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

尽管在基础和应用研究方面进行了大量的研究，但对功能蛋白进化的理解仍然是一个艰巨的挑战。大自然创造功能性蛋白质的能力仍然是无与伦比的，这就提出了一个问题，即我们可以在哪些方面改进定向进化中的仿生努力，使实验室进化更加强大。一个关键的区别是进化发生的背景：实验室进化通常将酶作为单一物种处理，但天然酶通常嵌入在受调节的网络和相互依赖的途径中。这个项目试图建立一个简单的网络，在这个网络中，偶联报告基因的表达标记大肠杆菌细胞成功地表达了一种新的、功能性的蛋白质，并使它们具有选择性。这种方法可以扩展可用于定向进化的反应类别，因为目前的实验室进化主要针对直接分析的功能，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有效，同时也为网络调节如何促进自然进化提供见解。

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.