2-Laser Cell Sorter

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We hereby apply for a cell sorter with 2 lasers. Such a device is capable of distinguishing individual cells by size, structure and fluorescence staining and sorting them based on predefined parameters. The device will be used to analyze and sort large libraries of bacteria and yeast (Escherichia coli, Saccharomyces cerevisiae) expressing mutants of RNAs and proteins. Here, selection is usually done by expression of a fluorescent reporter protein. The sorted cell populations are subsequently characterized by high-throughput sequencing. The device applied for here is capable of sorting more than 10 000 cells per second. This allows the construction of a "Flow-Seq" pipeline, which can be used to characterize extensive mutation libraries in a high-throughput format. It allows the generation of mutation fitness landscapes to infer important structure-function relationships (deep mutational screening). Furthermore, sufficiently large data sets are available through the pipeline to specifically improve proteins or RNA elements via deep learning approaches and thus raise design processes to a new level. The device can be applied to many biological problems. Specifically, it will be used to design and improve RNA-based switching elements (synthetic riboswitches) and specifically adapt them to their genetic context. Such RNA-based switches play an increasingly important role in synthetic biology in the design of synthetic genetic circuits for biosensing and biocomputing. Furthermore, the device will be used to develop allosterically switchable proteins. These are composed of, for example, an optogenetic receptor protein and an effector. Current work focuses on anti-CRISPR proteins for indirect control of CRISPR-Cas effectors with the goal of enabling spatially and/or temporally precise alterations of the cellular genome, epigenome or transcriptome. Furthermore, work on the stability of synthetic replicons, synthetic proteases as well as synthetic ion channels is planned.