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Growth media - OD at induction - growth phases...

tb growth phase induction od expression

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#1 map3k

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Posted 22 August 2013 - 06:05 AM

Hello everybody,

 

I have a rather basic question regarding protein expression (T7 based) in E. coli (I use strains Rosetta and BL21AI, if that matters). I usually induce at OD600 = 0.6, lower the temperature to 18 °C and express overnight (if I express at higher temperature, I have some issues with protein solubility).

 

OK, so this approach works reasonably well, but I\’m looking for possible improvements regarding some tricky candidates. Recently I thought about switching to TB medium (from LB) for those cases. TB allows me to reach higher ODs thus increasing the potential protein output for a given culture volume under non-time-limited incubation (overnight = culture reaches saturation), if I understand everything correctly.

 

Now, as I understand the increased buffer capacity and additional carbon source (glycerol) aids in reaching high ODs, and I assume cells grow to higher ODs because reaching the stationary growth phase is delayed. So this means the log phase is longer, right? If that is correct, I assume that I could as well induce at higher OD600 than the standard 0.6, as ”longer log phase” would mean that the ”early log phase” which is the sweet-spot for induction would be extended as well.

 

Is my reasoning correct and has anybody any practical experience that would be applicable to my case?

 

Thanks a lot!



#2 hln

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Posted 24 August 2013 - 10:48 PM

Yes, you are correct. TB will allow growth in shake flasks of OD up to about 10. You can induce at higher OD - I sometimes go to 1.0-1.2. TB will usually allow several fold higher protein production than LB. I have been told by several colleagues that in some cases, the lower protein production in LB or even minimal media can give you more functional and soluble protein, but I have not experienced this myself.



#3 labtastic

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Posted 29 August 2013 - 05:07 PM

You are right that TB is a richer media and therefore the log phase of growth is longer. Having said that though, whether you induce at 0.6 or 0.8 or 1.2 OD's in TB for overnight expression at 18C doesn't much matter...the final OD will be the same. I regularly grow expression strains at 37C until any one of these OD's, hit with IPTG and express at 18C overnight.  The final OD of the cells for each protein is always the same every time. Always use baffled flasks for max aeration. At those OD's, the bacteria will deplete O2 is seconds if not properly aerated. Never let O2 be the limiting factor for your expressions.

 

@hln- the final OD is absolutely dependent on the protein your expressing. Some of my proteins finish expression with an OD in TB at ~30, some finish around 3. This is repeatable and consistent every time depending on protein. 

 

Bottom line OP, don't stress about whether or not you induce at 0.6 or 1 or 1.2. You'll pretty much get the same thing at the end.

 

Also, consider autoinduction media. Works great, tops out at reasonable OD's (~7-10, but protein dependent), and time of induction isn't too critical (16-40hrs depending on protein is usually sufficient). Plus once the media is made all you have to do is inoculate and then go do something else. No need to worry about hitting with IPTG at the exact OD; the media does all the work for you.







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