2-Laser Cell Sorter

我们在此申请一台带2个激光器的细胞分选机。这种设备能够通过大小、结构和荧光染色区分单个细胞，并根据预定义的参数对它们进行分类。该设备将用于分析和分类表达RNA和蛋白质突变的细菌和酵母(大肠杆菌、酿酒酵母)的大型文库。在这里，通常通过表达荧光报告蛋白来进行选择。随后，通过高通量测序来表征分类后的细胞群体。这里申请的设备能够每秒分选10000多个细胞。这允许构建一条“Flow-Seq”管道，该管道可用于表征高通量格式的大量突变文库。它允许产生突变适合度图景来推断重要的结构-功能关系(深度突变筛选)。此外，通过管道可以获得足够大的数据集，通过深度学习方法专门改进蛋白质或RNA元件，从而将设计过程提高到一个新的水平。该装置可应用于许多生物学问题。具体地说，它将被用来设计和改进基于RNA的开关元件(合成核糖开关)，并特别使它们适应其遗传背景。这种基于RNA的开关在合成生物学中发挥着越来越重要的作用，在生物传感和生物计算的合成遗传电路的设计中。此外，该设备将用于开发变构可切换蛋白质。例如，它们由光遗传受体蛋白和效应器组成。目前的工作主要集中在间接控制CRISPR-Cas效应子的抗CRISPR蛋白上，目标是能够在空间和/或时间上精确地改变细胞基因组、表观基因组或转录组。此外，还计划对合成复制子、合成蛋白酶以及合成离子通道的稳定性进行研究。

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.