What are some suggestions for expressing a toxic gene?
Establish the initial construct in a non expression strain with no T7 RNA polymerase gene (like NEB 10-beta NEB #C3019). • Incubate the plates and inoculate the culture (from a fresh colony) at 20-30°C (not at 37°C) • When you are ready to express, try using a strain which is designed for the expression of extremely toxic genes. Use of BL21(DE3)/pLys may result in tighter control of expression. Stratagene has a specific system called Lambda CE6 induction kit, which is based on BL21 cells and bacteriophage CE6. In this case T7 RNA polymerase is introduced via CE6. BL21 cells alone do not carry the T7 RNA polymerase gene. • In the case of solubility problems (see 4.9.5): • Induce expression at low temperatures to reduce solubility problems (15°C overnight) • Following sonication, check both crude cell extracts and clarified cell extracts by SDS-PAGE and Western analysis to figure out if there is a solubility problem • Use the intein-mediated protein ligation [See Part 6: Applications: Pr
