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expression problem with pBAD33...similar experience?


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#1 Lynn Shay

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Posted 08 April 2013 - 06:45 PM

we recently try to clone a gene into pBAD33 for controllable expression with Venus as a reporter. we first cloned Venus into the XbaI and HindIII sites and named it pBADV, then we cloned the other gene in front of Venus between SacI and XbaI and named it pBADVY. we checked almost everything we could, there are correct RBS, the gene sequence is just as we expected, the first RBS is at least 30-40 bps from the end of the promoter. however, in the same strain, under the same condition, pBADV produced very week fluorescence while pBADVY produced much stronger, at least 50 times stronger fluorescence. this bothers me because i'm not sure if the gene in front of Venus was expressed as I thought...basically i want to use Venus as an indicator of the expression level of the other gene but now I'm not sure if it's gonna work. any of you guys have similar experience?
I don't understand why the expression level of Venus is suddenly enhanced by 50 times once there is another gene inserted in front of it. pBAD33 has been a classical vector for a very long time and i thought it should be easy to use...I would appreciate any suggestions.

#2 phage434

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Posted 09 April 2013 - 08:12 AM

Your other gene could have cryptic promoter sites. It could also have a far better RBS, and translational readthrough could be higher than in the original context.

#3 Lynn Shay

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Posted 11 April 2013 - 07:17 AM

not likely...because without induction, the cells are not bright. if there is a cryptic promoter, the cell should be bright all the time. I'm not sure about the RBS, but Venus with the same RBS in another plasmid under a constitutive promoter works quite well...
thanks a lot for your reply!




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