Signaling molecule AHL

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

N-3-Oxo-Hexanoyl-l-Homoserine Lactone belongs to the family of Acylated Homoserine Lactones (AHL). In our project , we use the LuxI-LuxR quorum sensing system to drive the signal from the sender to the receiver cells. The LuxI sender construct produces AHL. The AHL diffuses in the agar to reach the receiver cells containing LuxR which in turn triggers the hydrolase expression in the receiver colonies. The receiver cells comprise promoters that are tuned to express specific hydrolases depending on the amount of incoming AHL. The AHL diffusion is very important in that it drives the enzyme (hydrolase) expression in the non-mines depending on different high pass filters. The AHL concentration processed by the receiver cells depends on the number of mine colonies. Through an enzyme-susbtrate reaction that generates a colored product the player obtains information about the number of mines surrounding a non-mine.

Diffusion tests using AHL and a spiral receiver cell set-up

Figure 2.1 Spiral diffusion experiment after 5h of incubation using the J09855 (plac LuxR pluxR) construct and a GFP reporter. On the left image (1) shows the picture at 395nm excitation wavelength, and on the right image (2) the greyscale of image (1). Origin of diffusion is on the central colony.

The experimental data of Figure 2.1 and 2.2 show a diffusion test using receiver cells (J09855 (plac LuxR pluxR) with a GFP reporter) plated in a spiral pattern. The origin of diffusion : a drop of AHL with the according concentration was pipetted on the central colony. The experiment shows the diffusion over time, Figure 2.1 after 5h of incubation and Figure 2.2 after 23h of incubation at 37°C. The progression of the AHL throught the agar became visible thanks to the GFP reporter. This was one of the first experiments to characterize the diffusion of the signaling molecule in the agar as we need it later in the final set-up.(If you want to know more about the methods please click here.)We could conclude some information about the diffusion speed. But we don't know about the secreted AHL concentration from sender cells and the amount of AHL sensed by the receiver cells.

The next step was to carry out the same experiment using a sender colony instead of a drop of AHL in ther origin of diffusion. (see below Diffusion tests using sender-receiver set-up).

Figure 2.2 Spiral diffusion experiment after 23h of incubation using the J09855 construct and a GFP reporter. On the left image (1) shows the picture at 395nm excitation wavelength, and on the right image (2) the greyscale of image (1). Origin of diffusion is on the central colony.

Diffusion tests using sender-receiver set-up

Diffusion tests of AHL in double layer agar

Figure 3.1: Scanned image of GFP Fluorescence of AHL Diffusion with GFP receiver cells with two senders

In our biological circuit design, the diffusion of AHL from the sender to the receiver is vital in order to express the different orthogonal hydrolases. This is because the hydrolases are expressed under the control of plux promoters which are induced by AHL.
In order to visualize the AHL diffusion, experiments were carried out, using GFP as reporter. We started with using the part BBa_J09855 cloned with GFP as our receiver. We tested the sender with part BBa_K805016 under a constitutive promoter. double-layer agar tests (See protocol) were performed with the GFP receiver cells spread evenly as a top layer agar, and the 1.5 μl sender cells as source of signal AHL. The diffusion pattern was measured over 12 hours with a molecular imaging system. The distance of diffusion was noted as 1.5 cm as the radial diffusion distance across the sender colony. The time and distance data from these experiments were used for the spatio-temporal model of the AHL diffusion.

Figure 3.2: Scanned image of GFP fluorescence of AHL Diffusion test to check diffusion time and distance

Figure 3.3: ImageJ analysis of fluorescence peak showing radial diffusion around one sender colony

The image to the left shows the GFP fluorescence in a double layer agar experiment after 11 hours. In order to quantify the time and distance of diffusion of AHL from the sender to the GFP receiver, we analyzed the scanned image of the plate with the image processing program ImageJ . The analysis from ImageJ is shown in the picture to the right. The picture shows the GFP fluorescence in gray scale with distance in inches. The distance of diffusion can be seen as 1,2 inches, that is nearly 3 cm. Hence, for the further experiments, the colonies were placed apart from each other at a distance of 1,5 cm in a hexagonal manner.

Diffusion of Synthetic molecule AHL in agar

We thank our sponsors: