A genetic replacement system for selection-based engineering of essential proteins.
A genetic replacement system for selection-based engineering of essential proteins.
复制标题
DOI：
10.1186/1475-2859-11-110
复制
发表时间：
2012-08-16
影响因子：
6.4
通讯作者：
Panke S
中科院分区：
文献类型：
--
作者：
Billerbeck S;Panke S
必需基因代表生存所需的生物功能的核心。对必需性的分子理解以及合成细胞系统的设计包括必需蛋白质的工程。这项工作的一个障碍是缺乏适合定向进化方法的基于生长的选择系统。我们建立了一种简单的策略，用于在转化过程中用变体（文库）对必需基因进行遗传替换。该系统使用三种不同的必需基因和质粒组合进行了验证，并且可重复地显示每微克 DNA 107 个转化体的转化效率，没有任何可识别的假阳性。这使得能够从至少 105 倍过量的非功能变体中可靠地恢复功能变体。这种方法比传统漂白菌株的选择效果好至少两个数量级，其中功能性和非功能性变体之间的重组即使在recA阴性菌株中也会干扰可靠的恢复。我们认为，该选择系统非常适合评估工程必需蛋白的大型文库，从而在干净的菌株背景中可靠地分离功能变体，该变体可轻松用于体内应用以及用于体外研究的表达和纯化。
Essential genes represent the core of biological functions required for viability. Molecular understanding of essentiality as well as design of synthetic cellular systems includes the engineering of essential proteins. An impediment to this effort is the lack of growth-based selection systems suitable for directed evolution approaches. We established a simple strategy for genetic replacement of an essential gene by a (library of) variant(s) during a transformation. The system was validated using three different essential genes and plasmid combinations and it reproducibly shows transformation efficiencies on the order of 107 transformants per microgram of DNA without any identifiable false positives. This allowed for reliable recovery of functional variants out of at least a 105-fold excess of non-functional variants. This outperformed selection in conventional bleach-out strains by at least two orders of magnitude, where recombination between functional and non-functional variants interfered with reliable recovery even in recA negative strains. We propose that this selection system is extremely suitable for evaluating large libraries of engineered essential proteins resulting in the reliable isolation of functional variants in a clean strain background which can readily be used for in vivo applications as well as expression and purification for use in in vitro studies.
登录
查看更多内容
影响因子：
5.6
作者：
EGAN, SM;SCHLEIF, RF
通讯作者：
SCHLEIF, RF
影响因子：
6.4
作者：
Lee JH;Sung BH;Kim MS;Blattner FR;Yoon BH;Kim JH;Kim SC
通讯作者：
Kim SC
大肠杆菌K-12的构造框架，单基因敲除突变体：Keio Collection。
影响因子：
9.9
作者：
通讯作者：
--
影响因子：
9.9
作者：
通讯作者：
--
DOI：
10.1073/pnas.120163297
发表时间：
2000-06-06
影响因子：
11.1
作者：
Datsenko, KA;Wanner, BL
通讯作者：
Wanner, BL