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

Culture Condition 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 GFP+ Bacillus subtilis in three different conditions to see if they 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 0.2
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

To characterise our parts we let the strains containing them growing to an OD of 0.2 that is before subtilis or coli exponential phase to be able to well see it, an then put them in the plate. That characterisation allowed us to know if our systems were working. Indeed we managed to put a fluorescent marker below each system therefore we could measure the evolution of fluorescence during growth, and more specifically during exponential phase(phase of the nanotubes).

Team:Paris Bettencourt/Experiments/Methodologies/Tecan

From 2011.igem.org

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