I am in desperate need of advice.
I did transformation of BL21(DE3)pLySs, Rosetta 2, Rosetta gami and T7 Express LysSIq cells last year with the my genes of interest cloned in pET20+b vector. After almost 4 months of trial and error, I finally got the positive colonies which I confirmed with WB. This year I started to work on purification of my protein. I took small amount from my glycerol stocks and streaked them on fresh LB plates with 100ug/mL of carbenicillin and picked 5-6 colonies each time. Did pre-culture and then started the process of large scale culture and purification. I didn't get any protein. Then I started to check the expression in 3 ml cultures from colonies picked from plates and now I have almost checked all my bacteria and none of them show expression. I used IPTG from 0.4mM to 1mM and did different time and temp conditions. Any advice?
Can someone help with loss of expression in transformed bacteria?.