2-Laser Cell Sorter

我们特此申请一个有2个激光器的细胞分选机。这种装置能够通过大小、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，选择通常通过荧光报告蛋白的表达来完成。分选后的细胞群随后被高通量测序表征。这里应用的设备每秒可以分选超过1万个细胞。这允许构建“Flow-Seq”管道，可用于以高通量格式描述广泛的突变库。它允许生成突变适应度景观来推断重要的结构-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，从而将设计过程提升到一个新的水平。这种装置可用于解决许多生物学问题。具体来说，它将用于设计和改进基于rna的开关元件（合成核糖开关），并专门使它们适应其遗传环境。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，用于生物传感和生物计算的合成遗传电路的设计。此外，该装置将用于开发变构可切换的蛋白质。例如，它们由光遗传受体蛋白和效应器组成。目前的工作重点是用于间接控制CRISPR-Cas效应子的抗crispr蛋白，目的是实现细胞基因组、表观基因组或转录组在空间和/或时间上的精确改变。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.