A Directed Evolution Protocol for Engineering Minimal Transcription Factors, Based on CIS Display

Overview

Journal Methods Mol Biol

Specialty Molecular Biology

Date 2024 Mar 5

PMID 38441754

Authors

Lin Qi

Emily Bennett

Mark Isalan

Affiliations

Soon will be listed here.

Abstract

Directed evolution is an efficient strategy for obtaining desired biomolecules. Since the 1990s, the emergence of display techniques has enabled high-throughput screening of functional proteins. However, classical methods require library construction by plasmid cloning and are limited by transformation efficiencies, typically limiting library sizes to ~10-10 variants. More recently, in vitro techniques have emerged that avoid cloning, allowing library sizes of >10 members. One of these, CIS display, is a DNA-based display technique which allows high-throughput selection of biomolecules in vitro. CIS display creates the genotype-phenotype link required for selection by a DNA replication initiator protein, RepA, that binds exclusively to the template from which it has been expressed. This method has been successfully used to evolve new protein-protein interactions but has not been used before to select DNA-binding proteins, which are major components in mammalian synthetic biology. In this chapter, we describe a directed evolution method using CIS display to efficiently select functional DNA-binding proteins from pools of nonbinding proteins. The method is illustrated by enriching the minimal transcription factor Cro from a low starting frequency (1 in 10). This protocol is also applicable to engineering other DNA-binding proteins or transcription factors from combinatorial libraries.

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