recombinant ecoli in log phase overnight media suggestion - (Nov/17/2010 )
We express our protein (temp inducible) in E.coli K12 containing pUC 18 vector. our seed media is grown at 30š C overnight 16 hrs and then inoculated in fermentation media next day. currently we are using LB media with ampicillin for starter culture and O.D reaches to about 4 and its pH also becomes alkaline. there is no further increase in O.D indicating that this may be its stationalry phase.
Can you please recommend any media that will maintain our culture in log phase overnight. We see protein expression even in earlier case, but we would like to increase the yield. we incoulate 10% of starter to Fermentation media.
That is not an easy request, but here are my thoughts.
1. Reduce the incubation temperature.
This will reduce growth rate, and thus increasing the duration that the culture spends in log phase.
Reduce growth rate will also reduce the oxygen demand of the culture, and cause the bacteria to shift to a more efficient metabolism. The end result is a higher cell density at stationary phase.
2. Increase culture volume(with concurrent increase increase in oxygenation) or reduce the number of cells used to inoculate the culture..
3. use a richer medium.
That would increase cell yield, but maintaining the culture in log phase overnight would probably not be achieved.
4. Use a fermenter which can constantly add and removes culture... a constant density fermenter.
As the cell density increases, culture is remove from the fermentor and new fresh medium is added. If the outflow and inflow are balance correctly, the culture can be kept in log phase. The new medium added also keeps concentration of growth inhibitory products from building up. Cells are pelleted from the culture removed from the fermenter.
5. Added the starting culture early in the morning and harvest the cells in the evening.
Thus the problem of maintaining log phase overnight culture is removed.
Hola, LB hasnīt any sugar, so bacteria has to deaminate aminoacids to get hidrocarbon structure, for that the liberated ammonia increases the ph. You could add glucose at 0.2-0.5 g/L and 100mM phosphate pH 7-7.5 for buffering as terrific medium has, or use this TB medium for inoculum, it has 4g/l of glycerol and get 10 ud OD. If you use glucose donīt pass of 1g/l because it will form acetic acid and damage the culture. But the passage of 4 ud to 10ud is 20 min-1hour depending of oxigenation, so you will have statinary cultures in the morning too. So you can decrease the temperature at 25-28šC decreasing the generation time. But in general people seed fermenters with stationary cultures, and is in the fermenter when induce in log phase. Good luck