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I am designing a synthetic gene construct to express genes in E. coli driven by either Ptet or PLacO. The construct would look like:

-Ptet-(Gene1)-PLacO-(Gene2)-

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.

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Nice question! First of all make sure that you have multiple transcription stop / terminator sequences at the end of your first gene . This is quite standard procedure. Also in your case the phenomenon called transcription interference (TI) comes handy. Here's a review on TI. In short:

the term TI usually refers to the direct negative impact of one transcriptional activity on a second transcriptional activity in cis.

Although TI is mostly defined as two transcription events affecting each other, at the low level the basis of it is that two proteins bound to the DNA affect each other's activity. Now since LacI's function is to inhibit transcription, thus it is strongly bound to the DNA and I'd say even if a polymerase somehow slips through the polyA signals at the end of the first gene it is likely to be stopped by the LacI already bound at the second promoter.

Alternatively you could put a weak promoter opposing the first gene and drive its expression, so it would collide with the oncoming polymerase from the first gene thus stopping it before reaching the second gene. Also here is another article on TI, that proposes a gene regulation network based on TI.

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  • $\begingroup$ PolyA addition occurs in eukaryotic nuclei, but E. coli is a prokaryote. I think what @poka.nandor is referring to are transcriptional terminators, which are very well characterized in E. coli and its viruses. $\endgroup$ – mdperry Apr 23 '15 at 14:51
  • $\begingroup$ Oh, right, my bad. I got my head wrapped around TI and stuff we work with. I 'll edit my answer immediately. Thx for point that out! $\endgroup$ – Nandor Poka Apr 23 '15 at 15:22

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