Inspiration & description

Genetic tools and circuits to control various functions of living cells have been a top priority in the field of synthetic biology. However, there has been little focus on developing methods for dynamically manipulating biological systems. Mimicking nature's ability to control the internal dynamics by varying both protein levels and expression timing would deepen our understanding of how cellular pathways function and could prove useful biotechnological applications.

Optogenetics is an emerging field and the only outside of neurosciences, specializing in the development of regulatory switches that facilitate the dynamic regulation of target gene expression. Optogenetic approaches demonstrate precise control over cellular functions by light. The variability of the stimulus light allows for a specific triggering of cellular events in a non-invasive and highly resolving spatiotemporal fashion. The integration of light-regulated systems into synthetic biology opens up a way for creative and innovative applications.

The problem lies in the fact that there is a huge need for well-working optogenetic tools to control bacteria for the applications in synthetic biology.

Optogenetic tools in bacteria have two distinct groups. Currently, most of the transcription regulators are two-component systems. Such systems consist of two proteins for light activation and signal transduction. What is more, most of the two-component systems require additional proteins for the synthesis of light-responsive prosthetic groups, leading to a massive amount of cellular resources wasted, thus restricting the application for synthetic biology. On the other hand, one-component systems only require a singular protein, which, when excited by light, changes the conformation or the degree of oligomerization, leading to an increased affinity toward DNA. However, such systems have a low dynamic range between the induced and the uninduced states. Therefore, there is a need for a new class of light-modulated proteins to fulfill the full potential of optogenetics in E. coli.

Blue-light activators are the most common, and a large number of systems incorporate blue-light responsive optogenetic tools. Therefore, there is a need for tools, sensitive to wavelengths other than blue, for example, green or near-infrared.

However, currently, the most significant gap in optogenetic regulation is a lack of post-transcriptional control. At the moment, there are ZERO tools for optogenetic regulation of protein expression in bacteria other than at the transcriptional level. Tight optogenetic regulation, such as plasmid-copy number regulation or post-translation control, could start a new regulatory era for bacteria and would be a "super tool" for synthetic biologists.

The inspiration to develop such a complex project as Colight, came to us when we saw the untapped potential and the vast amount of problems we could adress in the field optogenetics. To address the shortcomings and open new avenues in bacteria regulation, we decided to develop our project Colight - a multi-level collection of optogenetic tools for modular bacteria control.

Our collection consists of:

1) Green light transcription regulator (CarH)

One-component optogenetic systems are the goal of bacterial light-modular control. However, all currently existing light-inducible one-component systems have a drawback of low dynamic range between the induced and the uninduced states. As all the one-component systems are activator-based, which results in huge leakage, we hypothesized that a repressor-based design would highly increase the dynamic range and would be the perfect optogenetic system for bacteria control.

2) NIR light transcription regulator (BphP1)

Near-infrared light regulators are highly desirable optogenetic tools because they are orthogonal to most photoproteins and the low NIR light phototoxicity. Moreover, NIR switches have massive potential in bacterial control inside the mammalian body. For example, these switches can be used with microbiota, or for tumor site-specific drug release in bacterial cancer therapy. Currently, the dynamic range with n this wavelength was 2.5 times. For this reason, we decided to investigate the bacteriophytochrome photoreceptor 1 (BphP1) and truncated variants of the DNA-binding protein PpsR2 from Rhodopseudomonas palustris.

3) Light modular plasmid copy number regulation

Plasmid copy number control provides global gene regulation. A dynamic plasmid copy number control would open up the possibilities to investigate new avenues of dynamic gene network characterization and make a global parameter for multiple genes. What is more, such a system would offer novel biotechnological techniques, as our system would allow maintaining a low copy number of plasmids at the growth stage and a high copy number at the production state. For the development of our system, we used the El222 blue-light responsive protein and applied it to the Synori Vilnius-Lithuania iGEM 2017 framework.

4) Post-translation regulation

Wouldn't it be nice to regulate and control bacteria functions directly in real-time and not affect the transcriptional level by disrupting other endogenous bacteria functions? The solution might be quite a simple one, but not so readily available, that is a system based on protein degradation. We decided to design and create a system in prokaryotes that uses light as a switch for protein degradation control. Tunable protein degradation with the possibility of protein steady-level control could become a versatile and useful instrument for synthetic biologists to create sophisticated gene networks without affecting or disrupting transcriptional levels in bacteria.