Team:ETH Zurich/Process/Validation

From 2011.igem.org

Revision as of 21:05, 21 September 2011 by Langmo (Talk | contribs)

Menu image preload Menu image preload Menu image preload Menu image preload Menu image preload Menu image preload


Systems Validation
Diffusion-only System A
Text goes here

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).

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).
Retrieved from "http://2011.igem.org/Team:ETH_Zurich/Process/Validation"