Exploring the Potential of Networked Directed Evolution Based on Novel LacI/effector Pairs

探索基于新型 LacI/效应器对的网络化定向进化的潜力

• 批准号: BB/J008214/1
• 负责人: Florian Hollfelder
• 金额: $ 41.32万
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2012
• 资助国家: 英国
• 起止时间: 2012 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FJ008214%2F1
• 关键词: Exploring; Potential; Networked; Directed; Evolution

Understanding of the evolution of functional proteins remains a daunting challenge, despite intense research efforts in basic and applied research. Nature's ability to create functional proteins is still unrivalled, raising the question in which respect we can can improve biomimetic efforts in directed evolution to make laboratory evolution more powerful. One key difference is the context in which evolution occurs: laboratory evolution usually deals with enzymes as single species, but natural enzymes are typically embedded in regulated networks and interdependent pathways. This project attempts to build simple networks in which expression of a coupled reporter marks E. coli cells that successfully express a new, functional protein and renders them selectable. This approach could expand the reaction classes that are accessible for directed evolutino, as current laboratory evolution is primarily targeted at functions that are assayed directly, e.g. by generating a fluorescent product. We hope to develop a method that will make directed evolution more versatile and efficient, but also provide insight into how network regulation might have contributed to natural evolution.

尽管在基础和应用研究方面进行了大量的研究工作，但了解功能蛋白的进化仍然是一项艰巨的挑战。大自然创造功能性蛋白质的能力仍然是无与伦比的，这提出了一个问题：我们可以在哪些方面改进定向进化的仿生努力，以使实验室进化变得更加强大。一个关键的区别是进化发生的背景：实验室进化通常将酶视为单一物种，但天然酶通常嵌入受调控的网络和相互依赖的途径中。该项目试图构建简单的网络，其中偶联报告基因的表达标记大肠杆菌细胞，成功表达新的功能性蛋白质并使它们具有选择性。这种方法可以扩展定向进化可访问的反应类别，因为当前的实验室进化主要针对直接测定的功能，例如通过产生荧光产物。我们希望开发一种方法，使定向进化更加通用和高效，同时也提供有关网络调控如何促进自然进化的见解。

项目成果

A single mutation in the core domain of the lac repressor reduces leakiness.

lac 阻遏物核心结构域的单个突变可减少泄漏。

• DOI: http://dx.10.1186/1475-2859-12-67
• 发表时间: 2013
• 期刊: Microbial cell factories
• 影响因子: 6.4
• 作者: Gatti