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Citrobacter freundii Characterisation
Growth in sea salts
Since synthetic biology is a rapidly developing field, we think that it will see a lot of innovation in the years to come. We also think that the use of fresh water for large scale experiments and industrial applications will eventually become limited as fresh water supplies will be scarce, or salt water will be more accessible to people. This is why we think that a novel chassis should be able to grow in seawater/salt water. The following experiments serve to characterize the ability of Citrobacter to grow in water containing varying concentrations of sea salts. Note that the average salt concentration of seawater is between 20 and 40 g/l.
Methods (expand)
As we have mentioned previously, we urge everyone to talk about their failed experiments, as if nobody talks about them, a lot of time could be wasted by different people trying to do the same thing to no avail. As such, the following experiments can be considered an example of how we started out with a failure but then modified the circumstances to get meaningful results.
3.1 First, we wanted to asses growth of C. freundii on media plates. For this purpose, M9 minimal medium was made up, using glucose as a carbon source, and varying amounts of sea salts were added to these media. Unfortunately, at higher (>40g/l) sea salt concentrations, the agar did not solidify so growth above this concentration could not be assessed. The plates that did solidify had 100 μl C. freundii spread on them and were put into the 37°C incubator overnight.
3.2 Liquid M9 medium was then made up using varying amounts of sea salts. These media were not clear as expected but what whitish precipitates floating in the medium, so we speculate that the M9 salts did not react well to the presence of sea salts. The precipitate is probably magnesium and calcium phosphate, as M9 is very high in phosphates. Nonetheless, we inoculated these bottles with overnight cultures (grown in LB) to see if any growth would occur. After overnight incubation at 37°C on a shaker, the ODs were measured (using the appropriate sea salt concentration M9 as blank for each culture to prevent interference of the precipitate with the readings).
3.3 Next, LB medium was made up, using yeast extract (5g/l) and tryptone (10g/l) and varying concentrations of sea salts (replacing the normal NaCl). These bottles of media were inoculated with overnight cultures, incubated overnight at 37°C with shaking and the ODs measured as described above.
4.4 Finally, we wanted to compare the ability of C. freundii to grow in the presence in sea salts with that of E. coli, so we set up 250ml flasks with 25ml of LB or LB with 40g/l sea salts, inoculated them with overnight cultures of E. coli MG1655 or C. freundii and measured OD every 30 minutes over the course of a day.
Close the method.
Results
1.1 All the plates that were inoculated showed a lawn of cells, suggesting that C. freundii can grow well in the presence of sea salts. Unfortunately, the sea salts + M9 salts formed a white precipitate, so the plates could not be photographed in a way that would actually show that there were cells growing on them.
1.2 A graph showing the OD readings taken from bottles of M9 + varying concentrations of sea salts can be seen in Figure 1.
Figure 1 - C. freundii growth in M9 minimal medium + varying concentrations of sea salts
These results show that C. freundii does not grow well in minimal medium + sea salts, as the OD keeps decreasing as the concentration of sea salts increases.
1.3 A graph showing the OD readings taken from bottles of LB + varying concentrations of sea salts incubated with C. freundii overnight can be seen in Figure 2.
Figure 2 - C. freundii growth in LB + varying concentrations of sea salts
These results show that C. freundii can happily grow in LB + sea salts even when the salt concentration is higher than that of most seawaters. Concentrations of 10-20g/l seem to be more optimal but an OD above 1.8 is maintained throughout. This suggests that our novel chassis would indeed be able to be grown without having to waste freshwater on it.
1.4 Figure 3 shows the OD readings taken every 30 minutes from flasks containing either LB or LB + sea salts inoculated with either E. coli or C. freundii.
Figure 3 - C. freundii and E. coli growth over time in either LB or LB with sea salts
These results show that C. freundii can grow with and without sea salts at a similar rate to E. coli, so there would be no hindrance in using C. freundii for synthetic biology (and any biology) work over E. coli in sea salt medium.
