We studied a quorum sensing (QS) response to the ComX signaling peptide in Bacillus subtilis PS-216. First of all, we optimized the protocol for synthesis of signal peptide ComX from Escherichia coli ED367 to reach bigger yield. Bacteria were firstly incubated in rich medium to reach high cell density and then followed IPTG induction of cell culture in minimal medium. Furthermore, we also checked stability of pure ComX signal peptide over time stored in SS buffer with BSA at 4 °C. ComX signal peptide in buffer was relatively stable (DT50~100 days). To study the response of B. subtilis PS-216 to ComX, we used biosensors that do not produce the peptide and carry a fluorescence reporter (yfp) fused to the surfactin promoter (PsrfAA), known to be under the QS control. We used two different mutant strains B. subtilis PS-216 to compare quorum sensing response. One strain had inactivation of comQ gene with antibiotic marker (BM1456), the other strain had deletion of comQ gene with markerless technology (BM1455). Our results show oversensitivity of biosensor 1, BM1456 (k= 2,7 ± 0,2) to biosensor 2, BM1455 (k=3,5 ± 0,2). Cell response to ComX was measured at 3 different incubation times – 3 h, 4 h, and 6 h. We observed cell response to ComX after 3 h incubation (exponential growth phase), with stronger response to ComX at late growth phase (4 h) and at stationary phase (6 h).
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