From 2011.igem.org

Here is the design page, in wich is sum up all the potential designs we may do designs. This page is private for the moment, but it may become available on the open wiki soon.

Designs for a direct observation

The principle of the Step 0 is to directly observe what passes from a producer cell to a receptor cell through the nanotubes. There is no signal amplification in this step.

We currently have 2 designs for the step 0:

• The Antibiotics resistance experiences: The principle is to put two strains of bacteria that present different antibiotic resistances in the same biofilm. After a growth time together, they are exposed to the two antibiotics corresponding to the resistances. They can resist together through a cooperative effect involving the exchange of antibiotic resistance enzymes through the nanotubes.
• The GFP-LacI fusion: The principle is to diffuse a GFP-LacI fusion protein from one cell that produces it to a neighboring cell that is without color and contains a plasmid with numerous LacO operons. The GFP that enters the neighboring cell via the nanotubes will then be concentrate on the plasmid giving a more intense fluorescence.

Designs for nanotube characterization

The principle of the step 1 is to characterize the nanotube communication. The idea is to pass molecules of different sizes and to measure the interval of time between the apparition of the two monitors corresponding to the diffusion time through the nanotube.

General plan for the experiment design to characterize the nanotubes

First, you put the two constructions in the same cell, and induce their expression with different quantities of IPTG. Then you measure the time between the apparition of the monitor 1 (RFP) and the monitor 2 (GFP). You repeat the experiment, but with the two constructs separately introduced into two differents cells. There should be an increase in the delay of apparition of the second monitor due to the necessary diffusion time through the nanotube.

We expect the time to increase with the size of the molecule as the diffusion coefficient is D = K/R where K is a constant and R the Stoke radius.

We have two different families of designs depending on the type of amplifier used for detecting the signal. There are non reversible amplifiers (T7 self amplifier) and reversible amplifiers, in which the property of reversibility is not exploited (Toggle Switch). Here are the designs classified according to the amplifier types.

Non reversible systems

• The T7 diffusion: The principle of this experiment is to pass T7 through the nanotubes, this T7 activating the T7 amplifier in the receptor cell.
• The amber suppressor tRNA diffusion: The amber suppressor tRNA diffusion: The principle of this design is to produce in one cell a amber supressor tRNA that will diffuse through the nanotubes. The receptor cell holds the gene for a T7 with amber stop codons that cannot be translated into a functional protein as long as the tRNA amber suppressor is not present in the cell. Once expressed, the T7amber will trigger the T7 amplification system.
• The Xis protein diffusion: Xis is a small partner of an exisase. The latter will exise a stop codon on the DNA strand that is preventing the expression of the GFP.

Potentially reversible systems

• The CI(ind) diffusion: The indestructible CI diffusion will change the toogle switch state
• The RecA* diffusion: The RecA diffusion will help the change of the toogle switch state

Two-component system designs (for. B. Subtilis - E. Coli systems only)

• The MBP diffusion: We need a CRP+,MBP- E. Coli mutant. We produce the MBP protein in Bacillus subtilis and make it diffuse through the nanotubes. As long as the MBP has not reached the E. Coli periplasm, the cell cannot digest the maltose in the medium. The indirect induction of MalR by MBP triggers the expression of the reporter GFP.
• The OmpR diffusion: We need a OmpR- Receptor* E. Coli mutant. We produce the OmpR protein in Bacillus Subtilis. As long as the OmpR has not diffused from the B. Subtilis, the signaling cascade cannot be actived. With the rescue by Bacillus Subtilis of the OmpR protein, the expression of the reporter gene is activated.

Designs for a Master-Slave system

The principle of Step 2 is to build a Master-Slave system, where the Master slave control the state of the Slave cell in a monodirectional exchange.

Summary of the principle of the Master-Slave experiments

Such a design imply to have all the sub-system reversible and the actuator and the amplifiers in particular, which makes the things non trivial.

There are several possibility of designs and they are summed-up below:

• The diffusive RecA push-on push-off system: In this design we re-use the 2010 Pekin iGEM team system of a push-on push-off system, but instead of triggering the change by UV, we trigger it making an always active RecA mutant through the nanotubes. We hope the emiter cell can control the change of the stage in the slave cell
• The transcient amplifier system: The system rely on a transcient amplifier that trigger a GFP pulse in the receptor cell

Designs for a bidirectional communication

If we succed in establishing a monodirectional communication, we may go on and try to build a bidirectional communication system. Here is the general scheme.

Summary of the principle of the bidirectional cummunication

The genetic design combine the two previously described amplification system. The complete sumup is the following:

Complete design

Hoping we can get there some day...