Team:Bordeaux/Results/Production

From 2014.igem.org

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At first, we used to produce ELP20 and EP40 in a LB medium supplemented with glycerol and ELP60 in a LB medium supplemented with glucose.

The biomass is produced during the exponential phase of growth at 37°C. After induction with IPTG at the end of this phase, the temperature is lowered at 25°C. This temperature has been lowered down to 18°C in our last batches.

In order to inoculate the cultures in the beginning of the exponential phase to limit at maximum the latency phase and to allow induction at the right time, we determined at which OD the inoculation or the induction would be the best. To do so the growth has been monitored to determine the growth curve.

Then the different growth phases have been determined.

The following results are obtained:
Latency phase => 0-1h
Speeding up phase => 1-3h
Exponential growth => 3-4h
Slowing down phase 1 => 4-6,25h
Slowing down phase 2 => 6,25-8,25h
The exponential phase begins after 3h of culture (OD=0.2). Inoculation has been performed with the right volume of preculture to get 0.2OD in culture flasks.
The exponential phase ends after 4h of culture which corresponds to an OD of 0.8. The induction is done at this OD value.

We then decided to increase the production time of the protein of interest (meaning after the induction). At first we let the cells grow for 5h after induction, but this time has been increased up to 21h. Another medium (TB instead of LB) has been used for all the next batches. The production yields are far better with this medium which is richer than LB. In the end we were producing ELP in TB medium with up to 4L culture each day, following this schedule: