Question: Specifically regarding Ampicillin; When growing cells in TB (terrific broth) for protein expression - when should I expect the ampicillin to be gone due to degradation by b-lactamases? (and how much Ampicillin do people add in addition to standard 100 ug/ml)

Background: I have heard rumours that all Ampicillin (100 ug/mL) is degraded within few hours (2-3) due to the enormous amount of b-lactamases secreted by the bacteria.I have also heard that some are not concerned about this, because 2-3 hours is anyway enough for the culture to outcompete anything else that will grow after the ampicillin is gone. On the other hand, some people are also writing that they add more ampicillin every 2-3 hours to maintain selectivity; however, I dont really see the point in maintaining selectivity as long as I have plenty of protein I will anyway lyse the cells and not use them for anything else.

My growth conditions: Grow at 37 degrees till OD600=1 (takes around 3h), then induce with IPTG and grow at 25 degrees for 18h, 200 rpm the entire time.

End comment: I know you can generally change to the more expensive carbenicillin which is degraded slower, but i'm more interested in knowing "how bad" Ampicillin really is for my general knowledge (and I will anyway keep on using it). Also: Any general thoughts or references to literature on this topic, specifically for Ampicillin is appreciated.

rather interesting question. Selection pressure is very important for any type of work. And it is true, amplicilin will degrade in media due to the secretion of beta-lactumase. This is a limitation that we must face, though there are ways of getting around this.

For example, when cloning, on a plate sometime you will notice "smaller" colonies growing around a central large one. This is due to beta-lactumases. They are not a problem if you are going to take the central colony and place it into fresh media with new amplicilin. They will simply die out. No harm done.

In liquid culture it is good to not "oversaturate the culture". Usually, do not let it get over OD600 = 1. In addition, do not use old stocks, as this causes problems with selection and use higher concentrations of ampicillin if you are still having troubles selecting!

Generally speaking, 100ug/ml is standard for most selection with cloning, and should be sufficient for what you are doing. It seems that your protocol should work fine for protein isolation.

Last remarks -- it is a problem that most microbiologist do not think of. Ampicillin has its limits!

  • Thanks, although this is all true - is there still an answers to how bad ampicillin is? Is it really gone from the media within 2-3 hours? Also: when using TB you really should not expressed before the OD600 reaches over 1, because IPTG will not be taken into the cell before the glycerol is used up (TB can theoretically support growth up to OD600=16) – CuriousTree Aug 13 at 6:42
  • Also, are you saying that the Ampicillin is likely gone after OD600=1? – CuriousTree Aug 13 at 7:03

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