In order to connect the c-di-GMP level to the reporter gene, we chose the signaling system FleQ protein from P. aeruginosa in which the FleQ protein functions as both a repressor and an activator to control gene expression from the pel operon promoter in response to c-di-GMP. However, how to make this system work in other bacteria is a big challenge. We have experienced three engineering cycles to construct a preliminary functional biosensor.

Theoretically, the gene under pel promoter can be constitutively expressed in the bacteria that have no repressor FleQ. To test this hypothesis, we directly fused pel promoter to gfp and we had our first plasmid, pHYD-1. (BBa_K4242011) Then we transferred the biobrick into two module organisms, Escherichia coli and Shewanella oneidensis. However, no detectable fluorescence was observed, which suggests the promoter Ppel is non-functional in these two bacteria.

Fig. 1 a|Structure of pHYD-1 and control plasmid b|Colony RPC result of pHYD-1

To overcome the difficulty we met in cycle1, we designed a tandem promoter P_cI/pel in which the transcription start site of the constitutive promoter PcI is immediately followed by the FleQ binding site of the promoter Ppel. (BBa_K4242012) Transcription of the reporter gene gfp is promoted by cI promoter in the absence of FleQ. We found that the fluorescence of recombinant E. coli/pHYD-2 and S. oneidensis/pHYD-2 was drastically higher than that of the strains harboring pHYD-1 (Fig. 4) , indicating that the tandem promoter is capable of constitutively promoting gfp expression in the absence of FleQ in both strains.

Fig. 2 a|Structure of pHYD-2 b|Colony RPC result of pHYD-2

To test whether FleQ can repress gfp expression under the control of the tandem promoter, we cloned fleQ gene from the genome of P. aeruginosa and inserted it into pHYD-2, generating the plasmid pHYD-3 (BBa_K4242013) that contains the complete c-di-GMP biosensor. FleQ represses the transcription of gfp though inhibition of the RNA polymerase to access DNA by binding to Ppel when the level of c-di-GMP is low. At high intracellular c-di-GMP, FleQ undergoes a conformational change when it binds c-di-GMP, which relieves FleQ from pel promoter and leads to gfp gene expression under the control of the tandem promoter. As FleQ represses the expression of gfp by binding to the promoter Ppel, the fluorescence of E. coli/pHYD-3 and S. oneidensis/pHYD-3 expressing the repressor FleQ (Fig. 4) was significantly decreased compared to that of the strains with pHYD-2 that has no FleQ. Taken together, the results showed the tandem promoter is effective in promoting expression of the reporter gene(gfp), and the transcription factor FleQ is able to decrease gfp expression level.

Fig.3 a|Structure of pHYD-3 b| Colony RPC result of pHYD-3

Fig.4 a|engineering vectors b| Normalized fluorescence intensity in E.coli BL21 c| Normalized fluorescence intensity in S. oneidensis MR-1