Hey everyone,
I am trying to do protein expression with BL21(DE3) cells and I am growing my cells in TB. I ordered the TB from RPI and added 4mL glycerol before autoclaving for 30 min. After innoculation with starter culture, the cells seem to be doubling fine (~20-25 min doubling) and I induce at OD of 0.6. I then come back to my cells and each time the OD is around 2-2.5 after 3 hours of induction at 1mM IPTG. I did a control where I did not add the IPTG and the cells only made it to OD of 2.8 after 3hrs induction. I am not sure if there is something wrong with my media, but it doesn't feel like a toxicity problem because the cells seem to be dividing fine during the earlier growth phase (leading up to OD 0.6). Do you guys have any idea what could be causing this issue?
I am growing 1L at 37C in a baffled 4L flask with rotation speed of 250 rpm.
Any suggestions are appreciated!
Best,
Joe
Hi Joe,
If you want to reach stationary phase, you should change your media, or you can start less bacteria number like (0.1 OD) or same number with less media. Because TB contains physiological buffer components which means that TB can tolerate the acidic metabolites producing from bacteria during the growth phase. When the bacteria are in growth phase, there is an acummulation of acidic metabolites which can stops the bacterial growth to lead all bacteria to enter the stationary phase. However in your case, buffer component in TB eliminate this acidic metabolites , so you could not see the stationary phase.
Best
Hi there,
For rich medium culture in flask, stationary phase occurs for the range of OD you mentioned (between 2 and 3).
Thanks for the replies:
Cigdem,
I do not want stationary phase, I am trying to express a protein at high yields so I was hoping that TB would allow my culture to get to around 15 OD, as this seems possible for TB.
Dominique,
Why would you say in a culture flask I can only reach between 2 and 3 OD? This may be true for LB but I don't believe it is true for TB which is a much richer media.
Any other suggestions?
Sorry I could not understand your question with "Stationary phase at ~O.D. 2.0 with Terrific Broth?" then
So you want to reach 15 OD, but the bacteria did not grow further. So, the preparation of your media could be wrong. How did you prepare it and sterilized it?
The other possibility could be that your protein of interest is toxic to your bacteria
- Badges
- Science topic