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Citrobacter freundii Characterisation:
Valencia Biocampus BioBrick Characterisation
In the spirit of iGEM, we have collaborated with the Valencia Biocampus team and characterised several of the BioBricks they made as part of their 'Talking Life' project.C. freundii and E. coli JM109 cells were transformed with the following three plasmids and the transformations were plated onto LB + chloramphenicol plates.
Bac2
Plasmid construction: groE promoter + AsRed2. This promoter is activated after a heat shock (keeping cells in a 44°C waterbath for 5 minutes), as it normally controls the expression of groE, a heat shock protein that helps degrade proteins that have misfolded due to the high temperature.
Method (expand)
Colonies were picked from the Citrobacter plate and inoculated into 3ml LB liquid medium + chloramphenicol40 (1μl/ml) and grown overnight at 37°C on a shaker. The following day, the ODs were measured and a normalized amount of these cultures was used to inoculate bottles of M9 minimal medium (containing glucose as a carbon source) and left to grow at 37°C until the culture reached an OD of 0.15-0.25. Half of the bottles were put into a 44°C waterbath for 5 minutes while the other half were not. The fluorescence of the cultures was then measured every 10 minutes for 70 minutes.
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Results
Figure 1 - Bac2 characterisation. The bars show standard error.
The results in Figure 1 show that there is a significant difference between the start and end fluorescence of the heat shocked cultures, whereas the control cultures' fluorescence remained more or less the same throughout the testing period.
The fact that the fluorescence of the heat shocked cultures is lower than that of the controls may be due to some of the cells dieing after heat shock or due to slight variation in cell number (even though every effort was made to keep cell numbers even).
Bac3
Plasmid construction: Anaerobiosis promoter + ZsGreen1. This promoter is activated when oxygen concentrations in the medium are low, as this is when its transcriptional regulators become active.
Method (expand)
Colonies were picked from Citrobacter and E. coli + Bac3 plates and inoculated into LB + chloramphenicol40 (1 μl/ml) liquid media and grown overnight (aerobically) at 37°C on a shaker. The following day, the ODs were measured and a normalized amount of these cultures was used to inoculate bottles of LB + chloramphenicol. Half the samples were topped up with LB in order to exclude any air and were incubated for two days at 37°C on a shaker, while the other half were incubated under the same circumstances (without adding the extra LB) and they were uncapped and shaken in order to reareate the culture one day after inoculation.
After two days, the fluorescence and the OD of the cultures was measured in LB. 1ml of the cultures was also transferred to Eppendorf tubes which were spun down and the LB was discarded and the cells were resuspended in water. The fluorescence and OD of these cultures was also measured. This was done to minimize any background fluorescence.
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Results
The fluorescence readings were divided by the relevant ODs in order to normalize the results. The resulting bar charts can be seen below.
Figure 2 - Anaerobiosis promoter characterisation in LB. The bars show standard error.
Figure 3 - Anaerobiosis promoter characterisation in water. The bars show standard error.
The results in Figures 2 and 3 clearly show that the colonies that were grown anaerobically produce a lot more fluorescence than those that were grown aerobically. This suggests that the anaerobiosis promoter is regulated in a very similar fashion in both E. coli and C. freundii. It also confirms that the BioBrick functions as expected.
Bac5
Plasmid construction: RecA promoter + GFP. This promoter is activated when the bacterial SOS response is needed. This response occurs when DNA damage is detected in the cell and is responsible for repairing this DNA so it can still be replicated, although the repair process is error-prone.
Method (expand)
Colonies were picked from Citrobacter and E. coli + Bac5 plates and inoculated into LB + chloramphenicol (1 μl/ml) liquid media and grown overnight at 37°C on a shaker. The following day, the ODs were measured and a normalized amount of these cultures was used to inoculate bottles of LB + chloramphenicol. The bottles were left to grow at 37°C with shaking until the culture reached an OD of 0.15-0.25 After this, 1 ml of culture was aliquoted into cuvettes and the cuvettes were exposed to UV radiation (254 nm wavelength) at a distance of 60 cm for 20, 40 or 60 seconds (unirradiated cuvettes were used as controls). The fluorescence was measured over the course of an hour using a blue filter.
This experiment was repeated but the distance was reduced to 10 cm.
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Results
The results from the first experiment (UV exposure at 60 cm over 20, 40 or 60 seconds) can be seen in Figures 4 and 5 below.
Figure 4 - Fluorescence of E. coli + Bac5 plasmid after exposure to UV (254 nm) for various periods of time at 60cm
Figure 5 - Fluorescence of C. freundii + Bac5 plasmid after exposure to UV for various periods of time at 60cm
These results suggest that there is no real difference in the level of fluorescence or in the rate of increase of fluorescence of the irradiated and unirradiated cultures. This may be due to the fact that no detectable amount of DNA damage was done due to the UV source being too far from the cultures.
The results of the experiment with the UV source being only 10 cm from the cuvettes can be seen in Figures 6 and 7 below.
Figure 6 - Fluorescence of E. coli + Bac5 plasmid after exposure to UV for various periods of time at 10cm
These results are slightly strange as we are unsure what caused such an increase in fluorescence of the unirradiated control after 20 minutes. It can be seen that the culture irradiated for 60 seconds starts to show increased fluorescence around 10 minutes after UV exposure and this level of fluorescence is maintained for a long period of time afterwards.
The cultures irradiated for less time do not show such a quick increase in fluorescence, nor does the fluorescence reach such high levels, but it is maintained throughout the period of measurement.
Overall, we think that these results are inconclusive as to whether or not the promoter works properly.
Figure 7 - Fluorescence of C. freundii + Bac5 plasmid after exposure to UV for various periods of time at 10cm
The C. freundii results suggest that the promoter takes around 20 minutes to activate, as a marked increase in fluorescence can be detected after this time and the level of fluorescence continues to increase for a period of time.
The culture that was irradiated for 60 seconds shows a jump in fluorescence after the irradiation, presumably because the promoter gets hyperactivated due to intense DNA damage. This culture then shows a little increase in fluorescence, after which it starts to decrease, probably due to the cells being unable to cope with the amount of DNA damage and dying.
The culture irradiated for 40 seconds also seems to die towards the end (but the drop in fluorescence could also indicate that the DNA damage had been mostly repaired, as the doubling time for E. coli is ~20 minutes so after two rounds of replication the DNA may have been restored).
The largest increase in fluorescence can be seen with the culture that had been irradiated for 20 seconds. This length of time is apparently more optimal for this experiment as it gives a good activation of the promoter without killing the cells (too much).
Conclusion
This construct seems to show some activity as there is a difference in fluorescence of irradiated and unirradiated cultures in both E. coli and C. freundii but further tests are needed to fully characterise it and to debug our tests.
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