Team:ETH Zurich/Experiments

Tranformation and cloning constructs

Here we will paste some circuit designs

Enzyme-substrate reactions

We have cloned fluorescent receiver systems as backup for our circuit in case the hydrolase reaction do not work properly.
The enzyme substrate reactions take less than 5 minutes and are visible by eye.

Our minesweeper become better and better so keep on track for updates !

Fig.1: Magenta butyrate on Aes expressing colony

Fig.2: NPP on PhoA expressing colony

Fig.3 Magenta glucuronide on GusA colony

Fig.4: X-Glucnac on NagZ colony

Fig.5: X-Gal on LacZ colony

Fig.5: Beta-green-X-gal on LacZ colony

Figure 6: Enzyme-substrate test matrix

Figure 7: Expected colorimetric response

An enzyme-substrate test matrix (Figure 6) was established to test each substrate against each enzyme. The results were as expected (Figure 7) and no cross reaction is visible. The NagZ-X - glucosaminide X-Glunac reveal some difficulties in the liquid culture as well as on the agar plate.

Native Acylated homoserine lactone diffusion tests

We perfomed simple tests in liquid culture with different AHL concentrations and saw different fluorescent intensities. We also did diffusion experiments on Agar to characterize the diffusion speed and distance depending on the concentrations. Those datas are used for the Model of the AHL diffusion.
The concentrations used in the experiments were based on either previous results or the plate reader experiment below.

On the one hand double layer Agar diffusion was tested which was not always succesfull. Double layer Agar consists of a first traditional 1.5% Agar layer. The specificity of the double layer Agar is the second 0.7% Agar layer containing receiver cells. In the experiments the AHL drop was placed in the middle and the diffusion was observed over everal hours.THe concentration we tested were [10uM];[100uM] and [1mM]

For the single layer Agar diffusion (the more realistic model and adapted to the applications for the GAME BOARD) we place receiver colonies on the Agar in a spiral pattern to avoid a "Shaddow" behind the cells. An AHL drop of 2uL was placed in on the central colony an the diffusion was observed over several hours. The concentration we tested were [10uM];[100uM] and [1mM] as well as a negative control.


(pictures of the results)

Native Acylated homoserine lactone tests in liquid culture using the plate reader Tecan infinite M2000 PRO

C6-oxo-AHL sensivity curve. The AHL concentration range is plotted against the standarized fluorescence (au)

I order to select mutated pLuxR promoters we need to know about the sensivity of the Wild-Type type. The test range was inspired form the paper about Evaluation of a focused libraryu of N-Acryl L-Homoserine lactone reveals a new set of potent quorum sensing modulators.The paper shows sensivities of the pLuxR promoter to different sets of AHL molecules in Vibrio fisheri. We need to readjuste the ranges for our E.coli DH5alpha strain in a second run and finally get our sensivity curve over the linear range of [0nM],[0.25nM],[0.5nM],[1nM],[2nM],[3nM],[4nM],[5nM],[10nM],[20nM],[30nM],[40nM]and[50nM],.

The experimental set-up included a 96-well plate filled with 180uL LB media , 10uL of receiver cells and 10uL of different AHL concentrations, everything in triplicates. (+ blank and negative control). The experiment runs for 16 hours in order to monitor the evolution of fluorescence and later choose the steady state to create the sensivity curve.The datas are analyzed with GraphPad Prism 6.0.