Hi there,
Hope you are all doing well. I am very new to synthetic biology, and have a very silly question. I would like to have a tight, promoter regulating the expression of a guide RNA, somebody suggested me to try PRhaB, rhamnose inducible promoter. However, the plasmid that I have right now contains a arabinose-inducible promoter, PBad regulating the expression of another protein. I am just curious is it possible to have both PBad and PRhaB regulating the expression of different proteins/RNA in a single plasmid expression system, would it crosstalk or cause leakiness in either of the expression system. Also, I should mention that I do not aim to induce both PBAD and PRhaB at the same time. Thanks
Should be fine to use those two promoters, I don't believe there is any crosstalk. It should not matter whether you induce them independently or concurrently.
Hi Yang,
You can definitely use multiple inducible promoters in the same plasmid. The one thing I would be careful of is finding out if either promoter you're using is leaky. I have used the pBAD promoter and I don't think it was very leaky, but I have no experience with pRhaB. Best practice is to put the construct with the least "leaky" promoter at the 5' end of your multiple cloning site and the leakier one at the 3' end so that you have a lower risk of readthrough. Another option for preventing readthrough would be to put your two constructs on opposing strands of the plasmid.
Good luck!
Ragan
I second everything Ragan wrote. Except I would add, consider not working at all with a jury-rigged derivative of whatever plasmid you have "right now". Instead, I would encourage you to have a synthesis company make the exact construct you want, with nothing in it that you don't want. All things considered, this is likely to be both faster and cheaper than trying to clone it yourself from whatever pieces you happen to have.
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