promoters (Plpp, Ptat, PlysS, PldcC, Pspc) with the method of bioluminescence-based measurement. They found that Promoter strength decreased in the order of Plpp > Ptat > PlysS > PldcC > Pspc during exponential phase whilst Ptat was stronger than Plpp during stationary phase. Stationary phase was observed from 12 h for all the strains and remained constant up to 48 h

BBa_K3995001

Usage and Biology

BBa_K3995001 is a coding sequence of atzR. Cyanuric acid combining with atzR can activates Pprovoin5 promoter. So atzR can act as a sensor.

BBa_K4030002

Usage and Biology

It is an transcription terminator derived from the E.coli rrB rRNA operon.

Experimental approach

Construction of recombinant plasmid

Figure 4. The electrophoresis results of pUC57_mini_atzR enzyme digestion by KpnI and MluI..

pUC57_mini plasmid and gene atzR were enzyme digestion. And then pUC57_mini backbone and atzR-fragment were connected by T4 ligase. Finally we did double enzyme digestion and sequencing for recombinant plasmid pUC57_mini_atzR to identify. Finally, we got correct plasmid. Transformation of plasmid A and plasmid B Plasmid A (pUC57-Pprovoin5-amilGFP) and plasmid B (pUC57-atzR) were transformed into a common competent cell to get bacteria C. And the bacteria C was used for the later functional test. Figure 4 shows the the bacteria C colony after transformation and selected by two antibiotics.

Figure 5. The bacteria C colony after transformation and selected by two antibiotics..

Proof of function

Table 1. Fluorescence intensity when the concentration of CYA equals to 30uM and the duration is 4 hours.

Figure 6. Histogram of the fluorescence intensity when the concentration of CYA equals to 30uM and the duration is 4 hours..

As seen from figure 6, comparing to the blank control, bacteria C presents an obvious higher fluorescence reaction to the cyanuric acid, the derivative from Atrazine. In such a case, it could indicate that our engineered bacteria could work for detecting cyanuric acid.

Table 2. Fluorescence intensity of bacteria C when when the duration is 4 hours under different concentration of the cyanuric acid.

Figure 7. Histogram of the fluorescence intensity of bacteria C when the duration is 4 hours under different concentration of the cyanuric acid..

Figure 8. Histogram of the fluorescence intensity of bacteria C when the duration is 6 hours under different concentration of the cyanuric acid..

In order to analyze the relationship between the concentration of cyanuric acid and the fluorescence intensity, we designed the control groups and collected the data as showing above. According to the histograms (Fig. 7 and Fig. 8), the fluorescence intensity shows a decreasing trend with the increase of concentration of cyanuric acid when we used the bacteria C for tests. Therefore, we speculate that the cyanuric acid might affect the growth of strains so that the higher the concentration of the cyanuric acid, the worse the growth of the bacteria, the less of the amount of the effective “biosensor”. In order to fully eliminate this impact, we introduced the concept of cell-free extraction and cell-free expression in the next stage of our project.

Figure 9. Curve of the fluorescence intensity of bacteria C against hours..

In order to analyze the relationship between the fluorescence intensity and the induction hours, we collected the data and drew the curves under various concentrations of cyanuric acid (CYA) as showing above.

In figure 9, we can see that basically there is an increasing trend of the fluorescence intensity of bacteria C as the induction hour increases. In addition, the curves also indicate that the appropriate detection hour for our biosensor to detect cyanuric acid would be 4 hours later where several curves tend to balance.

References

4.Liu, Xiangyang, et al. "Design of a transcriptional biosensor for the portable, on-demand detection of cyanuric acid." ACS synthetic biology 9.1 (2019): 84-94.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 548
  Illegal EcoRI site found at 1091
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 548
  Illegal EcoRI site found at 1091
  Illegal NheI site found at 124
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 548
  Illegal EcoRI site found at 1091
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 548
  Illegal EcoRI site found at 1091
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 548
  Illegal EcoRI site found at 1091
  Illegal AgeI site found at 938
  
• 1000
  COMPATIBLE WITH RFC[1000]