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    • T7 polymerase diffusion
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    • Integration in B.subtilis
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Project abstract

Mankind is only beginning to grasp the complexity of living organisms. New discoveries often challenge our understanding of life. We believe that synthetic biology can be used as a powerful and reliable tool to help us comprehend and characterize the phenomena we just encountered.

As an iGEM team, we decided to work on one of the most intriguing microbiology discovery of the last decade: the existence of nanotubes communication routes in Bacillus subtilis!

Summary:

Our project is based on an article published by Dubey and Ben-Yehuda [1] in the Journal Cell. In this paper, they show an extraordinary new form of communication between Bacillus subtilis cells and even exchanges with E. coli

The existence of the nanotube network discovered by Dubey and Ben-Yehuda is still discussed. We want to use synthetic biology to provide new evidences supporting the existence of a new cell-to-cell communication in Bacillus Subtilis and between Bacillus Subtilis and E.coli Then, we want to characterize this communiction as best as we can using carefully tuned genetic designs. We also aim at proposing new applications combining synthetic biology and the nanotubes network.

Each step of our project corresponds to a new level of understanding of the nanotube network inner mechanisms.

Direct observation

We want first to prove de novo what the authors found. Although some microscopy images prove solidly the existence of these so-called nanotubes, we aim at using synthetic biology to get a definite proof of the existence of nanotubes. We will simply re-do the experiments done in the paper with simple observations.

Characterization

Our second aim is to characterize the nanotubes: what passes through them and what are the typical diffusion times through the network. We will examine if RNA, proteins of different sizes and/or metabolites can pass through and with which ease and rate. The idea is to pass different molecules so that we can caracterize the speed and the transfer mechanism process. For that purpose, we are going to engineer, using synthetic biology approaches, [2] different designs build on this logic:

• An emitter cell that produces a messenger (RNA, protein etc.)
• This messenger passes through the nanotubes and into the receiver cell
• The emitter cell has specific promoters that activates an amplification system
• This amplification system in turn trigger a detection mechanism we can measure (fluroescence, others)

As a general outline we will first investigate the inter-species (subtilis-coli) connection to take advantage of all the existing biobricks for E .coli We will then move on to an intra-species (subtilis to subtilis) connection and develop new parts specific to subtilis

Master-slave system

Once the nanotubes will be caracterized, we would like to try the emitor cell to control the response of the receiver cell. The idea is that when an emittor cell meet a receiver cell, it triggers a change in the state of the latter, change, that is reversible. For that reason, we need to build a reversible amplification system. This will be acheive usinf the ComK/ComS system (see desing page)

What are the process involved in the nanotubes?

The results from the paper suggests that there can be an active process that makes the transfert of the cell constituents from the irst one to the secon faster thant the simple diffusion. This statement is to take with caution. A completely active process would involve specific transporters whereas the variety of the molecules transported indicate thate there are probably no specificity in the transport. To go farther in the research of what is really happening at the molecular scale, we runned several simulation and formal calculation to demonstrate the feasability, and calculate the speed of the mixing of the cells constituents through the nanotubes. To go farther, we propose a new mechanism, that would be driven by membrane tension relaxation, using a statistical physic approach. Is the communication directional or non directional? This is one of the key that would make the difference between the different mechanism proposed. To see more about our modeling, we strongly recommand you to go on the modeling wiki page.

You can find out more about the different steps in the Projects section.