Induction by IPTG of T7-RNA polymerase in BL21 strains : time lag? - (Feb/07/2008 )
I would need to know how long it takes for the T7-RNA polymerase contained into the chromosome of E coli in BL21 strains to be fully expressed after induction by IPTG?
Would anybody know the answer?
Thanks for your help!
I think the default protocol says 1hour, but it depends on the protein. But you can make a small culture and take several time points to check in a gel. After that you can grow a larger culture and collect it at the time point you determined to be the best.
I used BL21(DE3) pLysS to express proteins (from pET3d vector) by induction with IPTG. (This strain carries a low-level expression plasmid that encodes T7 lysozyme, which binds to T7 RNA polymerase thus inhibiting transcription by this enzyme. Therefore transcription of pET encoded proteins is very minimal during the initial period of bacterial culture). In this system the optimal harvest of the proteins to be expressed was usually achieved after 4 hours post IPTG induction.
I am not sure when the RNA polymerase would be fully expressed but I usually saw expression of my proteins beginning at 2 hours.
I used 1ml (of 1 litre) samples from the culture pre induction, 1 hour-, 2 hours-, 3hours-, 4 hours- and 5 hours post induction and run them on a SDS-PAGE gel to establish the time when expression of my proteins was optimal.
Maybe you can use this method to check for RNA polymerase (e.g. antibody stain in Western Blot)?
I used to lyse samples before loading onto the gel as follows:
Lysis of culture supernatant samples was performed in PBS containing 1% SDS at room temperature for 30 minutes followed by removal of undissolved material by centrifugation at 13,000 rpm prior to running on the gel. An equal volume of protein or lysis samples was added to loading buffer [50 mM Tris-HCl pH 6.8, 2% (w/v) SDS, 0.1% (w/v) bromphenol blue, 10% (v/v) glycerol and 100 mM DTT (Sigma)] and denatured at 98˚C for 3 minutes.
You may find answers in the file attached.