Proof-of-principle

Diffusion tests in hexagonal grid with sender-receiver constructs

Figure 3: Scanned imgae of GFP Fluorescence in a hexagonal grid

In single layer agar diffusion experiments we tested the diffusion from the sender colonies to the receiver colonies in different possible configurations. Different configurations of mines ( one, two or three) radially around the non-mines will lead to more OHHL that 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 GFP fluorescence image 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. 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 surprisingly gave exact prediction in the model.

GFP diffusion with mutated pluxR promoter variants

See first : High pass filters

In order to test the GFP fluorescence pluxR promoters that were mutated by site-saturation mutagenesis , two layer agar experiments were performed with mutated and wildtype promoters. In every case, the expression was compared with a negative control without the sender (pconst-luxI) which will not induce the GFP expression. We followed the grid pattern with the different configurations of mines in a hexagonal pattern.The figure to the left shows the expression in the wildtype pluxR with the negative controls to the right of the picture. The figure to the right shows the expression in the mutated promoter with two point mutations in the sequence. The negative controls without the sender cells are to the right of the picture.

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