Team:MIT/Coculture
Project
Preliminary Co-Culture
Methods for Preliminary Coculture
The preliminary co-culture experiment served to determine how unmodified C. hutchinsonii and E. coli grow in mono-cultures versus co-cultures over time. Five cultures with co-culture media, filter paper as a carbon source, and variations of E. coli and C. hutchinsonii populations were prepared, with replicates of each, as shown in Table 1. The conditioned media was co-culture media that had contained filter paper and C. hutchinsonii for three days. The cultures were incubated in a 30ºC incubator shaking at 250 rpm. Samples were taken before incubation and then every 3 hours for the first 12 hours and once every 24 hours on days 4-8. Cells from each sample were isolated from the samples and the supernatant via the co-culture protocol. We use the flow cytometer to measure the size of cell populations from the glycerol stocks of the cells, as detailed in Measuring Relative Populationed.
| Contamination control in unconditioned media | Only E. coli in unconditioned media | Only C. hutchinsonii in unconditioned media | E. coli and C. hutchinsonii in conditioned media | E. coli and C. hutchinsonii in unconditioned media | |
| Media | 20ml unconditioned co-culture media and filter paper | 20ml unconditioned co-culture media and filter paper | 20ml unconditioned co-culture media and filter paper | 8ml conditioned co-culture media, 12ml unconditioned co-culture media, and filter paper | 20ml unconditioned co-culture media and filter paper |
| C. hutchinsonii | None | None | |||
| E. coli | None | None |
Table 1
Results
The data from the preliminary coculture showed the sugar levels (from the conditioned media) initially sharply dropping and the E Coli population increasing and overtaking the C Hutch population. This is expected, because E Coli naturally divides more quickly than C Hutch. However, the E Coli population levels out after around 10 hours, due to running out of sugars, meanwhile the C Hutch population begins to increase around this time as it starts breaking down the filter paper and consuming the sugars it releases from that. C Hutch soons overtakes the E Coli and the carbohydrate level increases again as the filter paper gets degraded into polysaccharides and simple . The E Coli population then increases more slowly, as it is able to feed off the simple sugars that escape the C Hutch, but it cannot consume the polysaccharides.
These results show that the C Hutch is very efficient at consuming the sugars it generates before it can diffuse into the media, leaving little left for the E Coli.
