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Team:ETH Zurich/Experiments
From 2013.igem.org
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| + | [[File:Substratecrosstest.png|right|300px|thumb|Figure 1 : Enzyme-substrate test matrix]] | ||
| + | <p>An enzyme-substrate test matrix (Figure 1) was established to test each substrate against each enzyme. The results were as expected (Figure 2) 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.</p> | ||
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| + | [[File:Expectedsubstrate.png|right|200px|thumb|Figure 2 : Expected colorimetric response]] | ||
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<b>Native <i>Acylated homoserine lactone</i> diffusion tests</b> | <b>Native <i>Acylated homoserine lactone</i> diffusion tests</b> | ||
<hr> | <hr> | ||
| - | + | 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.<br>The concentrations used in the experiments were based on either previous results or the plate reader experiment below.<br><br> 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]<br><br>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.<br><br><br> (pictures of the results)<br><br> | |
<b>Native <i>Acylated homoserine lactone</i> tests in liquid culture using the plate reader <i>Tecan infinite M2000 PRO</i></b> | <b>Native <i>Acylated homoserine lactone</i> tests in liquid culture using the plate reader <i>Tecan infinite M2000 PRO</i></b> | ||
<hr> | <hr> | ||
| - | |||
<b>Enzyme-substrate reaction tests</b> | <b>Enzyme-substrate reaction tests</b> | ||
<hr> | <hr> | ||
Revision as of 07:14, 22 August 2013
Tranformation and cloning constructs
| Sender cells | Receiver cells | |
|---|---|---|
| AHL expressing system | Fluorescent reporter system | Enzyme reaction-based reporter system |
| Pcons-LuxI 1 (Strong) | Plac-LuxR-Plux-GFP | Plac-LuxR-Plux-LacZ |
| Pcons-LuxI 2 | Plac-LuxR-Plux-YFP | Plac-LuxR-Plux-Aes |
| Pcons-LuxI 3 | Plac-LuxR-Plux-CFP | Plac-LuxR-Plux-GusA |
| Pcons-LuxI 4 (Weak) | Plac-LuxR-Plux-PhoA | |
| Plac-LuxR-Plux-NagZ | ||
| Biobrick | Registry part |
|---|---|
| LuxI | K805016 |
| Pcons 1 (Strong) | J23100 |
| Pcons 2 | J23118 |
| Pcons 3 | J23110 |
| Pcons 4 (Weak) | J23114 |
| Mutagenized Plux ( low [AHL]) |
Optimized Biobrick ! |
| Mutagenized Plux ( middle [AHL]) |
Optimized Biobrick ! |
| Mutagenized Plux ( high [AHL]) |
Optimized Biobrick ! |
| pLuxR | R0062 |
| Plux | |
| Plac | |
| GFP | E0840 |
| YFP | E0030 |
| CFP | E0020 |
| LacZ | I732006 | GusA | New Biobrick ! |
| PhoA | New Biobrick ! |
| Aes | New Biobrick ! |
| NagZ | New Biobrick ! |
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 !
| Hydrolase | Complementary substrate / IUPAC name | Visible color | ||
|---|---|---|---|---|
| LacZ | Beta-Galactosidase | X-Gal | 5-Bromo-4chloro-3-indolyl-beta-galactopyranoside | Blue |
| LacZ | Beta-Galactosidase | Green-beta-D-Gal | N-Methyl-3-indolyl-beta-D_galactopyranoside | Green |
| GusA | Beta-glucuronidase | Magenta glucuronide | 6-chloro-3-indolyl-beta-D-glucuronide-cycloheylammonium salt | Red |
| PhoA | Alkaline phosphatase | pNPP | 4-Nitrophenylphosphatedi(tris) salt | Yellow |
| Aes | Carboxyl esterase | Magenta butyrate | 5-bromo-6-chloro-3-indoxyl butyrate | Magenta |
| NagZ | Glycoside hydrolase | X-glucosaminide X-Glunac | 5-bromo-4-chloro-3-indolyl-N-acetyl-beta-D-glucosaminide | Blue |
An enzyme-substrate test matrix (Figure 1) was established to test each substrate against each enzyme. The results were as expected (Figure 2) 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
Enzyme-substrate reaction tests
