The goal of our project is to construct a broadly applicable c-di-GMP biosensor to indicate intracellular c-di-GMP level. The main challenge is to convert the c-di-GMP concentration to a easily detected output, such as fluorescent signals. To achieve the aim, we used gfp as reporter gene and chose a c-di-GMP responsive transcription unit from P. aeruginosa, including the promotor Ppel and its repressor protein FleQ^[1]. By binding to c-di-GMP, the conformationally changed FleQ release from promoter Ppel, starting transcription of gene at the downstream of Ppel. In our test, we found the Ppel is a specific promotor that doesn't work in E. coli and S. oneidensis alone. To solve this problem, we constructed a tandem promotor PcI/pel by fusing a constitutive promotor PcI with Ppel. This new promotor (BBa_K4242002) works well in both bacteria. Then we introduced fleQ and verified whether its binding to the Ppel can reduce the gfp expression.

In the engineering cycle, we successfully built the biosensor (BBa_K4242013) and transferred it to different bacteria. Next, we tested the performance by changing intracellular c-di-GMP level at different levels via the overexpression of c-di-GMP hydrolase YjhH. We confirmed that our biosensor can response to different c-di-GMP concentration, and biofilm dissociating drugs that can decrease c-di-GMP level resulted in the decreased fluorescence intensity.

To make our biosensor functional in different bacteria, we tested the performance of tandem promotor PcI/pel and the inhibition function of FleQ in two different chassis, E. coli BL21 and S. oneidensis MR-1. We chose these two organisms after various considerations. First, we don't have facilities of P2 or higher biosafety laboratory, so we decided to use a common model bacteria E. coli instead of the pathogen. To test whether our biosensor can be used in different bacteira, we test it in another bacteria, i.e., S. oneidensis. Why we choose S. oneidensis? This is because S. oneidensis is another model organism in the field of biofilm study. Besides, its biofilm is related metal corrosion that contributing to billions of dollars in corrosion damage to industrial applications each year^[2]. One of proposed applications of our biosensor is to use our biosensor to develop some chemicals controlling S. oneidensis biofilm. To test the performance of c-di-GMP biosensor, we changed the intracellular c-di-GMP level of two organisms by overexpressing c-di-GMP hydrolase gene yhjH. For more detail about this part of work please check

The repression of FleQ to the promotor pel can be derepressed when its conformation is changed by binding to c-di-GMP^[3]. The principle of this system can be found in the design page (https://2022.igem.wiki/cug-china/design). This means the expression level of reporter gene gfp is proportional with the c-di-GMP level. To test this theory, we improved the composite part (BBa_K2471001) and built the part (BBa_K4242017), which encodes the c-di-GMP hydrolase YhjH under the IPTG-inducible promotor Ptac. Then we transferred this part into the strain E.coli BL21/pHYD-3 and S.oneidensis MR-1/pHYD-3. By adding different concentrations of IPTG during the cultivation, we have the strains that containing a decreasing c-di-GMP concentration gradient. After measuring the fluorescence intensity of these strain, we confirmed that the fluorescence produced by our biosensor decreased with the decrease of intracellular c-di-GMP level (Fig. 1).

Fig.1 a|Structure of the pSB3C5-Ptac and pSB3C5-YhjH b|Fluorescence in S.oneidensis MR-1/pHYD-3. c|Fluorescence intensity of E.coli BL21/pHYD-3 with different c-di-GMP levels.

One of the major applications of our biosensor is to develop drugs that have the potential to disperse biofilm and restrain biofilm formation. Thus, we choose the reported biofilm-dispersing agents^[4] to prove whether fluorescence produced by our sensor can be reduced in the presence of these agents. During the culture of both strains with biosensor, we separately added several reported biofilm-dispersing agents, detected the fluorescence and measured c-di-GMP level. The results show that these biofilm-dispersing agents caused the decrease in intracellular c-di-GMP levels, and contributed to reduce the fluorescence of the biosensor (Fig. 2).

Fig.2 a|Fluorescence intensity of MR-1/pHYD-3, cultured with different dispersal agents. b|Fluorescence intensity of BL21/pHYD-3. c|C-di-GMP level in MR-1/pHYD-3(measured by HPLC). Two-sided Student's t test was used to analyze the statistical significance (*P< 0.05)

Supporting information

Table.S1 Experimental concentrations of the biofilm-dispersing compounds and perturbation of FRET efficiencies in two E. coli strains..^[4]

[1] Baraquet C, Murakami K, Parsek M R, et al. The FleQ protein from Pseudomonas aeruginosa functions as both a repressor and an activator to control gene expression from the pel operon promoter in response to c-di-GMP[J]. Nucleic Acids Res, 2012,40(15):7207-7218.

[2] Zhou E, Li F, Zhang D, et al. Direct microbial electron uptake as a mechanism for stainless steel corrosion in aerobic environments[J]. Water Res, 2022,219:118553.

[3] Cotter P A, Stibitz S. c-di-GMP-mediated regulation of virulence and biofilm formation[J]. Curr Opin Microbiol, 2007,10(1):17-23.

[4] Ho C L, Chong K S J, Oppong J A, et al. Visualizing the perturbation of cellular cyclic di-GMP levels in bacterial cells[J]. Journal of the American Chemical Society, 2013,135(2):566-569.