The 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). We make use of the abbrevations OHHL and AHL, but in both cases we mean the native N-3-oxohexanoyl-l-homoserine lactone expressed by the LuxI system.

Native N-3-OxoHexanoyl-l-Homoserine Lactone diffusion tests

We performed simple liquid culture tests using different OHHL concentrations to measure and compare fluorescent output. We also did diffusion experiments on agar plates to characterize speed and distance of diffusion , depending on the concentrations. Those data were used for the 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.

In the double layer agar experiments we did, a second 0.7% Agar layer, containing receiver cells is added onto a normal 1.5% agar plate. Then OHHL was placed in the middle of the plate and the diffusion was observed over a time span of several hours. The OHHL concentrations we tested were [10 uM], [100 uM] and [1 mM].

In single layer agar diffusion experiments we placed receiver colonies on the Agar in a spiral pattern(Figure 1.2 and Figure 2.2) This set-up reflects more realisticly the later GAME BOARD. We also could test the influence of colonies onto the diffusion by placing them behind each others. We also included these findings into the model. 2 uL of OHHL were placed onto the central colony an the diffusion was observed.
After 5 hours of incubation we can interpret two things from images and grey values. First of all the background noise is relatively high and the two highest concentrations diffuse at the same distance until 5 hours of incubation. The lowest concentration (10 uM) diffuse less far than the higher concentrations, confirmed by the images and grey value analysis.</p>

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 (due to leacky expression ) is stil relatively high bvut the triggered GFP expression is clearly visible, espacially in the grey scale 365nm wavelenght 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

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