I have a situation with bacterial growth in M9 media with two plasmids
Wild Type (WT) and Mutant. BL21DE3-PLysS cells.
procedure followed is as follows
1. Incoulated 5ml LB primary with a single colony (~12:00 noon)
WT after 9 hours OD = 0.6
Mutant after 6-7 hours OD = 1.0 (Stored in fridge when the OD reached 1.00)
2. spin down cells resuspend in 1ml M9 and add to 100ml of M9 primary
and incubate at 37C.(night 9.30pm)
3. After about 12 hours (morning 10.00am))
OD of M9 primary = 0.52
OD of mutant = 1.0
4. Add entire 100ml M9 primary to 1Liter of M9 secondary. (at 10:00am)
5. WT @ 7:00pm the OD was only 0.4. i.e OD increased from 0.1 to 0.4
in about 9 hours.
Mutant @ 4:30pm OD=0.7.
*In both the cases Ampicllin = 100mg/ml and chloramp = 34mg/ml.
*Both media were prepared together and divided into two halfs hence
there is no difference in the media quality or ingredients. In primary
as well as secondary.
Is there a way to make the WT cells grow faster? What could be the reason for long generation time of the bacteria.
Thank you for reading
Your suggestions and input are appreciated
@Pierre Beguin Trace metals were added to the M9 media as given in the article Studier, F. W. (2005). Protein production by auto-induction in high-density shaking cultures. Protein Expression and Purification, 41(1), 207–234. http://doi.org/10.1016/j.pep.2005.01.016
Also MgSO4 and Vitamin mix are added.
What carbon source are you using?
Second question, I presume you have made a typo with your antibiotic concentration, because if you are truly using 100mg/ml ampicillin and 34 mg/ml chloramphenicol then you are 1000 fold too high. Assuming that is the case you might try reducing the antibiotic concentration by 2 fold. Those concentrations are appropriate for rich medium but could be too high for minimal.
@Michael....I use glucose as carbon source....sorry for the type error it is 100ug/ml amp and 34ug/ml for chloramp.
Try either reducing the antibiotics by half or just removing the chloramphenicol during the large outgrowth phase. Also be aware that growth may dramatically slow down or stop upon induction.
@Micheal Benedik...Thank you for the reply. I tried reducing chloramphenicol to 50% its normal concentration i.e 17ug/ml.....and the growth rate improved....but it is still about 8 hours....to reach an OD of 0.6 from 0.1....which is rather long....However the mutant of the same protein did a faster growth even with full chloramphenicol concentration (it took only ~6-7 hours to reach an OD of 0.6 from 0.1 for the mutant even with full chloramphenicol concentration)....Hence the mutant and wild type have different growth rates under same conditions.....I did not expect one point mutation to have such a big difference.....
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