I express using BL21 DE3 e-coli cells and use the ZYM 5052 media. Some colleagues who also use this media have suggested adding IPTG to increase the protein expression. Would that make any sense for an autoinducing media?
Additionally, would it be added at OD 600 of 0.6 or it wouldn't matter as much exactly when I add it?
The original auto-induction paper by Studier can be found at the following link: http://wolfson.huji.ac.il/expression/local/bacteria/studierAutoInduced.pdf
When glucose is present in the media the expression of the lactose permease is inhibited and lactose will not be transported into the cell. After approximately 12 hrs of growth the glucose will be consumed and the lactose permease will begin to express thereby transporting lactose into the cells.
In strains such as BL21 the T7 RNA polymerase has been inserted into the chromosome and is under control of the lacUV5 promoter and is therefore inducible with lactose. Genes cloned into vectors containing the T7 promoter (eg. pET 2 and Flexi®3 vectors) will not be expressed until all of the glucose in the media has been consumed. This is very useful for protein production and generally results in significantly higher protein yields than is obtain in conventional media.
IPTG is a synthetic and structural analogue of alloactose which can be used as a substrate but cannot be metabolized.
So to answer your question, it will work for expression and possibly it will increase the yield but the all point of using ZYM medium is probably that your protein require that special medium for a specific reason. I will suggest you to follow the original protocol.
Hi Engi
As already pointed out IPTG is a synthetic analog of galactose, but it is also toxic for cells. I never mixed up autoinduction and classical induction by IPTG. I am not sure if this will work, since in autoinduction medium you have glucose as carbon source and it should repress the expression of the promoter.
Best
Paco
Hi there,
The point of autoinducing media is to avoid the use of IPTG and to get a smooth transition from off to on. So adding IPTG to autoinduction media is just a waste and furthermore there will be competition between lactose and IPTG for lactose permease. For IPTG induction any rich media devoided of lactose is OK.
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