Team:ETH Zurich/Process/Validation

Experimental Setup

Figure 1: Experimental setup for the diffusion test in agarose filled tubes.

To validate that we can create a gradient of a small molecule along our agarose filled tube, we filled a tube (2 mm diameter, 7 cm long) with agarose and E. coli (see Figure 1). The E. coli cells were engineered to express an IPTG-inducible GFP. The cells were incubated at 37 °C overnight. One end of the tube was connected to a sample medium (1 ml) containing 10 mM IPTG solution.

Results

Fluorescence pictures of the tube (see Figure 2) showed a clear gradient of the fluorescence signal over approximately 5cm of the tube. After 5cm, the signal strength dropped under the background noise.

Figure 2: GFP gradient in tube: E. coli with IPTG-inducable GFP were incubated in a tube (2 mm diameter, 7 cm long). GFP expression was assessed under the fluorescent microscope after overnight incubation, with a excitation wavelength of 480 nm and a emission wavelength of 510 nm. The 15 microscope photos were reassembled into one using [http://research.microsoft.com/en-us/um/redmond/groups/ivm/ICE/ the Microsoft Research Image Composite Editor].

Analysis

We quantified the fluorescence signal using a moving average of 80×80 pixel, which moved along the symmetry axis of the tube (see Figure 3).

The fluorescence distribution of this experiment has a similar shape as the distributions predicted by the model (see modeling section). The difference in the experimental results compared to the simulations can be explained mainly due to the different diffusing molecule. The simulations where obtained for acetaldehyde, whereas the experiments were carried out with IPTG. We expect the different values of the diffusion as well as degradation constants of the two molecules two be the main reason for the differences in the experimentally obtained curve as compared to the theoretically obtained.

Figure 3: Quantification of the gradient in Figure 2: The light intensity of the IPTG-induced GFP signal was quantified by a 80×80 pixel moving average. The peak at around 1.2cm is due to an air bubble in the channel (see Figure 2).