I performed a fresh transformation (with sequenced plasmid) into commercial BL21 competent cells. I scrape a colony into a starter culture w/ antibiotic overnight. I check the O.D. of the starter culture in the morning and it looks promising/turbid (O.D. ~2.0). I add the 25mLs into freshly autoclaved 1L LB that has 1mL AMP (100mg/mL). There seems to be an initial period of growth but them decline or asymptotic plateau at ~0.2 O.D. Why would the culture not grow up to induction pt. (0.5-0.6)? Are the cells spitting out the plasmid?
What size flask are you using to grow the 1L culture? That's quite a bit of media- I suspect it may be possible that your culture is unable to grow due to lack of gas exchange/nutrient distribution. You might try using multiple flasks for that volume to increase surface to air ratio, and make sure you're shaking them at a suitable rpm. Hope this helps!
Bad aeration typically leads to linear instead of exponential growth so taking a growth curve should confirm this or rule it out. However, as E. coli can grow anaerobically as well it won't stop growth completely, just slows it down.
Problems with the medium are also unlikely, as LB is a complex medium and you probably use the same to grow your preculture, which reaches OD 2.
How are the shaking flasks cleaned? Once we had this problem because they were contaminated with some chemicals from other stuff that was in the dish washer together with the flasks for bacterial cultures.
Some proteins can be toxic for E. coli. As the T7 polymerase in BL21 is under control of the lac operon which is leaky, you might have a low expression of your toxic protein of interest which then inhibits growth. As your preculture growth this option is also unlikely.
Do other people in your lab have the same problem from time to time? If so, someone could have contaminated your lab with a phage which needs proper decontamination.
Thanks for your replies everyone. I use 2L flasks w/ a 1L volume of LB. Yes, others have also encountered this problem in our lab. As for aeration, is tin foil (autoclaved) not enough to provide some circulation? In regards to phage, I just washed flasks, scrubbed with 10% bleach, 1% SDS, and then autoclaved with water in the flask. I don't think it's a proteotoxicity issue as I haven't been able to reach an inducible O.D. (levels out at 0.2-0.3). Any other decontaminant tips?
For ideal aeration you should load the flasks with 10% of their volume meaning 200 mL in 2 L. So reducing the volume in your cultivation is a good idea.
Maybe you have residual bleach / SDS in your flasks which inhibits binding? We typically autoclave the flasks after cultivation to kill the bacteria, then put it in the dish washer and afterwards heat them overnight at 180°C I think.
To test for phages you can plate E. coli very densely (use a stock of bacteria from a time where the problem with not growing bacteria did not exist) and then drop your overnight culture on defined spots on this plate. If the bacteria are lyzed it's a phage.
This doesn't sound like an aeration problem to me, although 1L in a 2L flask is pushing it, if the flask is not baffled.
I have had this problem when using plastic flasks that have been disinfected with Bacdown. My guess is that the Bacdown was not getting rinsed away completely in the wash, and was slowly leaching into the media during growth (I have heard of similar problems with bleach). The cells would grow for awhile and then just stop. I suppose it could have also been something leaching out of the plastic itself. We solved the problem by replacing the old flask and changing our disinfection procedure.
It could definitely be T1/T4 phage as well. If this is the case you should be able to detect it on plates with a plaque assay using the supernatants from your cultures. Also, you could try tonA- Bl21 strains. I believe all of NEB's expression stains are phage resistant.
Hi Luke!
I got to spend all summer doing this exact procedure. First note, make sure that you have rinsed your flasks entirely several times with water. Any residual SDS or bleach will kill your culture pretty quickly. Second, 1L is a sufficient amount of broth, but the first thing to check is your culture. Are your cells growing on agar? If they aren't, that's a good start. When autoclaving, make sure that you are reaching a sterile temperature for an adequate amount of time. In my case, the autoclave was aborting prematurely so any culture that I added was no longer being selected rather it was forced to compete with a bunch of unknown cultures.
I agree with Stephen Smith on this one, it doesn't seem like an aeration issue. You could also test the amount of bacteria in a different spectrophotometer if you want to cross-reference. If there's one thing I've learned in research it's that you can never assume something isn't a variable. Is your AMP working? Is your autoclave properly sterilizing? Are your flasks free of bleach residues? Are your bacteria expressing well on your plates?
Of course, it could also be an issue of an exaggerated lag phase, which could result from a drastic temperature change from plate to flask.
OH!
One thing that my professor had me do was to grow up a small colony of my bacteria in a 250mL flask with 100mL sterile LB overnight which would lead to a quicker growup. Not sure if you've been doing this but it also might help.
So stoked to see you're thriving at Scripps, Fox misses you!
Blessings and look forward to your response/resolution.
Hi Luke,
Inducing at OD 0.5-0.8 ensures that the cells are healthy and growing (since in the exponential phase) to produce sufficient amount of your proteins. If the cells cannot reach that OD it means that you either have contamination, excess antibiotic, sometimes the LB bunches have problems or you need a tider expression systems if your protein is toxic. For the last one try cell lines like BL21 pLysS.
Good Luck.
- Badges
- Science topic