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I have a insertion fragment that I wish to express from pUC19 in Escherichia coli. The insertion fragment is a sub-section from a larger operon sequence and contains just the last two cistrons from this operon.

I plan to insert this fragment into pUC19 under the control of its single lac promoter. Therefore, there will be two cistrons under the control of a single promoter (which is of course, very similar to how it would be expressed in the WT operon).

In my mind, expressing both the cistrons under the control of the single pUC19 lac promoter should allow both genes to be translated; once the mRNA is transcribed, both cistrons have their respective Ribosome Binding Sites upstream of their ORFs, and should be translated.

My question is, has anyone ever had experience expressing poly-cistronic fragments from plasmids using just a single promoter for the entire fragment? I cannot see any glaring issues with this in theory, but I thought i'd ask before I went ahead. Ultimately, I'd rather express both cistrons from a single plasmid, rather than express each cistron on a separate plasmid.

Cheers! Izaak

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If I understand correctly, you are seeking option for polycistronic expression. Have you considered using T2A or P2A sequences? These come from virus and essentially break polypeptide into two parts, so-called self-cleaving peptide T2A. Real mechanism of its work is that ribosome stalls on the sequence and unable to create peptide bond, effectively re-starting translation with next after T2A amino acid.

Of course, you lose independent control over expression, both products will be expressed at same level (but degradation might be different).

So, you should consider creating construct that looks something like that:

lac:gene1-T2A-gene2-stop

Thing is that a) it might not work in E.coli and b) it might not be necessary, because primary purpose of T2A is equal expression level of two polypeptides. And, if your 2 genes are big, for bacteria it is easier to handle two separate plasmids, rather than one but much larger.

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  • $\begingroup$ Ahh, intresting! I don't T2A is necessary though; the genes themselves have their natural transcription termination sequences, so the ribosome should be able to 'parse' the ending of each ORF separating the products. The genes are prokaryotic, so I think the natal RBS's of the operons should work. I just wanted to check it over on here to see if there is any big no no's. $\endgroup$ – user3473083 Apr 26 '15 at 10:34
  • $\begingroup$ The genes form a hetrodimer together so equal expression is what I'm after ;) cheers for the help. I didn't know about T2A, but its a useful one to know! $\endgroup$ – user3473083 Apr 26 '15 at 10:35
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    $\begingroup$ your guess seems correct. I never bothered with several-genes expression, so idk. however, since there is a plasmid for what you describe, it should work: snapgene.com/resources/plasmid_files/… $\endgroup$ – aaaaaa Apr 26 '15 at 10:57
  • $\begingroup$ never bothered with expression several genes in bacteria $\endgroup$ – aaaaaa Apr 28 '15 at 3:35
  • $\begingroup$ pETDuet is close, but its not exactly what I want to do. pETDuet requires me to split the PCR fragment (containing both genes) so each of the two genes will be expressed under one of the two promoters on the pETDuet plasmid. There are two promoters and two separate MCS in this plasmid. I want expression under just one promoter. $\endgroup$ – user3473083 Apr 28 '15 at 4:45

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