• Foundational achievements of this project open several avenues for synthetic biology but also for other fields of science and for diverse applications.
• Several new valuable tools and collections of parts have been developed in the project, from a toolset of orthogonal proteases to an in vivo mechanosensing system, which could be further implemented with other settings and parts.

 Protease-based signaling

Although the specificity of viral proteases have been described before, this is the first time that functional split protease variants are produced and characterized. We envision that this toolset and moreover innovative synthetic protease-based signaling pathways will find their use for engineered cells. This system is based on modules which already enabled us the design of several two-input logic functions in vivo, which suggest that more complex circuits could readily be constructed. Additionally, it is likely that the number of orthogonal proteases could be further expanded. Point mutations of known proteases, such as the TEVpE and TEVpH demonstrate that the recognition sites could further be engineered.
We demonstrated by simulation that the activation of selected logic functions can be relatively fast, while the system needs to be reset to the resting state considerably slowly, comparable to the transcriptional type of regulation, that is within hours. None of the active proteases was toxic to human cells, so their implementation for signaling pathways does not seem to pose any problems to further developments. Release of proteins or peptides from cells has been demonstrated and the capacity of the secretory machinery for secretion will have to be investigated in details in comparison to the secretion via exocytosis, which could however also be triggered via influx of calcium, which is regulated by mechanosensors.

nbsp;Mechanosensing - following the footsteps of Optogenetics

The project was very successful in the implementation of designed mechanosensitive genetic circuits. We have demonstrated cell activation by ultrasound, direct touch, and flow of liquid, which demonstrates that other direct or indirect mechanical forces, such as the osmotic stress, pressure and others could be detected in this system. Mechano-sensing plays a very important role in many biological senses, such as hearing, proprioception but also in senses that are developed only in some animals, such as the lateral line of fish. It is likely that the systems based on our building modules might be able to detect gravity, acceleration and pressure. Delivery of the mechano-sensing platform could be accomplished through viruses, which would probably be more appropriate for in vivo applications, where the focused ultrasound will spatially limit the activation of targeted cells.

nbsp;Research applications

• Following on the tracks of optogenetics such as investigation of cell activation including in vivo experiments
• Investigation of the molecular mechanisms of mechanosensing
• Refactoring or rewiring of biologicals senses

Potential therapeutic/diagnostic applications

• Ultrasound-regulated therapeutic activation of neuronal, immunological or other cells
• Development of sensors of mechanical stress or shear forces in the tissue
• In situ production of bioactive peptides/proteins regulated by the ultrasound (e.g. insulin, neurotransmitters or other bioactive molecules)
• Complex signaling pathways and logic functions based on the proteolysis

Envisioned long term applications

• Ultrasound-controlled therapeutic device
• Integrated electronic-biological devices
• Cell-based interface - such as a touchpad, more tightly integrated than for example the Temporary Tatoos by the MIT Media Labs

• Repair/enhancement of the sense of touch
• Introduction of new types of senses, such as proprioreception, gravity, flow...