Latest revision as of 03:47, 29 October 2011

Team IGEM Paris 2011

Kinetics

What we call the Tecan® is the Infinite 200 PRO multimode reader from Tecan®.

This machine can read fluorescence and absorbance in a 96 well plate, within cycles that are programmed to be repeated, and the kinetics can last for hours. It allows to look at evolution of fluorescence during all the growth of a bacterial colony.

In our case as the receiver and the emitter of each of our system have specific fluorescence, we tuned the Tecan® to measure fluorescence every 5 or 10 minutes for 3 to 12 hours depending on what we were studying. To characterize a part, we usually do an overnight experiment. To test wether nanotubes are forming or not, 3 to 4 hours are enough since we need B.subtilis to be in exponential phase.
The sensitivity of the Tecan® allows precise quantification of the fluorescence emitted by our various designs.

Nanotube experiment

Optimization

What we want to measure is the appearance of fluorescence in receiver system (switch off -> on). But to switch the receiver system on, we needed to make sure we could reproduce the conditions of nanotubes formation in the 200 microliters wells of the 96 plate. We therefore first tested the growth of Bacillus subtilis in three different conditions to see if it could grow normally :

cells in rich media (LB), concentrated at the bottom of the well by centrifugation
cells in rich media (LB), with on the bottom of the well a thin layer of LB agar, concentrated by centrifugation again
cells in rich media without centrifugation

Our negative controls were LB without bacteria, and LB agar and LB without bacteria. We fine tuned the Tecan sensitivity for each experiments. At the end of experiments we resuspend the cells and measure the optical density. We are hence able to compare the cells growth in each conditions.

As you can see from the experiment result's, the LB agar is not important for the cell's growth.

Tested conditions

Once the experimental set up optimized, we systematically tested in triplicate our designs with different ratios of emitter versus receiver strains.

We usually organized our plate as below.

As the ratio 1:1 emitter/receiver seemed to be the most favorable according the original paper, we decided to have most wells in these optimal conditions to increase our chances of observing nanotubes.

Protocol

let grow the strains in LB until the OD reaches 0.8 (if subtilis) or 0.5 (if coli)
fill the plate with the different ratios and negative controls
measure the OD in the whole plate
centrifuge at 1500rpm for 7 minutes
resuspend some wells for negative controls
let them in the Tecan® with a measure of fluorescence of interest every 5mn for 4 hours.
when the kinetic is over resuspend the cells
measure the OD again

Characterisation experiment

All our system were designed for the emitter cell to express one fluorescence and its receiver an other one. Moreover most of the time the "answer" of the receiver system when getting the signal is to express a new fluorescence. Therefore most of our parts rely on fluorecent reporter, and we could realize some of our characterisation, with that machine.

Protocol

let the strains grow in rich liquid media(LB) until the OD reaches 0.2
dilute them with LB with or whithout inducer/inhibitor
fill the plate wiith those solutions
add mineral oil on the top of it to avoid evaporation
tune the tecan to realise OD and fluorescence measures every 10minute for 12 hours

@@ Line 3: / Line 3: @@
 =Kinetics=
-What we call the Tecan is the Infinite 200 PRO multimode reader from Tecan®.
+What we call the Tecan® is the Infinite 200 PRO multimode reader from Tecan®.
 [[File:Tecan_Picture_Paris.jpg|right]]
-This machine can read fluorecence and absorbance in a 96 well plate, within cycles that are programmed to be repeated, and the kinetics can last for hours. It allows to look at evolution of fluorescence during all the growth of a bacterial colony.<br>
+This machine can read fluorescence and absorbance in a 96 well plate, within cycles that are programmed to be repeated, and the kinetics can last for hours. It allows to look at evolution of fluorescence during all the growth of a bacterial colony.<br>
-In our case as the receiver and the emitter of each of our system have specific fluorescence, we tuned the Tecan to measure fluorescence every 5 or 10 minutes for 3 to 12 hours depending on what we were studying. Indeed, to characterize a part, we do the characterization overnight. If we wanted to do a nanotube experiment, it had to last the exponential phase of <i>B. subtilis</i> - 3 to 4 hours were enough.<br>
+In our case as the receiver and the emitter of each of our system have specific fluorescence, we tuned the Tecan® to measure fluorescence every 5 or 10 minutes for 3 to 12 hours depending on what we were studying. To characterize a part, we usually do an overnight experiment. To test wether nanotubes are forming or not, 3 to 4 hours are enough since we need <i>B.subtilis</i> to be in exponential phase.<br>
-The advantage of the Tecan over the other techniques is the quantification of the fluorescence observed.
+The sensitivity of the Tecan® allows precise quantification of the fluorescence emitted by our various designs.
 ==Nanotube experiment==
-What we want to measure with the Tecan is the appearance of fluorescence in receiver system (switch off -> on). But to switch on the receiver system we need the conditions of the nanotube experiment to be reproduced in the 200 microliter well of the 96 plate. We first tested the growth of GFP+ <i>Bacillus subtilis</i> in three different conditions to see if they could grow normally, the conditions were :
+===Optimization===
-*cells in rich media (LB) centrifuged
-*cells in rich media, with on the bottom of the well a thin layer of LB agar, centrifuged
+What we want to measure is the appearance of fluorescence in receiver system (switch off -> on). But to switch the receiver system  on, we needed to make sure we could reproduce the conditions of nanotubes formation in the 200 microliters wells of the 96 plate. We therefore first tested the growth of <i>Bacillus subtilis</i> in three different conditions to see if it could grow normally :
+*cells in rich media (LB), concentrated at the bottom of the well by centrifugation
+*cells in rich media (LB), with on the bottom of the well a thin layer of LB agar, concentrated by centrifugation again
 *cells in rich media without centrifugation
-Our controls were LB without bacteria, and LB agar and LB without bacteria.
+Our negative controls were LB without bacteria, and LB agar and LB without bacteria.
-We tuned the Tecan for it to measure at the good level even when there was agar in the bottom and ran the experiment for 12 hours. At the end of the 12 hours we resuspended the cells and measured the optical density to be able to compare if the cells grew approximatively the same according to the conditions.
+We fine tuned the Tecan sensitivity for each experiments. At the end of experiments we resuspend the cells and measure the optical density. We are hence able to compare the cells growth in each conditions.
 [[File:Curves_GFP+_Paris.jpg|center|800px]]
-According to the curve the LB agar wasn't important for the cell's growth.
+As you can see from the experiment result's, the LB agar is not important for the cell's growth.
+===Tested conditions===
+Once the experimental set up optimized, we systematically tested in triplicate our designs with different ratios of emitter versus receiver strains.
-We then realized in parallel of the microscopy team all the experiments they were doing with the same systems, but as we had 96 wells to do so we tried different ratios of strains. For each ratio we did at least one triplicate.<br>
 We usually organized our plate as below.<br>
 [[File:Tecan_nanotube_plate_Paris.jpg|center|700px]]
-We realized so much wells with a ratio 1:1 to get the closest possible to the conditions of the article.
+As the ratio 1:1 emitter/receiver seemed to be the most favorable according the original paper, we decided to have most wells in these optimal conditions to increase our chances of observing nanotubes.
-To reproduce the nanotube experiment, we let grow the strains to reach the exponential phase and then we dilute them in the wells for them to continue the exponential growth, for the 3 to 4 hours that last the measure.
 <br>
+===Protocol===
+*let grow the strains in LB until the OD reaches 0.8 (if <i>subtilis</i>) or 0.5 (if <i>coli</i>)
+*fill the plate with the different ratios and negative controls
+*measure the OD in the whole plate
+*centrifuge at 1500rpm for 7 minutes
+*resuspend some wells for negative controls
+*let them in the Tecan® with a measure of fluorescence of interest every 5mn for 4 hours.
+*when the kinetic is over resuspend the cells
+*measure the OD again
 ==Characterisation experiment==
+All our system were designed for the emitter cell to express one fluorescence and its receiver an other one. Moreover most of the time the "answer" of the receiver system when getting the signal is to express a new fluorescence.
+Therefore most of our parts rely on fluorecent reporter, and we could realize some of our characterisation, with that machine.
+===Protocol===
+*let the strains grow in rich liquid media(LB) until the OD reaches 0.2
+*dilute them with LB with or whithout inducer/inhibitor
+*fill the plate wiith those solutions
+*add mineral oil on the top of it to avoid evaporation
+*tune the tecan to realise OD and fluorescence measures every 10minute for 12 hours
-To characterise our parts we let the strains containing them growing to an OD of 0.2 that is before <i>subtilis</i> exponential phase to be able to well see it, an then put them in the plate.
-If our system requires an induction to switch on, we use strains that are induced before being put in the wells and non induced ones that we would induce in the plate itself.
 <br><br>

Team:Paris Bettencourt/Experiments/Methodologies/Tecan

From 2011.igem.org

Latest revision as of 03:47, 29 October 2011

Contents

Kinetics

Nanotube experiment

Optimization

Tested conditions

Protocol

Characterisation experiment

Protocol