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  Challenge 1: Noninvasive activation of cells in the tissue

Among the signals that can be used to activate engineered cells, light seems to have the best properties – it can be instantly turned on and off, it can be restricted to the selected location and its intensity can be adjusted, which makes it in many ways superior to stimulation by chemicals, temperature or pH and is responsible for the popularity of optogenetics. Its only apparent disadvantage is that most tissues are poorly transparent to light, which can therefore penetrate only short distance. Therefore, to stimulate cells in deep tissue, such as in the brain, invasive procedures have to be used. We envisioned, as an attractive alternative signal, induction of cells with ultrasound, which can readily penetrate deep into tissues, has been proven harmless unless used at very high power, can be regulated in duration and intensity similar to light, and can also be focused. Ultrasound has been reported to activate mechanoreceptors; therefore the challenge was to increase the sensitivity of cellular mechanosensing and then detect the activation of mechanosensors and couple it to selected signaling processes. Achieving these milestones would usher in the era of mechanogenetics/sonogenetics.

  Challenge 2: Engineering fast cellular response

One of the important challenges of synthetic biology is to achieve faster response of cells to different stimuli. While most engineered genetic circuits are based on transcriptional regulation, which has a delay due to the transcription/activation, cells are also able to respond faster using signaling pathways based on protein interactions or their modifications. Fast response is particularly important, for example, for the release of hormones such as insulin or neurotransmitters, where the response is required within minutes. Engineering new orthogonal signaling pathways without interfering with the normal cellular processes may be quite demanding, particularly for a complex response combining several inputs. Simple transfer of a signaling pathway from another organism may be feasible; however, engineering the desired response by adapting an existing signaling pathway may be difficult. The challenge was therefore to build a modular protein interaction/modification-based signaling and logic processing pathway that would provide a faster response than transcriptional regulation.

  Challenge 3: Fast secretion of target protein from cells

Fast signal processing may not be sufficient to result in a fast benefit to the biological system. In addition to detection by reporters cells need to rapidly demonstrate a new phenotype or send output signals to the environment. Therefore a fast response should not depend on the new synthesis of molecules but rather on release or modification of already synthesized molecules. The challenge was therefore to design a system where the already synthesized proteins would be released from the cells upon introduction of a signal. This has been reported before to function through the addition of chemicals to disassemble protein aggregates, however we wanted the complete system to be genetically encoded.