Synthetic biology opens exciting perspectives to control cells, for applications ranging from industrial processes to cell-based therapy. However, the large majority of designed cellular circuits are based on the transcriptional regulation, which may be too slow for many therapeutic or diagnostic applications, for example delivery of insulin or detection of a metabolite. Several medical doctors and researchers that we consulted stressed that a fast but controllable response is high on their wish list of expectations from synthetic biology. Additionally, noninvasive stimulation of selected tissues in the organism would also be highly desirable. While light is extremely useful as a rapid, spatially-restricted input signal, it cannot penetrate deep into the tissue. On the other hand, ultrasound combines several advantages of light with the added ability to penetrate tissue.

In our project we enhanced the sensitivity of mammalian cells to ultrasound or other mechanical stimulus by introduction of bacterial or engineered mammalian mechanosensors. Additionally, the response to ultrasound and touch was strongly increased by expression of the two components of bacterial gas vesicles, GvpA and GvpC. Mechanosensing was detected by the calcium-induced calmodulin-M13 complex reconstituting split cyclic luciferase, highly applicable for the emerging field of mechanogenetics. This enabled us to draw on cells using touch, where we engaged in collaboration with an artist.

For the rapid response of cells to multiple stimuli we designed proteolysis-based signaling pathways. For this purpose four orthogonal split proteases were generated, each recognizing its own motif of seven amino acid residues. Based on cleavage of coiled-coil dimerizing domains we demonstrated the ability to implement proteolysis-based signal pathways and logic functions in mammalian cells. Based on the cleavage of an ER retention peptide by a protease, input signals lead to protein secretion without the slow step of induced protein synthesis.

We believe that this project introduced several foundational advances that could be very useful to synthetic biology far beyond iGEM and for the benefit of humanity for therapy, diagnostics and potentially many other advanced applications.

Synthetic biology aims to control cells so they can obey our commands and do what we want, for example to produce drugs when needed. In our project we made cells respond to ultrasound or touch. When we touch the cells they light up, which can be recorded on a camera. Ideally we want cells to respond to our commands as fast as possible, because sometimes we can’t wait an hour before the cells produce the medicine and release it. That is why we gave cells a novel mechanism of processing information. We achieved this by combining several enzymes that recognize very specific parts of proteins and cut them, which changes their function. This allowed us to combine different signals, like sound, touch, light or chemicals, to obtain the desired cell response. The new enzymes can also cut the anchor with which medicines are attached to cells after the cells make them. Among many possible uses of our inventions, we can imagine activating cells in the brain by ultrasound, which means that we don’t need to use surgery to help people with Parkinson’s disease, or can trigger fast production of insulin in the body, to help people with diabetes.

• Mammalian cell sensitivity to ultrasound and mechanical stimuli was increased by ectopic expression of bacterial or human cation permeable channels and functional reconstitution of bacterial protein gas vesicles from two protein components (GvpA and GvpC)
• A custom-made ultrasound generator device was used to stimulate mammalian cells
• A mechano-sensory luciferase reporter sensitive to an influx of free calcium ions was introduced into mammalian cells, which enabled rapid light emission of mammalian cells in response to mechanical stimuli and enabled painting on cells by touch with exciting potentials for other applications
• A circular proteolysis-activated luciferase reporter was experimentally verified and introduced into the iGEM collection
• A set of four different orthogonal site-specific proteases was designed and tested as split proteins in mammalian cells
• New orthogonal protease-based signaling pathways and information processing platform was designed and several logic functions based on the combination of multiple input signals were tested experimentally
• Proteolysis of the ER retention signal was introduced as the trigger for the fast release of proteins from cells aimed to enable fast therapeutic responses such as required for the release of peptide hormones, neuroactive peptides etc.

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