Dear all, I am working on a synthetic biology project and will be using a titratable expression system (pLac). Since this system gives leaky expression, I would like to introduce a lacI repressor gene on the vector (to suppress the system so that there is no expression before induction by IPTG). However, I am worried that if the repressor represses the system completely and upon induction by IPTG, there will be a relatively low increase of protein expression (say 2-5 folds from the baseline: I have to do a large-scale protein production at the end).
Does anyone have a similar experience in introducing a repressor to a plasmid and later compare the protein expression (induced and non induced)? Thank you.
Hello,
Wild type lacI works well with lac promoter. You can replace it with lacIQ but for weaker lac promoter it will be an overkill. To suppress leaky expression, please add 1% glucose in the medium while culture is being outgrown. Once it reaches to mid log phase of growth (0.5 to 1 OD600), pellet the cells and replace the old medium (containing 1% glucose) with fresh medium containing selection marker and inducer but no or 0.1% glucose. You'll be good.
Hello Syed,
Thank you so much for your suggestion. I have thought about adding glucose in the medium but have no experience in doing so to repress my expression system. Therefore I was a bit skeptical about this approach. Previously I used a commercial vector which already has a lacI gene on it, so to build one by myself, I have to consider every possibility that my system would go wrong. Will definitely try this and hopefully everything goes well.
To my knowledge, there is no problem with the lacI or lacIq repressor once you add IPTG, at least if you use a T7 polymerase-based expression system (e.g. pET vectors). If you are worried about leakage of expression in the absence of inducer, use BL21AI (arabinose-inducible) from Invitrogen (now part of Thermo Fisher), provided that your plasmid carries a T7 promoter and induce wth 0.2 % L-arabinose + 1 mM IPTG (if your vector carries a lac operator
Hi Pierre,
Thank you for your recommendation. However, I am not using a T7 expression system but what you suggested could be used in my future works
Hi there,
Introducing the lacI gene at the plasmid level is a usual strategy for a better shutdown of lac promoter in absence of inducer (pET vectors, pGEX...), so I don't see why it should not work in your case...
Hi Dominique, thank you for your opinion. My concern is that over killing the promoter will results in a slow increase of protein production upon induction. There is a probability that very high amount of repressors in a cell require higher amount of inducers to relieve its repression.
The the literature is teaming with information on lacI -mediated repression of lac promoter. I guess all that is needed to be done is to clone the lacI gene in your vector and and put the system to test.
HI Syed, that could be done as well. Might build one with a repressor and another without. Comparing these two can help me to determine which one is better. =)
You have to make the difference between shutting down the lac promoter by overexpressing the lacI product (binding of LacI to DNA) and the raise of the lac promoter inhibition (binding of IPTG to LacI). The number of lacI molecules is important for the former but once you introduce the inducer (IPTG), the IPTG/lacI ratio within the cell is such that any lacI molecule is inactivated and therefore lac promoter is working perfectly OK.
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