pUB Vector with two promoters
Posted 09 August 2012 - 01:58 AM
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.
Posted 09 August 2012 - 06:31 AM
Posted 10 August 2012 - 01:38 PM
Posted 10 August 2012 - 09:44 PM
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).
Posted 11 August 2012 - 10:52 AM
Posted 12 August 2012 - 04:02 PM
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)
Posted 13 August 2012 - 04:26 AM
Edited by praveen921, 13 August 2012 - 04:26 AM.