H4 multi mode reader

To construct the green fluorescence gene (gfp) reporter plasmids, the original plasmid pSET152 was cut with BamHI and XbaI. The scyA1 promoter (P_A1) and the coding region of scyR1 were amplified from S. cyaneogriseus ssp. noncyanogenus NMWT1 genomic DNA with primer pairs pscyA1GFPF/R and ScyR1GFPF/R, respectively. The gfp and the strong constitutive promoter SF14 were amplified from pSET152:P_sbbAgfp:SF14sbbR with primer pairs GFPF/R and pSF14F/R, respectively (He et al., 2018 (link)). First, P_A1 and gfp coding region were ligated into pSET152 using the ClonExpress^TM MultiS One Step Cloning Kit (Vazyme) to obtain pSET152:P_A1gfp (Supplementary Table 1). Secondly, the plasmid pSET152:P_A1gfp was digested with NheI and then assembled with SF14 promoter and the scyR1 coding region to generate the corresponding reporter plasmid pSET152:P_A1gfp:SF14scyR1 (Supplementary Table 1), in which scyR1 was controlled by SF14 and the gfp gene was controlled by P_A1. These two plasmids together with the control vector pSET152 were introduced into DH5α, respectively, to detect the intensity of green fluorescence (excitation at 485 nm; emission at 535 nm, Synergy H4 Multi-Mode Reader). All fluorescence values were normalized to growth rates (OD₆₀₀).