Rapid and Efficient One-Step Metabolic Pathway Integration in E. coli
| Autor: | Vivek K. Mutalik, Dylan W. Domaille, Andrea L. Halweg-Edwards, Marcelo C. Bassalo, Adam P. Arkin, William C Grau, Andrew D. Garst, Ryan T. Gill |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Butanols 030106 microbiology Green Fluorescent Proteins Biomedical Engineering Biology Biochemistry Genetics and Molecular Biology (miscellaneous) Genome Homology (biology) Metabolic engineering 03 medical and health sciences Synthetic biology Genome editing Bacterial Proteins CRISPR-Associated Protein 9 Escherichia coli CRISPR Clustered Regularly Interspaced Short Palindromic Repeats Gene Genetics Escherichia coli Proteins Reproducibility of Results General Medicine Endonucleases MutS DNA Mismatch-Binding Protein Metabolic pathway Metabolic Engineering Mutation Genetic Engineering Genome Bacterial Metabolic Networks and Pathways RNA Guide Kinetoplastida |
| Zdroj: | ACS synthetic biology. 5(7) |
| ISSN: | 2161-5063 |
| Popis: | Methods for importing heterologous genes into genetically tractable hosts are among the most desired tools of synthetic biology. Easy plug-and-play construction methods to rapidly test genes and pathways stably in the host genome would expedite synthetic biology and metabolic engineering applications. Here, we describe a CRISPR-based strategy that allows highly efficient, single step integration of large pathways in Escherichia coli. This strategy allows high efficiency integration in a broad range of homology arm sizes and genomic positions, with efficiencies ranging from 70 to 100% in 7 distinct loci. To demonstrate the large size capability, we integrated a 10 kb construct to implement isobutanol production in a single day. The ability to efficiently integrate entire metabolic pathways in a rapid and markerless manner will facilitate testing and engineering of novel pathways using the E. coli genome as a stable testing platform. |
| Databáze: | OpenAIRE |
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