I am designing a synthetic gene construct to express genes in E. coli driven by either Ptet or PLacO. The construct would look like:
I want to express each gene using either aTc or IPTG, but I want to make sure that the transcription of each gene can be controlled independently.
Let say I only add aTc and transcription is initiated at the Ptet location, would RNA polymerase bump into LacI bound to the PLacO promoter and stop transcribing? Or would it knock-off LacI transcribe (Gene 2)? I am not sure whether or not need to include terminator sequence to make sure the expression of each gene is decoupled.