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Programmable BioMolecular Circuits: Emulating Regulatory Functions in Living Cells Using a Bottom-Up Approach

Periodic Reporting for period 4 - BioCircuit (Programmable BioMolecular Circuits: Emulating Regulatory Functions in Living Cells Using a Bottom-Up Approach)

Reporting period: 2021-02-01 to 2022-01-31

Introduction
Living cells form the basis of life. Cells have evolved to process an enormous amount of chemical signals using dedicated molecular networks. However, because of the high complexity of the living cell, unraveling the design principles of these molecular networks has proven impossible. In this project, we have built molecular networks outside of the living cell with the objective of studying the design principles of molecular networks using a bottom-up approach. Such an approach can provide detailed answers on the way molecular networks are able to process information. This is important for society as malfunctioning of molecular networks in living cells can lead to diseases such as cancer. In addition, these types of biomolecular networks could be used for sensitive diagnostics and molecular data storage.

Scientific Results
In the past 5 years, we have investigated several types of cell-free biomolecular networks, including i) cell-free gene circuits that can sense DNA and RNA inputs, ii) a DNA-based apoptosome mimic and iii) microcapsule colonies capable of scalable molecular communication. Our work, published in high impact journals like Nature Nanotechnology, Nature Catalysis and ACS Nano, has shown that elementary biological principles can be recapitulated under cell-free conditions. Because of the high level of control of these systems, fundamental design principles could be uncovered. For example, we have shown for the first time diffusive, multi-channel communication between semipermeable microcapsules (Nature Nano 2019). By building biologically inspired sender-receiver architectures, we have uncovered general design principles of cell-cell communication. In addition, we have also shown that these microcapsules can be used for DNA-based data storage. Next to this, we have also obtained fundamental insight on the working of the apoptosome, a large multi-protein complex that regulates cell death (Nature Catalysis 2020). Our experiments have revealed, for the first time, multivalent catalytic effects in the activity of crucial caspase enzymes.

Societal Results
Cell-free biomolecular circuits can be used in diagnostics. In the past five years, supported by an ERC PoC grant, we investigated their use in point-of-care miRNA diagnostics. In addition to this, we have discovered new materials that can be used to preserve DNA for DNA-based digital data storage.
First Period
1) Designed several genetic circuits and tested the response of these circuits in silico.
2) Designed and tested the microfluidic devices that will be used to test run genetic programs outside of the living cell.
3) Cloned DNA constructs and tested them in cell lysate.

Second Period
1) Constructed genetic circuits in the lab
2) Developed microcapsules for DNA-based communication
3) Conjugated DNA oligos to caspases

Third Period
1) Tested and analyzed the response of cell-free gene circuits capable of feedforward control (ACS Synthetic Biology 2021).
2) Demonstrated DNA-based molecular communication between microcapsules (Nature Nano 2019).
3) Demonstrated a DNA-based apoptosome (Nature Catalysis 2020).

Fourth Period
1) Tested and analyzed the response of cell-free gene circuits capable of weighted-sum operations (ACS Synthetic Biology 2022).
2) Demonstrated light-responsive DNA-based molecular communication (ACS Nano 2020)

Overall, the ERC StG has resulted in around 20 publications and invited lectures (around 5). Because of the ERC StG I was awared an ERC PoC which has allowed me to establish a collaboration with Microsoft Research in the area of DNA data storage and in-vitro diagnostics.
Scientifically, BIOCIRCUIT has pushed the field beyond the state of the art. For example, our publication on DNA-based molecular communication has been cited 130 times since 2019 and has been reviewed in around 7 review articles. In addition, our work on using DNA nanostructures to understand enzyme mechanisms has been cited 30 times and has been reviewed in 3 review papers.
A population of 3 compartmentalized DNA computers