Project Sonicell introduces exciting foundational advances to synthetic biology aimed to enable rapid cellular response to a combination of external stimuli such as sound, light or chemical compounds. This system is composed of a module for enhanced sensitivity of cells to ultrasound or other mechanical stimuli sensed by a calcium-dependent reporter, and a module for integration of a combination of several input signals into a signaling pathway based on the collection of orthogonal proteases. Finally, the proteases were designed to cleave an endoplasmic reticulum retention signal from target proteins, which results in a secretion of premade proteins.

Enhanced mechanosensing:
Sensitivity of mammalian cells to ultrasound or other mechanical stimuli was enhanced by the introduction of mechanosensitive ion channels and/or by the expression of protein gas vesicles from bacteria. Influx of calcium through channels is sensed by formation of a complex between calmodulin and M13 peptide that can result in a rapid light emission by cells (used for cell painting) or reconstitution of a split protease.

Protease based signaling
and information processing:
Combinations of proteolytic activities against specific targets resulted in activation of a reporter or another protease, which forms the basis for the design of a new type of rapid signaling pathways and construction of logic functions.

Orthogonal site-specific proteases:
A collection of orthogonal site-specific proteases that recognize different targets was prepared as split proteins, whose activity against selected targets can be induced by stimulation with an external signal such as light or chemicals.

Protease-triggered rapid secretion
of therapeutic proteins:
A rapid cellular response by secretion of a protein is triggered by the proteolytic cleavage of an endoplasmic reticulum retention peptide. After the cleavage the cargo protein is moved from the ER, and secreted as therapeutic protein.

Abstract for experts

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.

Abstract in plain English

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.