Why does pETDuet-1 only have one T7 terminator but two T7 promoters (vector map below)? Is there any advantage of having only one terminator? There are going to be two mRNAs transcribed from this vector: one polycistronic (with two genes) and one monocystronic. Also, presumably, there will be more RNA for the gene expressed from the second promoter compared to the gene expressed from the first. That could lead to higher expression of the second protein, provided that the RBSs are the same. Is there experimental evidence confirming/disproving that hypothesis?

Is there any advantage over using polycistronic mRNA vs moncystronic? Have Duet vectors been used for heterologous expression of long (>40kb) biosynthetic pathways?

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  • $\begingroup$ Sounds like the manufacturer could answer these questions much better than we can. $\endgroup$ – MattDMo Apr 25 '16 at 22:17
  • $\begingroup$ Not sure it can. But waiting from reply from them as well. $\endgroup$ – Gergana Vandova Apr 25 '16 at 22:23
  • $\begingroup$ One of the duet user manuals (emdmillipore.com/CA/en/product/…) says that they are useful for studying protein complexes. I'm assuming it allows for more optimal co-translational assembly processes, and possibly favours 1:1 stoichiometry. $\endgroup$ – Jory Apr 26 '16 at 0:03
  • $\begingroup$ @Jory But in that case why is there a second promoter? $\endgroup$ – Gergana Vandova Apr 26 '16 at 0:12
  • $\begingroup$ OK so I didn't really read up on this last time I answered. As you said the downstream ORF isn't translated as much, so it wouldn't favour 1:1 stoichiometry. I have a feeling the expression of the shorter transcript is to compensate for this effect, but I'm not sure. $\endgroup$ – Jory Apr 26 '16 at 2:31

I think the presence of a single terminator is related to the high processivity of T7 RNA polymerase.

T7 RNA polymerase will transcribe a circular plasmid repeatedly before dissociating. The T7 terminator after ORF2 ensures that the transcript terminates efficiently.

Based on your schematic, these are the potential mRNA transcripts from that construct:

Transcript from T7 promoter #1

 5'---RBS---ORF1---STOP----RBS---ORF2---STOP---[T7 terminator]---3'

Transcript from T7 promoter #2

 5'---RBS---ORF2---STOP---[T7 terminator]---3'

It is doubtful that this vector will produce precisely equimolar concentrations of each protein product, however, the topology ensures that there is at least some of each protein produced. Of course, this could be tested empirically.

I hope that helps!


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