Periodic Reporting for period 1 - DNA-ENC SYNCELLS (DNA Lattice-Encoded Information for Genotype-to-Phenotype Evolution of Self-Replicating Synthetic Cells) Reporting period: 2018-03-01 to 2020-02-29 Summary of the context and overall objectives of the project Living cells may be the most sophisticated example of functional machines operating on the nano- and microscale. But is it possible engineer synthetic analogues in the laboratory? In this fellowship, we propose a strategy to engineer cells de novo, by merging two precision technologies, microfluidics and DNA nanotechnology, to position and manipulate functional parts in space and time [1]. In particular, we use DNA as a near-universal linker to functionalize microfluidic compartments. Using this DNA handle approach, we demonstrate the stimuli-responsive attachment of natural and synthetic subcellular components. We further employ DNA to construct functional parts, including ion channels or a pH-responsive DNA-based cytoskeleton mimic, which serves as a stabilizing cortex. Following passive encapsulation, we actuate DNA nanostructures in microfluidic or lipid-based compartments to assemble dynamic systems with structural reconfigurability. By the integration of plasmonic probes we achieve real-time optical feedback to monitor the dynamics upon external stimulation. These unique tools bridging the mico- and nanoscale will enable synthetic cellular systems, which are not merely copies of nature’s own example of life, but prescribe a direction towards synthetic cellular machines, ultimately applicable as active probes inside the human body. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far First, I established a new method to form giant unilamellar vesicles as cell-like compartments in a simple one-pot reaction. This technique will directly benefit laboratories around the world (ACS Synth. Biol.). Second, I invented a near-universal method to functionalize microfluidic droplets via DNA handles – now filed to the European Patent Office. Having guided my Master student through the attachment of various functional groups and components, I am the co-corresponding author on the manuscript in Adv. Funct. Mater. I then employed proteorhodopsin-expressing bacteria in synthetic cells to trigger the reversible assembly and disassembly of a pH-responsive DNA-based cytoskeleton upon illumination. For the first time, bacteria are used as organelles in synthetic cells (to be submitted). Remarkably, I then demonstrated autonomous division of synthetic cells. I further established a small group with independent collaborations and laid out my view on engineering synthetic cells in a review featured on the cover of Trends in Biotechnology. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) We combined DNA nanotechnology and droplet-based microfluidics. Thereby, we established a new direction in synthetic biology, paving the way towards truely synthetic cells assembled de novo. Tangible applications were realized with a new technique to functionalize microfluidic droplets (patent pending). Functionalization of microfluidic droplets with DNA
