How to improve IPTG induction? - (Mar/14/2007 )
I agree with timjim,
Growing your cells without antibiotics could lead to a rejection of the plasmid. You have to force the cells to keep it.
What antibiotics are you using?
I used different antibiotics depends on the E.coli strains that I used.
For strains with pREP4 like M16 and SG13009, I have to use kanamycin for maintenance. So for the plasmid, I used ampicillin.
For others, I just use ampicillin.
By the way, I heard that carbenicillin is a more stable antibiotics compared to ampicillin. And thus it will be more selective compared to ampicillin. Is it true?
Just went back to the original post, in case we got a bit off-topic. (Who, us? Off topic?) A couple of quick questions...
1. Why did you choose the BL21 cells? I don't think there will be any adverse effects of using pGEX in BL21, because expression in pGEX uses the endogenous E. coli polymerase, so the T7 pol is superfluous.
2. Are you totally sure the vector has your gene? Things get lost all the time...
When using BL21, do you think it will cause leaking expression? Cause there is no control of expression. Not too sure though.
Expression will be slightly leaky, but not because you're using BL21. To control leakiness, you'd have to clone into a pET vector.
I thought to control leakiness, have to use the laclq gene.. or the prep4 plasmid that encode a certain protein to control the expression.
I used BL21 and I got leaky expression before inducing with IPTG. Kinda scary though...
You created some doubts in me now... without antibiotic? I am not getting good expression of the protein and u make me think that its because of the antibiotics. Since i am using 2. Ampi and chloramphenicol since it is a rosetta strain that carries a plasmid encoding extra tRNA. I thought to use both to keep selecting bacteria with the 2 plasmids but now maybe its to harsh for them to grow or express the protein
Personally, I would happily allow a reduced expression for the knowledge that my plasmids were still retained in the cell.
For pGEX you can semi control leakiness using glucose. But it only matters if the product is toxic and is killing the cells.
We also use pGEX for GST fusion protein and usually don't have problem with expression (given that the gene is inserted properly). If just to check which clone expresses desired protein, I just dilute o/n culture to OD600 = 0.6-0.8 (usually 1:4 or 1:5 will do) in total 1ml (with antibiotic). Then induce with 0.5-1mM of IPTG (0.5 is the norm) at 37oC for 2h, then take 20ul out and run gel, compared to uninduced sample side by side and see if I have any induced protein at the correct size. It should work normally. If the insert is big, maybe longer time of induction is needed. If neccessary, induce o/n at 30oC (not 37). For making lots of GST protein, the o/n 30oC induction may make a real difference. In my experience, if you don't have induced product, you may need to:
- Double check the cloning
- Check the IPTG stock
- And then induce for longer time at lower temp, since this step really should not be much problem at first checking. It is more important when you want to purify the GST protein with better efficiency