Manchester Institute of Biotechnology (MIB), The University of Manchester, Manchester, UK.
Department of Chemistry, The University of Manchester, Manchester, UK.
Methods Mol Biol. 2022;2461:175-193. doi: 10.1007/978-1-0716-2152-3_12.
Transcription factor-based biosensors are important tools in Synthetic Biology for the sensing of industrially valuable molecules and clinically important metabolites, therefore presenting applications in the bioremediation, industrial biotechnology, and biomedical fields. The directed evolution of allosteric transcription factors (aTFs) with the aim of altering effector specificity has the potential for the development of new biosensors to detect natural and nonnatural molecules, expanding the scope of available aTF-based biosensors. In this chapter, we delineate a general method for the directed evolution of aTFs. The theory of library design is discussed, along with the detailed methodology for an improved transformation of combined libraries, and the experimental search space by counterselection using fluorescence-activated cell sorting (FACS) is presented.
基于转录因子的生物传感器是合成生物学中用于检测工业有价值分子和临床重要代谢物的重要工具，因此在生物修复、工业生物技术和生物医学领域具有应用前景。具有改变效应物特异性的变构转录因子(aTFs)的定向进化有可能开发新的生物传感器来检测天然和非天然分子，从而扩展可用的基于 aTF 的生物传感器的范围。在本章中，我们描述了一种用于定向进化 aTFs 的通用方法。讨论了文库设计的理论，以及改进的组合文库转化的详细方法，以及通过荧光激活细胞分选 (FACS) 反选择来呈现实验搜索空间。
