Part:BBa_K3995004

Pprovoin5_amilGFP

Pprovoin5_amilGFP

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.

Figure1.Genetic design of biosensor.

Construct Design

amilGFP is key functional factors that show the signs of fluorescence which is controlled by a Pprovoin5 promoter (Figure 2). The Pprovoin5_amilGFP is inserted in the pUC57 mini vector to get plasmid A (Figure 3).

Figure 2. Pprovoin5_amilGFP box..

Figure 3. Schematic maps of Pprovoin5_amilGFP (reporter plasmid)..

Usage and Biology

This operator region has a leftward-facing repressible promoter and a rightward-facing activated promoter. AtzR remains bound to the DNA. In the natural system, the left-facing promoter is upstream of the atzR CDS, and atzR is autoregulatory; in the presence of cyanuric acid, the binding of AtzR changes to cause the right-facing promoter to become activated, allowing for the expression of the downstream atzDEF CDS. The presence of cyanuric acid "frees" AtzR from being bound to the atzDEF promoter so polymerase can now bind and transcribe following gene. AtzR remains bound to the atzR promoter region even in the presence of cyanuric acid. Note that high glnK concentrations are also needed, as glnK is a co-activator of downstream operator.

BBa_K592010

Usage and Biology

This part is useful as a reporter and it naturally exhibits strong yellow color when expressed.

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 enzyme digestion and PCR.Left:Lane pUC57-amilGFP: Plasmid pUC57-mini-vector-amilGFP digested by Bsa1 and the band at around 750bp (amilGFP: 703bp) was got.Lane pUC57-Pprovoin5: Plasmid pUC57-kana-mini-Pprovoin5 (2097bp) digested by Bsa1.Right:Lane pUC57-amilGFP: amilGFP (703bp) was got by PCR method...

This step is used to get the plasmids pUC57-Pprovoin5 digested by enzyme and gene amilGFP by PCR method for later in the process. Therefore, channel pUC57-Pprovoin5 plasmids were done enzyme digestion of BsaI. And channel pUC57-amilGFP were gene amilGFP got by PCR. Clean-up the product of pUC57-Pprovoin5 and pUC57-amilGFP to obtain pUC57-Pprovoin5 backbone and amilGFP-fragment.

T4 DNA ligase is used to connect pUC57-Pprovoin5 backbone with amilGFP-fragment.

Figure 5. The electrophoresis results of bacteria PCR..

Lane pUC57 -Pprovoin5-GFP-1 to 3: Bacteria PCR of monoclonals of amilGFP with size of 703bp. 1 to 3 were positive monoclonals.

Extract 1 to 3 plasmids for sequencing.

Figure 6. 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 7. Histogram of the fluorescence intensity when the concentration of CYA equals to 30uM and the duration is 4 hours..

As seen from figure 7, 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 8. Histogram of the fluorescence intensity of bacteria C when the duration is 4 hours under different concentration of the cyanuric acid..

Figure 9. 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. 8 and Fig. 9), 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 10. 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 10, 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

1. Zhang X, Huang Q, Zhao ZZ, Xu X, Li S, Yin H, Li L, Zhang J, Wang R. An Eco- and User-Friendly Herbicide. J Agric Food Chem. 2019 Jul 17;67(28):7783-7792. doi: 10.1021/acs.jafc.9b00764. Epub 2019 Jul 3. PMID: 31267752.

2. Zhu M, Wang L, Wang Y, Zhou J, Ding J, Li W, Xin Y, Fan S, Wang Z, Wang Y. Biointeractions of Herbicide Atrazine with Human Serum Albumin: UV-Vis, Fluorescence and Circular Dichroism Approaches. Int J Environ Res Public Health. 2018 Jan 11;15(1):116. doi: 10.3390/ijerph15010116. PMID: 29324720; PMCID: PMC5800215.

3. Silverman, Adam D., et al. "Deconstructing cell-free extract preparation for in vitro activation of transcriptional genetic circuitry." ACS synthetic biology 8.2 (2018): 403-414.

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.