Team:ETH Zurich/Experiments

From 2013.igem.org

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<b>Native <i>Acylated homoserine lactone</i> diffusion tests</b>
<b>Native <i>Acylated homoserine lactone</i> diffusion tests</b>
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<p> 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. <big>Those datas are used for the Model of the AHL diffusion.</big>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 <big>game board</big>) 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>
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<p> 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 <big>game board</big>) 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>
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<b>Enzyme-substrate reaction tests</b>
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Revision as of 14:54, 20 August 2013

Header2.png
80px-Eth igem logo.png

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


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