Overview

Context

Stéphane Pinhal, one of our multi-talented iGEM advisors, is currently a PhD student in microbiology and a self-proclaimed professional couch potato. At the beginning of our team’s iGEM adventure, we were lucky enough to get Stéphane to introduce us to the basic laboratory procedures required for running a project in synthetic biology, such as culturing bacteria or making them produce recombinant proteins. After several of these key training sessions, one main aspect that Stéphane kept stressing, much to his dismay I might add, was that working with living organisms sometimes requires great sacrifices. Coming into lab late on Sunday nights to inoculate your bacterial culture, or postponing your lunch break because your cells have just reached the right growth phase for transformation are classical examples of little inconveniences that are nearly incompatible with our beloved advisor’s lifestyle.

BUT… what if Stéphane could control his bacterial culture from home?

To make his dream come true, our team began to develop a bioelectronics system that enables full remote control of living cell concentration in shaken bacterial cultures… a device we call TalkE’coli.

Principle

Our system is based on KillerRed (KR), a fluorescent protein that produces Reactive Oxygen Species (ROS) upon illumination with green light [1]. ROS, such as hydrogen peroxide (H₂0₂) or singlet oxygen (1O₂), react with bacterial DNA and proteins, causing irreversible damages that ultimately lead to cell death (figure 2). Using KillerRed with illumination at different light intensities, we aim to control variations in the number of living E. coli bacteria of a liquid culture (Fig 1.).

Figure 1.
Light-mediated control of the living biomass. Increase, decrease or stabilization of the number of living cells occurs in response to light stimulations at a carefully selected intensity.

In this project, the expression of the KillerRed gene is placed under control of the Cph8/OmpC/pOmpC red-sensitive gene expression system [2]. Therefore, both KR production and ROS-mediated cell death can be triggered with appropriate light stimulations (Fig 2.).

Figure 2.
Overview on our genetic network (copy of the genetic network of an iGEM team from a previous edition of the competition. This sketch is quite simple and easy to understand. We could try doing something similar for presenting the whole network simply and clearly (Voigt’s system is complicated, and we probably don’t need to detail the complete set of genetic sequences involved in his sensors).

This optogenetic system enables us to interface the cell culture with TalkE’coli, an electronic device that can send orders to bacteria (“produce KR”, “die”) via a light source, all while monitoring their fluorescence via a photodiode.
In order to fine-tune the device, several initial biological experiments were performed in order to gather data that could be effectively modeled. Modeling these experiments allowed us to identify specific parameters that could be focused upon in further biological tests. This key interplay between biology and modeling pushed our project to the next level, allowing the team to improve our experiments at each phase and to strive for better and better results.

Genetic Network

Achievements

The iGEM Grenoble-EMSE-LSU Team reached several key goals over these past several months in order to construct our unique optogenetic system and electronic device.

In summary, iGEM Grenoble-EMSE-LSU: • Built two new biobricks for the KR Module: pLac-RBS-KR and pLac-RBS-mCherry • Prooved the expression of KillerRed (KR) in E. coli, while demonstrating that the number of living cells could be controlled with light illumination at different intensities • Developed a predictive model in order to derive the intensity function, which achieves the desired variation in the number of living cells • Performed additional qualitative characterization experiments based on the Voigt system • Built 3 new biobricks for the Voigt Module: Redsensor-mRFP, Redsensor-KR, Greensensor-GFP that may enable the dynamic and quantitative investigation of Voigt’s photosensitive systems • Built the biobrick pBad-sspB for the Degredation Module: This could be of interest in case of a high concentration of intracellular KR, which could prevent bacteria from growing in any light conditions • add Nicolas box • add the videogame • anything alse, sellable?!