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, 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 (Fig 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 KillerRed production and ROS-mediated cell death can be triggered with appropriate light stimulations (Fig 2.).

Figure 2.
Overview on our genetic network [2,3]

In response to green/white light stimulation, the KillerRed protein produces ROS that damage endogenous DNA and proteins, leading to cell death. The expression of the KillerRed gene is controlled via the cph8/OmpR/pOmpC red light-sensitive transcription system [2]. pLTetO-1 and pLac/ara-1 allow for the expression of the transmembrane protein cph8 in its phosphorylated ground state and of the phycocyanobilin chromophore, respectively [2]. In its phosphorylated state, cph8 triggers phosphorylation of the regulatory protein OmpR, which activates transcription of the cI repressor gene. cI can be considered as a not gate that represses the expression of the KillerRed gene.

Light stimulation at 650 nm enables dephosphorylation of cph8 and thus to bypass the repression system, ultimately leading to the expression of the KillerRed protein.

The use of optogenetic tools such as KillerRed and the cph8/OmpR/pOmpC gene transcription system enable us to interface our biological system with an optoelectonic device, equipped with a light source, that can send orders to the system ("die" or "produce KillerRed") 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.

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 KillerRed Module: pLac-RBS-KR and pLac-RBS-mCherry
• Prooved the expression of KillerRed 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
• Built the electronic interfacing device TalkE’coli, so that a user can maintain a desired concentration of living cells within a sample with our engineered bacteria. It is mountable in an incubator and allows you to stabilize the living cell density
• Developed an interactive video game that aims to raise awareness about Synthetic Biology and the iGEM competition
• 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

References

[1] M.E. Bulina et al., A genetically encoded photosensitizer, Nature Biotechnology, January 2006.
[2] J.J. Tabor et al., Multichromatic Control of Gene Expression in Escherichia coli, Journal of Molecular Biology, 2011.
[3] https://2011.igem.org/Team:TU_Munich/project/introduction