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Team:Paris Bettencourt/Experiments/List
From 2011.igem.org
Achievements
Preliminary experiments
In the Dubey and Ben-Yehuda paper [1] a set of simple experiments are presented in support of the existence of the nanotubes. We reproduced some of them in order to demonstrate the existence of these entities as a medium of communication between bacteria, and to be sure we are in the good experimental conditions to produce them when working with our detectors (see below).
We reproduced the two keystone experiments of the paper: the GFP diffusion, and the antibiotic resistance exchange, where our results point to an alternative explanation.
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The GFP diffusion experiment is a simple experiment. One Bacillus subtlis strain producing GFP is mixed with a wild type strain. If nanotubes are formed, the GFP would diffuse through the tubes and color the non fluorescent strain. We succeded in reproducing the results of the paper, showing GFP diffusion when gfp+ cells are close to gfp- cells. We invite you to visit corresponding the page to learn more about what we did and the results we had. |
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The antibiotic resistance exchange is a more tricky experiment in which bacteria are shown to exchange resistance enzymes through nanotube and allow the population to survive even though all the cells does not carry the resistance gene. We invite you to visit corresponding the page to learn more about what we did and the results we obtained that offer an alternative simple explanation that does not evoke use of nanotubes. |
Testing for the presence of nanotubes
The goal of all our designs was to test for the presence of and characterize nanotubes. You will find here the experiments we conducted to this end, carefully designed according to the results of our computational modeling.
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The YFP concentrator This design relies on a TetO-array which allow us to concentrate YFP-TetR fusion proteins. We were able to to a E.coli to B.subtilis diffusion through nanotubes experiment with this design. Preliminary results with coli-subtilis did not show any transfer in microscopy experiments. |
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T7 RNA polymerase diffusion In this design, we introduce the use of the T7 polymerase both as the transfer molecule and as the auto-amplification system. We were able to do E.coli to B.subtilis as well as B.subtilis-B.subtilis diffusion through nanotubes experiment with this design. We also tried to experiment with our microfluidic chip with this design. Preliminary results with coli-subtilis or subtilis-subtilis did not show any transfer in microscopy experiments. |
Building and characterizing new devices
Before testing the nanotubes, we had to design entirely these new devices. They are composed of an emitter, a receptor and an amplifier subunit.
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The YFP concentrator This design relies on a TetO-array which allow us to concentrate YFP-TetR fusion proteins. We were able to properly characterize it through microscopy images. |
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T7 RNA polymerase diffusion In this design, we introduce the use of the T7 polymerase both as the transfer molecule and as the auto-amplification system. We were able to properly characterize it through microscopy images and 96-well kinetics experiments. |
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tRNA amber diffusion The tRNA amber is the smallest molecule we are trying to get pass the nanotubes . At the receiving end: T7 polymerase with two modified amber codons coupled with the T7 auto-amplifier and T7-driven GFP expression. We have also characterized the pHyperSpank promoter. We were able to properly characterize it through microscopy images and 96-well kinetics experiments. |
Using bistable switches
During our brainstormings, we noticed several natural or artificial bistable switches that could serve both as a receptor and an auto-amplifier. One molecule carefully chosen could toggle the switch in another position. All we have to do is see if it diffuses through the nanotubes.
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ComS diffusion We took advantage of a switch already existing in B .subtilis (the ComK/ComS switch) and tried to see if we could toggle it from one state to the other using molecules diffusing through the nanotubes. |
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Sin Operon We took advantage of a switch already existing in B.Subtilis (the Sin operon switch regulating sporulation and biofilm formation) and tried to see if we could toggle it from one state to the other using molecules diffusing through the nanotubes. |
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Lambda switch We took advantage of an artificial switch in E.coli created by the PKU team of 2007. We tried to see if we could toggle it from one state to the other using molecules diffusing through the nanotubes. |
References
- Intercellular Nanotubes Mediate Bacterial Communication, Dubey and Ben-Yehuda, Cell, available here
