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.
Keywords: Allosteric transcription factor; Directed evolution; FACS counterselection; Transcription factor-based biosensors.
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