BN forum Marijn van den Brink

Synthetic cells provide a minimal model system to study fundamental principles of life. One approach to the construction of synthetic cells requires the stepwise integration of primary cellular functions, such as DNA replication, cell growth and division. We aim at accelerating the optimization and integration of these biological functions using in vitro evolution. By applying different screening and selection strategies, we enrich the best-performing genotypes, expressed in liposomes, from a large population of genetic variants. First, we exploited selection through DNA replication. Using MOSAIC [1], we generated a library of DNA-encoded self-replicators with mutated ribosomal binding sites (RBSs), leading to the enrichment of DNA variants with high protein translation rates. Second, we used fluorescence-activated cell sorting to screen large libraries of RBSs in a 4-gene construct and isolated the DNA variants that promote in vitro phospholipid synthesis. Last, we are developing a microscopy-based sorting method, which we integrate with real-time artificial intelligence-assisted image analysis for spatial and temporal phenotypic interrogation. This image-based technique can sort liposomes based on multidimensional features, including cell shape, protein localization and dynamic behaviours, as validated with Min proteins. We believe that these foundational technologies will enable the directed evolution of complex synthetic genomes, which is indispensable for the construction of a minimal cell with integrated functionalities.

[1] Van den Brink et al., MOSAIC: a highly efficient, one-step recombineering approach to plasmid editing and diversification. bioRxiv preprint https://doi.org/10.1101/2024.03.22.586135 

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