Quorum Sensing signaling molecule

Figure 1. N-Methyl-3-oxo-hexanoyl-L-homoserine lactone

N-3-OxoHexanoyl-l-Homoserine Lactone (OHHL) belongs to the family of Acylated Homoserine Lactones (AHL). In our project , we use the luxR-luxI quorum sensing model to drive communication from the sender to the receiver cells. The sender cells are designed to produce AHL from luxI. The AHL produced diffuses in the agar to reach the receiver cells consisting of luxR which inturn triggers the hydrolase expression in the non-mine colonies. The AHL diffusion is very important in that it drives the enzyme expression and via enzyme-susbtrate reaction gives colored product gives the player cue for the next logical move in the game.

Diffusion Tests of OHHL

Double layer agar diffusion tests

As part of our system design, the diffusion of OHHL from the sender to the receiver is vital in order to express the different orthogonal hydrolases. As our game mine grid is played on an agar petridish, diffusion experiments were carried out on agar plates with 1.5% agar to characterize speed and distance of diffusion , depending on different OHHL concentrations. The data from these experiments were used for the spatio-temporal model of the OHHL diffusion. The concentrations used in the experiments were based on the paper handling the evaluation of a focused library of OHHL and previous results with the plate reader experiment below.

Double layer agar experiments were carried out to check diffusion of different concentrations of the OHHL. On a normal 1.5% agar plate, a second thinner layer of 0.7% agar inoculated with 100ul receiver cells. A 1.5ul drop of OHHL was added on the middle of the plate and the diffusion was observed over several hours. The OHHL concentrations tested were [10 uM], [100 uM] and [1 mM].

Single layer agar diffusion tests

In single layer agar diffusion experiments we placed receiver colonies on the agar in a spiral pattern(Figure 1.2 and Figure 2.2) We tested the effects of diffusion from the sender cells to the receiver cells with the placement in different possible configurations. Testing several configurations gave us an idea of the diffusion behaviour in the actual game set-up. These results were then used for the spatio-temporal model of OHHL diffusion. 2 uL of OHHL were placed on the colonies and the diffusion was observed.
The plates were incubated for five hours and we then observed the diffusion patterns from fluorescent images and grey-scale images. It was noted that the background noise was relatively high and the two higher concentrations diffuse to similar distances after 5 hours of incubation. The lowest concentration (10 uM) diffused the least than the higher concentrations, as confirmed by the images and grey value analysis.

Figure 1.1 Evolution of Grey value over relative colony distance after 5 hours.The expectation of what we see under 365nm wavelength illumination is digitally comfirmed using the grey values for the different colonies

Figure 1.2 OHHL diffusion on agar plate after 5hours.Agar plate containing receiver cell colonies in spiral pattern. Image taken 23 hours after adding the drop of OHHL on the central colony. Concentration :[10 uM] (top left);[100 uM](top right) and [1 mM](Bottom left) as well as a negative control(Bottom right). On the left side an image at 365 nm wavelength, on the right side the same picture in greyscale

After 23 hours of incubation the difference is clearly visible in the 365 nm exposure images as well as from grey values. As expected, depending on the [OHHL],the GFP expression in different colonies are activated more or less far away from origin (central colony). The background noise is still relatively high but the GFP expression is clearly visible, especially in the grey scale 365nm wavelength images.

Figure 2.1 Evolution of Grey value over relative colony distance after 23 hours. The expectation of what we see under 365nm wavelength illumination is digitally comfirmed using the grey values for the different colonies

Figure 2.2 OHHL diffusion on agar plate after 23hours. Agar plate containing receiver cell colonies in spiral pattern. OHHL drop (2 uL) was added on the central colony. Concentration :[10 uM] (top left);[100 uM](top right) and [1 mM](Bottom left) as well as a negative control(Bottom right). On the left side an image at 365 nm wavelength, on the right side the same picture in greyscale

Sender - receiver cell set-up

Diffusion experiments were carried out with the sender-receiver constructs as well. With the hexagonal grid, we plated out 1.5ul of the sender-receiver cells from the log phase liquid cultures on a 1.5% gar plate. After incubaion for three hours, the diffusion of OHHL was observed for over a period of 11 hours until saturation of the GFP expression was observed in the receiver cells.