Team:HIT-Harbin/project/part3

    Biofilms are groups of cells at an interface cemented together by polysaccharides, protein, DNA and lipids. It enhances bacteria’s resistance to environmental stress and is able to perform more complex transformations. In our system, detecting part and killing part are separated into two different engineered E.coli populations. As we know, people in a small room communicate more conveniently than those in a big one. We assumed that signal transduction between E.coli is similar to people’s communication. In order to strengthen signal transduction between two communities, we construct an enhanced consortium biofilm.

    We introduce a gene yddV, which is under the regulation of Ptrc promoter (IPTG-inducible) (Fig. 1). yddV is a Diguanylate Cyclase-Genomic. The product of gene yddV has diguanylate cyclase (DGC) activity. DGC uses 2 GTP to form a Bisbis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP).

    C-di-GMP is a global second messenger in bacteria. Biofilm formation of E.coli is manipulable by varying c-di-GMP concentrations. When the c-di-GMP level stays low, there is little biofilm and the bacteria is dispersive. High concentrations of c-di-GMP promotes bacteria to form more cellulose and fimbriae, which enhances the biofilm formation and decrease the motility of bacteria.

    In order to check whether the construct is ligated to the plasmid, the biobrick region (1604bp) is PCR-amplified using primers B1(5'-GTTTCTTCGAATTCGCGGCCGCTTCTAGAG-3') and B2(5'-GTTTCTTCCTGCAGCGGCCGCTACTAGTA-3'). As shown in Fig. 2, the amount of the amplified sequence is almost same as expected. yddV is maximal when an exponentially growing culture (A600=0.3) was treated with 1mM IPTG for 2h. As shown in Fig. 3, an increase in the expression of yddV was evident when 1mM IPTG was added to the medium.

    Biofilm assays were carried out at 37℃. Cultures were grown in LB to exponential phase(A600=0.3). The culture of the E.coli cells carrying the plasmid pYddV was split in two test-tubes, each containing 5ml medium. 1mM IPTG was added to one culture, and the other was left untreated. At the same time, 5ml culture of the E.coli cells not carrying the pYddV was transferred into a test-tube. After 24h of treatment, visualization of attached cells was performed by removing the cell culture, staining the well with 1.0% crystal violet for 20min, and rinsing the well three times with distilled water. It was evident that a significant increase in biofilm formation in LB cultures of the E.coli cells carrying the pYddV in comparison with those E.coli cells not carrying the pYddV(Fig. 4).

    Next, we will determine the composition of two engineered bacteria in the biofilm we constructed. Moreover, we will further test whether the biofilm strengthen the intra- and inter-species signal transduction.