A Directed Evolution Protocol for Engineering Minimal Transcription Factors, Based on CIS Display.
| Autor: | Qi L; Department of Life Sciences, Imperial College London, London, UK., Bennett E; Department of Life Sciences, Imperial College London, London, UK., Isalan M; Department of Life Sciences, Imperial College London, London, UK. m.isalan@imperial.ac.uk. |
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| Jazyk: | angličtina |
| Zdroj: | Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2024; Vol. 2774, pp. 1-13. |
| DOI: | 10.1007/978-1-0716-3718-0_1 |
| Abstrakt: | 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 6 -10 7 variants. More recently, in vitro techniques have emerged that avoid cloning, allowing library sizes of >10 12 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 9 ). This protocol is also applicable to engineering other DNA-binding proteins or transcription factors from combinatorial libraries. (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.) |
| Databáze: | MEDLINE |
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