SYTO^TM 9 Green Fluorescent Nucleic Acid Stain

Company: Thermo Fisher Scientific

Catalog#: S34854

Purification of Photorhabdus Virulence Cassette (PVC) Protein Complexes from Escherichia coli for Artificial Translocation of Heterologous Cargo Proteins

Yueying Wang, Xinting Zhang, Xiao Feng, Xia Wang, Qi Jin and Feng Jiang

Published online: 2024-04-05

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Bacterial Microcolonies in Gel Beads for High-throughput Screening

Author:

Yolanda Schaerli,

Date:

2018-07-05

[Abstract] High-throughput screening of a DNA library expressed in a bacterial population for identifying potentially rare members displaying a property of interest is a crucial step for success in many experiments such as directed evolution of proteins and synthetic circuits and deep mutational scanning to identify gain- or loss-of-function mutants.

Here, I describe a protocol for high-throughput screening of bacterial (E. coli) microcolonies in gel beads. Single cells are encapsulated into monodisperse water-in-oil emulsion droplets produced with a microfluidic device. The aqueous solution also contains agarose that gelates upon cooling on ice, so that solid gel beads form inside the droplets. During incubation of the emulsion, the cells grow into monoclonal microcolonies ... [摘要] 在细菌群体中表达的DNA文库的高通量筛选用于鉴定显示感兴趣性质的潜在稀有成员是在许多实验中成功的关键步骤，例如蛋白质和合成回路的定向进化以及用于鉴定增益的深度突变扫描 - 或功能丧失的突变体。

在这里，我描述了一种用于高通量筛选凝胶珠中细菌（大肠杆菌）微菌落的方案。将单细胞包封成用微流体装置产生的单分散油包水乳液液滴。水溶液还含有琼脂糖，其在冰上冷却时凝胶化，从而在液滴内部形成固体凝胶珠。在乳液温育期间，细胞在珠内生长成单克隆微菌落。在从乳液中分离凝胶珠并通过荧光激活细胞分选（FACS）分选后，从凝胶珠中回收细菌，然后准备进行进一步的分选，诱变或分析。为了通过FACS分类，该方案需要荧光读数，例如荧光报告蛋白的表达。测量微小菌落的平均荧光信号降低了高表型细胞间变异性的影响，并且与单细胞分选相比提高了灵敏度。我们应用这种方法在ON和OFF状态下对pBAD启动子文库进行分类（Duarte et al。，2017）。

【背景】荧光激活细胞分选（FACS）具有> 10 7 事件/ h的无与伦比的筛选通量（Davies，2012）。然而，通过FACS根据其荧光分选单个细胞以筛选合成回路的文库（Schaerli和Isalan，2013）经常受到高表型细胞间变异性的阻碍。或者，可以对水凝胶珠中所含的小细胞集落（微集落）进行分类（Weaver ...

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SYTOTM 9 Green Fluorescent Nucleic Acid Stain

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