Fig. 1

The workflow for designing B.licheniformis genetic switches. A The design flow of genetic circuit was shown in figure, the inducer controls the indicated OFF/ON selections. OFF selection: in the absence of inducer, MtlR protein’s two PRDs were phosphorylation, made a low affinity for MtlR box, and blocked the expression of target gene. ON selection: in the presence of inducer, the PRD-II region that involved in the negative regulation is dephosphorylated, made a high affinity for MtlR box, then active the expression of target gene. B The sequences of MtlR box A and MtlR box B. MtlR box A, originated from B.licheniformis, sequence is ‘TTGTCAcacggctccTGCCAA’; MtlR box B, originated from B.subtilis, sequence is ‘TTGTCAcagtatgTGCCAA’. And the elements of eGFP expression cassette was shown. C The test of genetic switch- MtlR box in vivo. Three strains, BlpPSE, BlpPSAE (contain MtlR Box A), BlpPSBE (contain MtlR Box B) were cultured under three conditions (1. No inducer 2. + 1.5% mannitol 3. + 1.5% sorbitol). The eGFP fluorescence was test after added inducer 12 h. D The dynamic range of MtlR box A and MtlR box B. The dynamic ranges of BlpPSAE (contain MtlR Box A), BlpPSBE (contain MtlR Box B) were calculated. The dynamic range was defined as “ON value/ OFF value’. E Three fragments (Pshutle09, Pshutle09A, Pshutle09B) were selected as probes, EMSA was performed by MtlR protein in different concentration (0, 0.4 µM) for three fragments