Did you check the optical density (OD value monitored at 600 nm) after you resuspended the bacterial pellet into M9 minimal media?

TB is a highly enriched medium compared to LB. Therefore, the low protein expression yield could be due to that the bacteria density in M9 media was already too high to be grown.

In general situation, the protein expression should be induced at OD of around 0.6-0.8 as long as you ensure that the bacteria grow while the log phase after you transferred the bacteria pellet into the labeling culture.

Best,

Cloud Lee

Dear Donhyu

I'm agree with Yun-Tzai and Dadchi that the OD of the bacteria at the resuspension in m9 is essential.

In this case higher biomass do not mean high protein expression because if you resuspend bacteria at too high cell density, the cells will consume all the nutrients and all the oxigen n very short time and your induction will be not efficient and if it true that at the end you have many cells, it is also true that you will have low protien/cells and this make complex lisys and purification. If you protein is soluble and not toxic for E.coli, you need to maximize protein production/cells and not biomass and good protein expression require nutrients and good oxigenation.

I think that the biomass from 4 liter LB is already quite too high for 1 liter of M9. TB could be an alternative to reduce the pre-culture volume, but use 4 liter of TB seem to me really to much.

Generally i'm doing 500ml LB (O/N at 30°C in 2000l shake flask cup vented and baffled) and i resuspend the cells in the morning in 800ml M9, incubate the cells 30' in the new media at 37°C 180 rpm and than induce the proten expression by IPTG for 3h at 37°C or 5h at 25°C depending from the protein solubility.

Good luck

Manuele

Thank you very much!! Manuele, Yun-Tzai and Dhadchi

The O.D. was controlled to the same level (either TB or LB). Maybe the cell is in a different phase.

Best,

Allison

Dhadchi Jeyaharan

The M9 plate is great idea. I will give it a try some time!

Best,

Allison