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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:
2021-01-05
[Abstract] Cell suspension cultures have been studied for decades to produce natural molecules. However, the difficulty in generating stably transformed cell lines has limited their use to produce high value chemicals reproducibly and in elevated quantities.
In this protocol, a method to stably transform and maintain Arabidopsis cell suspension cultures is devised and presented in detail. Arabidopsis cell cultures were directly transformed with A. tumefaciens for the overexpression of the CORONATINE INSENSITIVE 1 (COI1) jasmonate receptor. Cell cultures were established after transformation and continuously maintained and tested for the overexpression of COI1. The protocol was also previously used to silence Arabidopsis peroxidases and allows for long ...
[摘要] [摘要]细胞悬浮培养已经研究了数十年，以生产天然分子。然而，产生稳定转化的细胞系的困难限制了它们用于可再现地和高产量地生产高价值化学品的用途。
在该协议中，设计并详细介绍了稳定转化和维持拟南芥细胞悬浮培养物的方法。直接用根癌农杆菌转化拟南芥细胞培养物，以过量表达可乐宁不敏感1（COI1）茉莉酸酯受体。转化后建立细胞培养物，并连续保持并测试COI1的过表达。该协议以前也曾用于沉默拟南芥过氧化物酶，并允许长期维持转化细胞。提供了在液体和固体培养基中培养维持的详细信息，以及蛋白质表达的证据来证实转化。
所描述的系统为合成生物学研究独立于发育控制的信号传递并获得生物技术和医学领域感兴趣的代谢物提供了强大的工具。
[背景]植物细胞悬浮培养为植物次生代谢产物的生产和去分化组织的研究提供了可行的替代方法（Wu和Ge，2004； Lee等，2010）。数十年来，人们对它们的使用兴趣不断提高，因为它们可以为人类使用的高价值生物技术产品提供连续生产系统。有各种各样的细胞培养物可产生大量具有商业和工业意义的次级代谢产物，例如花青素，甜菜碱，调味剂，甜菊苷等（Rao和Ravishankar，2002）。
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