Targeted mutagenesis and high-throughput screening of diversified gene and promoter libraries for isolating gain-of-function mutations
Targeted mutagenesis of a promoter or gene is essential for attaining new functions in microbial and protein engineering efforts. In the burgeoning field of synthetic biology, heterologous genes are expressed in new host organisms. Similarly, natural or designed proteins are mutagenized at targeted...
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Published in | Frontiers in bioengineering and biotechnology Vol. 11; p. 1202388 |
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Main Authors | , , , , , , , |
Format | Journal Article |
Language | English |
Published |
Switzerland
Frontiers Media S.A
17.07.2023
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Subjects | |
Online Access | Get full text |
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Summary: | Targeted mutagenesis of a promoter or gene is essential for attaining new functions in microbial and protein engineering efforts. In the burgeoning field of synthetic biology, heterologous genes are expressed in new host organisms. Similarly, natural or designed proteins are mutagenized at targeted positions and screened for gain-of-function mutations. Here, we describe methods to attain complete randomization or controlled mutations in promoters or genes. Combinatorial libraries of one hundred thousands to tens of millions of variants can be created using commercially synthesized oligonucleotides, simply by performing two rounds of polymerase chain reactions. With a suitably engineered reporter in a whole cell, these libraries can be screened rapidly by performing fluorescence-activated cell sorting (FACS). Within a few rounds of positive and negative sorting based on the response from the reporter, the library can rapidly converge to a few optimal or extremely rare variants with desired phenotypes. Library construction, transformation and sequence verification takes 6-9 days and requires only basic molecular biology lab experience. Screening the library by FACS takes 3-5 days and requires training for the specific cytometer used. Further steps after sorting, including colony picking, sequencing, verification, and characterization of individual clones may take longer, depending on number of clones and required experiments. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 89233218CNA000001; NL0032182; NL0035994; NL0037843; FWP LANLF32A USDOE Office of Energy Efficiency and Renewable Energy (EERE), Office of Sustainable Transportation. Bioenergy Technologies Office (BETO) LA-UR-23-21261 USDOE Office of Science (SC), Biological and Environmental Research (BER) Edited by: Wei Luo, Jiangnan University, China These authors have contributed equally to this work Reviewed by: Quanfeng Liang, Shandong University, China Amir Pandi, Université Paris Cité, France |
ISSN: | 2296-4185 2296-4185 |
DOI: | 10.3389/fbioe.2023.1202388 |