I dont know how much information i will need to provide, so i will give it a go: Im trying to characterise a GFP expression vector response on acetic acid overflow metabolism, therefore i incorporated a acetic acid gene that produce GFP when acetic acid is present and i use the GadA promoter. The strain is also Kanamycin resistant. But as of current they have a 24 hours lag phase with a OD of 1 in pre culture 2 and 12 hours lag phase in main culture. The growth is monitored through a software measuring biomass (940nm) and emission(465/515). I take the cells (50μL) from a glycerol stock solution at -80 C and add TB (10mL) and Kanamycin (10μL) for pre culture 1. For the media in pre culture 2 i use Wilms Mops mineral media(~15mL), Kanamycin (15μL), Trace Elements solution (15μL), Thiamin(15μL), MgSO4(150μL) and Glucose (20g/L). For the main culture all the parameter is adjusted to 30 mL and the glucose concentration is set to 4 g/L. The strain was retransformed 2 weeks ago in suspicion of E.Coli malfunction, but the lag phase of 24 hours and 12 hours remain. Any tips on what im doing wrong or something i have overlooked in the process?
The best way to minimize lag phase is to pregrow a culture in the same medium until the cells are well into exponential phase (but not too long, you don't want them to be in stationary phase for long time) and then use this as an inoculum for your main culture. The other thing you need to consider is whether your cultures are really experiencing a lag phase or is it an apparent lag phase? If you inoculate a culture at low cell density and then measure biomass by OD, you will have no signal until there is sufficient cell mass although the cells might be growing exponentially but just too low a density to detect. It may be your frozen stocks are lower viable cells than you think so the inoculum is less. Lastly, once the cells are growing, how fast are they growing in your medium? It might just be a reflection of the medium you are using or your strain.
I agree with Michael J. Benedik about pre-growing your cells. One approach to consider is streaking out your glycerol stock onto plates so you are starting with a nice single colony of live cells. The glycerol stock will have a mix of live and dead cells and the starting OD can't tell you live vs dead.
You can also speed up the cell growth a bit by pre-warming all the growth media to your growth temperature (guessing you are doing 37, that's pretty standard).
I haven't used that specific growth medium but it sounds pretty minimal, it might just take the cells a while to double in those conditions.
If you are concerned about your specific strain, see if a second kanR variety grows similarly in your conditions.
Dear Andriy Glushchuk
one question about your main culture media: which nitrogen source did you use? Since from the recipe that yhou share it seems missed.
best regards
Manuele
@Manuele Martinelli The Wilms Mops media got the nitrogen source: (NH4)2SO4 and (NH4)Cl
How to adjust mid log phase growth of e.coli dh5alpha using overnight culture? When i grow at 37 degree temperature, 200 rpm after 8 hours i get real OD 3. Then transfer inoculum to main production flask?
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