Team:Paris Bettencourt

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Team IGEM Paris 2011

Towards harnessing bacterial nanotubes by and for synthetic biology

The interest: A recent ground-breaking paper [1] described a new cell-to-cell bacterial communication system: nanotubes. Through excellent electronic microscopy images, antibiotic resistance transfer, faint fluorescence transfer, and cross-antibiotic resistance, previously unknown exchange channels were revealed between B.subtilis cells and even between completely different species. Results suggest that protein and/or RNA can travel through these tubes. This discovery may lead to a redefinition of individuality in bacteria. Given the many applications of known communication systems (e.g., quorum sensing, conjugation) in synthetic biology, harnessing the capacity of the nanotubes will open endless possibilities for new applications as amorph computing....

The challenge: Using synthetic biology to characterize the nanotubes: the structure, composition and control of which are still unknown. We decided to work on this challenging problem by providing other proofs to support this discovery.

See our work!

The project:

We bet-hedged our chances using different reporter systems by creating several devices relying on protein or RNA diffusion. Our devices rely on an emitter cell and a receiver cell that amplifies the signal. We chose to work with signaling molecules of different sizes and natures to test thoroughly the diffusion possibilities. Experiments were coupled with a thorough modeling approach tackling both the detection devices as well as diffusion, assisted transfer within the tubes and the membrane fusion within.

Our lab achievements

We worked very hard test the presence of nanotubes and characterize them. Come and find out more about the results of this scientific summer.

The values:

Ethics and safety are two main concerns when building genetically engineered organisms.

You can visit our Human practice page and our safety page.

New Achievements since Regional Jamboree

We have:

Characterized a fully functioning T7 autoloop (emitter and receiver) device in B. subtilis.(15)
Characterized a fully functioning sporulation device (KinA emitter and Sin operon receiver) device in B. subtilis.(16)
Integrated in the chromosome of B. subtilis the T7 polymerase diffusion entire system (emitter and receiver) and the YFP:tetR emitter system
Integrated in a episomal plasmid in B. subtilis and TetO Array
Tested nanotubes formation between species (B. subtilis and E. coli) with the system: TetR-YFP emitter in E. coli and TetO array receiver in B. subtilis. No evidence of nanotubes was found so far.(17)
Tested nanotubes formation between species (B. subtilis and E. coli) with the system T7 autoloop. No evidence of nanotubes was found so far. (18)
Created the working integrative plasmid pDCPKO(19)
Created and analyzed the collaboration map between iGEM teams throughout the years as a part of our Human Practice project (20)
A bonus track: Protocole - The song (21)
Survived a second wiki freeze night.

Achievements

We have:

Used rational design in combination with modeling to create 6 new couples of emitter/receiver constructs (1) that could help characterize nanotubes' properties, using synthetic biology approach.
Proofs of principle of 5 working emitter and receiver devices.(2) (3) (4)
Successfully reproduced the GFP experiment of the founding paper (5), indirectly proving the existence of the nanotubes under our microscopes.
Developed two original models (6) (7) that could explain the observed transport parameters through the nanotubes.
Built a microfluidic chemostat chip, to monitor single bacterial layers under controlled conditions.(8)
Reproduced the original antibiotic experiment (9), design complementary controls and proposed alternative explanations for the results observed.
Created 38 new biobricks, based on new charachterized parts, all are now at the registry (10) and devices for B. subtilis.
Specifically, created and characterized an efficient amplifier with positive feedback loop of T7 RNA polymerase on T7 promoter.(11)
Created devices aiming at hijacking two endogenic bistable switches in B. subtilis. (12)
Created and characterized a fully functioning fluorescent concentrator device.(13)
Collaborated with the Grenoble iGEM team for the human practice(14), building a scientific and citizen proposal on synthetic biology.
Most importantly, we have learned a lot during this iGEM phase of the competition. We were selected as one of the three finalists of the European Jamboree and advanced to the World Championship in Boston.

References

Intercellular Nanotubes Mediate Bacterial Communication, Dubey and Ben-Yehuda, Cell, 2011, available here

@@ Line 59: / Line 59: @@
 <li>Characterized a fully functioning <b>sporulation device (KinA emitter and Sin operon receiver) </b>  device in <i>B. subtilis</i>.<a href="https://2011.igem.org/Team:Paris_Bettencourt/Experiments/SinOp">(16)</a></li>
 <li><b>Integrated in the chromosome</b> of <i>B. subtilis</i> the T7 polymerase diffusion entire system (emitter and receiver) and the YFP:tetR emitter system</li>
-<li><b>Integrated in a episomal</b>plasmid in <i>B. subtilis</i>  and TetO Array</li>
+<li><b>Integrated in a episomal </b>plasmid in <i>B. subtilis</i>  and TetO Array</li>
 <li><b>Tested nanotubes formation</b> between species (<i>B. subtilis</i> and <i>E. coli</i>) with the system: TetR-YFP emitter in <i>E. coli</i> and TetO array receiver in <i>B. subtilis</i>. No evidence of nanotubes was found so far.<a href="https://2011.igem.org/Team:Paris_Bettencourt/Experiments/YFP_TetR_diffusion_experiments">(17)</a></li>
 <li><b>Tested nanotubes formation</b> between species (<i>B. subtilis</i> and <i>E. coli</i>) with the system T7 autoloop. No evidence of nanotubes was found so far.  <a href="https://2011.igem.org/Team:Paris_Bettencourt/Experiments/T7_diffusion_experiments">(18)</a></li>