Proof-of-principle

Diffusion tests in hexagonal grid with wild-type sender-receiver constructs

Figure 1: Scanned image of GFP fluorescence in a hexagonal grid with sender cells in green and wild type pLux GFP receiver cells

Single layer agar diffusion experiments were performed to study diffusion from the sender colonies to the receiver colonies. The colonies were placed in a hexagonal grid pattern such that every colony can have zero, one , two or three mine colonies adjacent to it. Depending on the number of mines around a non-mine colony, more OHHL will be processed in the receiver colonies; due to more mines, but not due to more diffusion. 1.5μl of the receiver and sender cultures were inoculated in a hexagonal grid pattern in an agar plate and incubated for three hours. We then observed the diffusion patterns by scanning the plated with a molecular imaging software. Images of the GFP fluorescence were taken at time intervals of one hour and half hour as fluorescence started. Images taken after 11 hours showed GFP saturation.

The picture to the right shows the scanned image of GFP fluorescence after 11 hours of incubation. The colonies are plated in a hexagonal grid. The green circles are the mine/sender colonies. The receiver colonies are those that process the diffused OHHL from the sender colonies. The numbers 1, 2 and 3 represent the number of mines around the non-mine colony. Interestingly, it can be seen that those receiver colonies that have more mines around have the highest fluorescence than those which have lesser mine colonies. The data from this experiment was also compared with the spatio-temporal model which gave a similar prediction in the model.

GFP diffusion with mutated pluxR promoter variants

Figure 2: Scanned image of GFP fluorescence in a hexagonal grid with sender cells in green and mutant pLux GFP receiver cells

Also the mutant promoter we obtained through site-saturation mutagenesis was tested in the hexagonal grid experiment with LuxI sender cells. The sensitivity for OHHL was too low thow and we did not see any induction, not even after 21h. These results are consistent with the model predictions.

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