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pUB Vector with two promoters


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6 replies to this topic

#1 praveen921

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Posted 09 August 2012 - 01:58 AM

Hello all,

I am cloning my gene into a PUB18 vector with a C-terminal RFP fusion. My gene has its own promoter for expression. If i clone the gene along with my promoter into the PUB18 vector, will it still express it as PUB18 has its own promoter as well.I was wondering if the gene expression will be normal in the presence of two promoters in the PUB vector.

Thanks for the help.

#2 phage434

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Posted 09 August 2012 - 06:31 AM

It will probably work if the promoters are oriented in the same direction. Otherwise, you could end up with problems, making anti-sense RNA, interfering with expression.

#3 Nayeem991

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Posted 10 August 2012 - 01:38 PM

First confirm the orientation of insertion. I think it will work if both the promoters are in same direction (as phage 434 said) and the gene will obviously express under the promoter of vector. If they are in opposite direction i think it will only work if the plasmid is transformed into the host from where your gene is originally belongs to. Because that host contains the particular RNA pol which will recongnise the promoter and it will express the gene inserted in either direction. But i guess in this case it wont fulfill your purpose.

#4 ascacioc

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Posted 10 August 2012 - 09:44 PM

Assuming that you are using your pUB18 vector in B subtilis (as far I can see pUB18 is used for B subtilis, correct me if I'm wrong because I am not familiar with this exact vector) and that your gene with its promoter is a B subtilis gene with a B subtilis promoter, then we can cotinue. If your gene is not B subtilis specific, you will get transcription only from the promoter of the vector. However, then you have the problem that you can have, by chance a mixture of nucleotides that are recognized as rbs between this promoter and the gene. To be more specific, you have your mRNA and the ribosome will start attaching to a rbs (not very specific sequences, high in Gs and As) and look for an AUG, 6-8 bases downstream of the rbs. It can find the correct AUG, then you are safe, but it might be the coincidence that there is another AUG, not in frame with the real AUG and then you have translation of the wrong protein.

If you have a B subtilis gene inserted and even if the orientation of the promoter is correct, you might have some troubles. After transcription, you will end up with a mixture of mRNAs that have the following structure ...pUB18 promoter - pUB18 rbs - original promoter - original rbs - AUGgene... and ...original promoter - original rbs - AUGgene... (rbs = ribosome binding site). It is true, that most probably the first one, transcribed from the pUB18 promoter, will be the most abundant since the pUB18 promoter is most probably stronger than yours. You might be lucky and ribosomes will bind to the original rbs and start the translation of the right protein, but you might be unlucky, and another coincidence rbs is detected in between the pUB18 promoter and the oroginal rbs, and it might be the case that there is another AUG out of frame and you get other things translated. Anyhow you can check whether everything is fine only by checking the red fluorescence in the end. However, you might have some additional N-terminal amino acids (best case scenario).

I suggest that you do not play with more promoters. Either you clone your thing in such a way that you deleted the promoter of the vector (tricky but doable) or you use only the promoter of your vector and get rid of the promoter of the gene (and its rbs, start from ATG).

Andreea

#5 phage434

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Posted 11 August 2012 - 10:52 AM

Sigma-70 coli and SigA subtilis promoters are actually quite similar; probably this would work, but the more conservative approach would be to use one from the organism you are expressing in.

#6 ascacioc

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Posted 12 August 2012 - 04:02 PM

@ phage434: I have a question regarding your answer: you basically say that the mRNA will be produced. But what will happen afterwards? Wouldn't the presence of 2 rbs's affect the translation? Moreover, wouldn't the fact that the first rbs is so far apart from AUG affect the translation? I mean, in order to have the right protein, we can only hope that the ribosome binds to the second rbs, or? (see my answer above yours) Am I wrong in being so 'paranoid' in my scenario?

I am asking because I never did this before (I mean, I put only one promoter per ORF) but now that the topic was opened, I am curious to hear also other opinions (no matter how hypothetical)

Andreea

#7 praveen921

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Posted 13 August 2012 - 04:26 AM

Thanks for all your answers. The promoters are oriented in the same direction after sequencing results and i am guessing it should work fine in that case. Posted Image

Edited by praveen921, 13 August 2012 - 04:26 AM.





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