Hello all,
I want to express a gene of wheat into BL21 DE3+ strain of E. coli. But there was some problem as my gene is not getting induced. I am working with a conc. of 1 mM IPTG at different time intervals 2 hrs, 4 hrs, 6 hrs, 8 hrs at 370C. I think my IPTG may not be working properly. Kindly suggest me how i can check wether the IPTG is working propely or not beside the blue white screening method.
You can check it by SDS_PAGE method. You will get the specific band if it positive
Hi Megha,
I have had similar induction related issues in the past. Try 0.1-1 mM IPTG, and induction times 2-6 hours. Alternatively, you may use the same conditions you are using now but with a known gene that has already been tested for expression by others (e.g. lab members or colleagues). Temperature is another parameter you may want to vary. Finally, run SDS-PAGE followed by Coomassie staining to look for expression of protein of your interest in BL21 DE3+, and make sure you include uninduced samples ("no IPTG" samples) in your gel.
A very quick and easy thing to do is to try out the same expression conditions you described in your question, using IPTG from a different source-- from another lab or a new purchase.
Good luck!
Regards,
Anoop
Dear Megha
do you think that is the iptg stok and not your specific protein that Has a problem? the Simple was to test it is use a positive Control as the gfp abd you van easly see od iptg work comparing the colour of The cells after centrifugation before and at the end of The induction. if you do not have the gfp or one other positive Control i reccomend to compare your clone with The same before the induction.
good luck
.Manuele
Hi!!
First, you may try reducing reduce the IPTG concentration. Then, reduce the temperature to 35 degrees. Then, increase the volume of culture broth. Even though SDS PAGE can show you the presence of protein, western blotting is better. You can see in WB: the thicker band for highly expressed protein.
I believe you sequenced your clone for the insert sequence before going for expression. Better recheck once again the cloned sequence.
The induction concentration is typically within a log of 1mM. If you are doubtful of your particular stock, you should have a known clone expressing something easy to visualize as an expression control.
It might also be an issue with the backbone of your clone. I have had sequence verified clones not express because of a backbone mutation. The selective pressures during cloning and expression can occasionally result in mutations. Screen several clones at a small-scale before investing your time in trying the larger-scale purification.
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