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Author:
Date:
2021-02-20
[Abstract] Precise genome engineering has become a commonplace technique for metabolic engineering. Also, insertion, deletion and alteration of genes and other functional DNA sequences are essential for understanding and engineering cells. Several techniques have been developed to this end (e.g., CRISPR/Cas-assisted methods, homologous recombination, or λ Red recombineering), yet most of them rely on the use of auxiliary plasmids, which have to be cured after the editing procedure. Temperature-sensitive replicons, counter-selectable markers or repeated passaging of plasmid-bearing cells have been traditionally employed to circumvent this hurdle. While these protocols work reasonably well in some bacteria, they are not applicable for other species or are time consuming and laborious. Here, we present ...
[摘要] [摘要]精确的基因组工程已成为代谢工程的一种普遍技术。同样，基因和其他功能性DNA序列的插入，缺失和改变对于理解和改造细胞也是必不可少的。几种技术已经发展到该端部（例如，CRISPR / CAS-辅助方法，同源重组，或 λ 红色重组），但其中大多数依赖于辅助质粒的使用，必须在编辑程序后将其固化。传统上已采用对温度敏感的复制子，反向选择标记或带有质粒的细胞的重复传代来规避这一障碍。尽管这些协议在某些细菌中可以很好地发挥作用，但它们不适用于其他物种，或者既费时又费力。在这里，我们提出了快速和通用的荧光假单胞菌荧光标记辅助基因组编辑协议，然后通过用户控制的质粒复制干净固化辅助质粒。一种荧光标记有助于鉴定基因组编辑的菌落，而第二种报道分子能够检测无质粒的细菌克隆。该协议不仅是用于假单胞菌物种的最快方法，而且可以轻松地适应任何类型的基因组修饰，包括序列删除，插入和替换。
图形概要：
带有可治愈质粒的假单胞菌的快速基因组工程
[背景]靶向，精确的基因组操纵技术已经大大推进了微生物工程领域。这样的方法不仅允许评估基因型与表型的关系，而且使微生物细胞工厂的复杂工程化成为可能。近年来，CRISPR / Cas9方法为真核生物的精确基因组工程铺平了道路。在细菌中，CRISPR / ...
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Author:
Date:
2020-07-05
[Abstract] This protocol provides a step-by-step method to create recombinant fluorescent fusion proteins that can be secreted from mammalian cell lines. This builds on many other recombinant protein and fluorescent protein techniques, but is among the first to harness fluorescent fusion proteins secreted directly into cell culture supernatant. This opens new possibilities that are not achievable with proteins produced in bacteria or yeast, such as direct use of the fluorescent protein-secreting cells in live co-culture assays. The Fluorescent Adaptable Simple Theranostic (FAST) protein system includes a histidine purification tag and a tobacco etch virus (TEV) cleavage site, allowing the purification tag and fluorescent protein to be removed for therapeutic use. This protocol is split into five ...
[摘要] [摘要] 该方案提供了一种逐步建立重组荧光融合蛋白的方法，可从哺乳动物细胞系分泌。这项技术建立在许多其他重组蛋白和荧光蛋白技术的基础上，但它是第一个利用荧光融合蛋白直接分泌到细胞培养上清液中的技术之一。这为细菌或酵母产生的蛋白质开辟了新的可能性，例如直接使用荧光蛋白分泌细胞进行活体共培养实验。该荧光适应性简单热（FAST）蛋白系统包括组氨酸纯化标签和烟草蚀刻病毒（TEV）裂解位点，允许去除纯化标签和荧光蛋白用于治疗用途。该方案分为五个部分：（A）感兴趣基因（GOI）和感兴趣蛋白（POI）的电子表征；（B）表达载体的设计；（C）表达载体的组装；（D）用表达载体转染真核细胞系；（E）检测重组蛋白。这种广泛的方案只需聚合酶链反应（PCR）和细胞培养训练即可完成。另外，协议的每个部分都可以独立使用。
[背景] ...
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