I have been looking at papers for protein expression and trying to follow the process. I'm using TB medium to do the process, and the paper says to grow to an OD600 = 1.8 at 37 degrees, then let it grow to an OD600 = 2.6 at 18 degrees before proceeding with induction. You might say that if I'm going to follow the paper, why not just do that, but it's the first time I've done induction at such a high OD value, so I'm worried. Since it's TB, is it okay to proceed at 2.6? Or am I misunderstanding something?
Generally induction is done when the cells are still growing and not when they are in stationary phase. So the correct OD for induction is going to depend upon the growth conditions, media, aeration, growth vessel etc. Secondly the accumulation of your desired protein is also going to vary with conditions and with the protein itself.
So in other words there is not any one "correct" value when you should do induction, I would do some trial experiments and see what works best for your situation.
Dear Hyunjin Kim
as Michael J. Benedik wrote the important is that the cells are induced during growth phase. If i remember well the TB can reach OD up to 10 so 2.6 could be ok in theory even if induction at 18°C can delivery strong reduction in expression yield.
If you can i suggest to you to try alternative high cell density media respect than TB as YGR and Enpresso those are more able to controll the bacterial growth rate even at higher temperatures and therefore guarantee a longer induction phase.
you can found informations about those media at the following links
https://www.blogger.com/video.g?token=AD6v5dwU8Sl-43BfVTSeIRFBcQiWXAeg80Ud5UCxiXqUj4AFUO7p1BOHHmV4NiV0MY32A41mTgUH2-nyouDVBhcVFOuGc1lJ5_1WgP3RrsvakB6iJHCW_JOnrD8kIeiUSimWYahOs-OP
https://www.blogger.com/video.g?token=AD6v5dzLWqhoYwVoIC07Aj1G4RRYNxwM3Ndyk09KE3rmvGIdgfhR6WOBAH6TE0sDThwNquI9hZ9u7vKUDdMsusqQ3IcouXA53Y37DqhPmlYX4e9_Jnz5YS-87K68gQcCTjBMvNE4MlI
best
Manuele
Dear @Hyunjin Kim
As @Michael J. Benedik suggested the induction should be done in log phase of the respective cells growth in order to maximise the induction probability, as the ratio of number of viable cells will be very high in log phase compared to the remaining phases. And I personally believe, OD of 2.6 is fine when you inoculate adequate inoculum during subculture(for say, loop full), if you do very small inoculum transfer during subculture yes 2.5+ would be late log phase or near stationary phase.
