Fig. 5.
Proof‐of‐concept test to show that all three SEVAtile destination vectors can co‐exist and individually express a gene of interest in P. putida KT2440 and P. aeruginosa PAO1. (1) Graphical overview of the proof‐of‐concept. The T7 transcriptional system was integrated in P. putida KT2440 and P. aeruginosa PAO1 by electroporating pSTDesXa/b·T7RNAP, pSTDesR·T7lysozyme and pBGDes·PT7‐BCD2‐msfGFP together in both hosts. pBGDes is genomically integrated in the host’s Tn7 landing site. In this set‐up, expression of T7 RNAP can be induced with 3mBz, which will cause expression of the msfGFP reporter from the T7 promoter. On the other hand, T7 lysozyme expression is induced with Rha and will inhibit the T7 RNAP. (2) After integration of the T7 transcriptional system in P. putida KT2440 and P. aeruginosa PAO1, both hosts were induced with all four combinations of 0 or 0.3 mM 3mBz and 0 or 10 mM Rha. The fluorescence intensity levels after 10 h of induction are displayed in the graph. Bars represent the mean value of four replicates, error bars indicate the 95%‐confidence interval. Full graphs of fluorescence intensity and OD600 are available in the Supporting Information (Figs S6 and S7).