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Chosing the ideal bacteria growing platform
In order to physically constrain the bacteria, Hasty used a trapping chamber as depicted in Figure Xsome below. The chamber had the dimensions of 1X1 micron. This limited the cell growth to forming a monolayer (sentence). Excess cells and AHL were flushed away through the main chanel. [REF]
To create a monolayer, the micro-sieve seemed promising. The cells could be drawn toward the sieve by manually applying an under pressure with a syringe. Since the Top10 e.coli strain used for our transformations does not form biofilms, additional cells would be flushed away once all the pores of the micro-sieve are blocked. However the resulting flow over this monolayer turned out to be in the wrong dimensional plane (how to say this?). The direction of the flow over the micro-sieve can be seen in figure Xsome+1.
This setup imposes the problem that the diffusion of AHL is much slower than the flow rate over the sieve. Therefore the AHL produced will always be flushed away before a uniform concentration can be established over the whole cell culture, thus preventing any synchronized behaviour to arise. The use of a micro-sieve in the course of our iGEM project was therefore discarded.
The problem described above does not arise when using the micro-dish. In the 40 micron deep wells the cells can be trapped and AHL will have a better chance to establish a uniform concentration througout the well. This will create a higher chance of synchronized oscillatory behaviour of the cells growing in a well. Special care has to be taken with the velocities of the fluid flowing over the wells. If the flow rate is to high, the cells will be spilled out of the wells. This behaviour was also observed under the microscope.
Since flowing over the wells(howtowritethis.) it's not necessary, so in the end only bottom flow was used. as seen in the picture below. As a result, the bacteria were bottom fed as seen in the scheme in figure Xsomeplussomemore. This allowed the measurements to be taken continuously for various hours, as nutrients can diffuse through the bottom of the well.
Fig.Xwhatever: Applying bottom feeding to keep the cells in the wells alive
As already mentioned in the design section of the device, the chamber was constructed in such a way that it was possible to place it under a fluorescence microscope for measuring GFP.
Top: Devices under the microscope
Right: Entire setup of the system around the fluorescence microscope
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Measuring oscillations
By chance an oscillatory behaviour of transfomed E.coli containing the streamlined construct was observed in one of the experiments performed with a plate reader. This suggested oscillations could occur even without applying any flow over the wells. Letting the modeling tool iterate over a range of cell desities while keeping the flow rate constant at 0 confirmed that oscillations could occur at high cell densities. Therefore the measurements for oscillatory behaviour of the construct were taken without applying any flow.
For the experiments, an overnight culture of the cells containing the relevant construct was spun down and resuspended in PBS. The resuspended culture was inoculated in the device and left in the chamber to settle down for a while. Since the bacteria were bottom fed with LB as seen in the set up section in figure X+2, only the bacteria which settled down in the wells survived, while the bacteria in PBS starved to death. This is shown in the short video below. The pictures were taken every ten minutes.
The video shows the micro-dish directly after inoculation, when the bacteria are still floating around everywhere in the chamber. After that the bacteria which are only in PBS start to die, whereas the bacteria in the wells survive.
After letting the bacteria grow in this manner for 2-3 hours, the PBS was removed with the same syringe that was used to inoculate. The chamber was then let to dry out over night. The two pictures seen below show the micro-dish with a ptet-GFP strain growing in the wells directly before and after removal of the PBS.
For the experiment seen above, the PBS was removed before all the cells died. The procedure varied depending on how well the bacteria grew in the wells. They were left to grow in the device for an additional night and the measurements were then taken in an 10 minute intervall during the next day. This was done for two reasons, for one the chamber had to be completely dried out before measurements could be taken, otherwise the remaining liquid would condense through the heat of the light and blurr the pictures. The second reason was that, as mentioned before, modeling suggested that, when applying no flow, the oscillations would occur only when starting with a high cell density.
