Microbes can exist as either planktonic or biofilm. A biofilm is a complete architecture including cells and a self-produced matrix of extracellular polymeric substances (EPS). EPS mainly contain polysaccharide, protein and extracellular DNA. Since biofilm presents the prevalent mode of microbial growth in natural, engineered and medical settings, the investigation of biofilm has become an attractive field of research.

Once pathogens biofilm is formed in the human body, it will be rather hard to eradicate. The three-dimensional biofilm structure formed by EPS can effectively protect bacteria and become a natural barrier to prevent antibiotics from penetrating the biofilm. Approximately 10-1000 times more antibiotic doses are typically required to kill pathogen biofilms compared to planktonic cells. Therefore, we need to investigate ways to inhibit/disperse biofilm formation, one of which is to develop drugs that inhibit the growth of biofilms or disperse existing biofilms. By degrading biofilm in advance, we can raise the efficiency of antibiotics.

Some chemicals often affect biofilm formation by interfering with chemical signaling systems essential in biofilm formation. One of essential signaling networks, mediated by intracellular secondary messenger bis-(3'-5') cyclic dimeric guanosine monophosphate (c-di-GMP) which regulates important events in biofilm formation and dispersal in a wide range of bacteria, has attracted much interest as a target for biofilm control. There have been numerous studies showing that maintaining a low level of c-di-GMP efficiently attenuates biofilm formation and provoking a decrease in intracellular c-di-GMP levels can disperse preformed biofilms. Therefore, the development of biofilm-inhibiting/dispersing agents targeting c-di-GMP signaling would efficiently combat biofilm formation or induce the dispersal of preformed biofilms.

Detecting c-di-GMP concentration is an essential entry point for the identification of biofilm-inhibiting/dispersing agents that target c-di-GMP metabolism. Two commonly used methods for c-di-GMP measurement are phenotype-based screens for motility and mass spectrometry(MS) analysis of cell extracts. However, the first method shows low sensitivity for c-di-GMP levels and the MS-based method is challenged by the complex sample preparation and long analysis time.

In this year, we construct a c-di-GMP biosensor that contains a transcription factor FleQ originated from Pseudomonas aeruginosa, a tandem promoter P_cI/pel being composed of a constitutive promoter P_cI and a c-di-GMP responsive promoter P_pel, as well as a fluorescent protein GFP as a reporter. The results show that the biosensor is able to indicate the decreased c-di-GMP level by providing a fluorescent readout in Escherichia coli BL21 and Shewanella oneidensis MR-1. Furthermore, we show that the reporter can identify the agents that reduce the intracellular level of c-di-GMP in BL21 and MR-1.

This project will construct the first c-di-GMP biosensor that is able to work in different bacteria. It has potential to effectively screen c-di-GMP-targeted drugs. This will promote the study regarding the regulation of biofilm formation and improve the field of biofilm engineering, which has important theoretical implications and technological value for the development of biofilm study.