Robust counterselection and advanced λRed recombineering enable markerless chromosomal integration of large heterologous constructs
| Autor: | Dmitrii M Bubnov, Tigran V Yuzbashev, Andrey A Khozov, Olga E Melkina, Tatiana V Vybornaya, Guy-Bart Stan, Sergey P Sineoky |
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| Přispěvatelé: | Royal Academy Of Engineering |
| Rok vydání: | 2022 |
| Předmět: |
Biochemistry & Molecular Biology
Science & Technology EFFICIENCY 05 Environmental Sciences SALMONELLA-TYPHIMURIUM PROTEIN DNA 06 Biological Sciences DNA MIMICRY Bacteriophage lambda ESCHERICHIA-COLI GENOME BACTERIAL ARTIFICIAL CHROMOSOMES PLASMID R1 Operon Genetics Escherichia coli DELETION MUTATIONS GENE REPLACEMENT 08 Information and Computing Sciences Life Sciences & Biomedicine SYSTEM Genome Bacterial Developmental Biology |
| Zdroj: | Nucleic acids research. 50(15) |
| ISSN: | 1362-4962 |
| Popis: | Despite advances in bacterial genome engineering, delivery of large synthetic constructs remains challenging in practice. In this study, we propose a straightforward and robust approach for the markerless integration of DNA fragments encoding whole metabolic pathways into the genome. This approach relies on the replacement of a counterselection marker with cargo DNA cassettes via λRed recombineering. We employed a counterselection strategy involving a genetic circuit based on the CI repressor of λ phage. Our design ensures elimination of most spontaneous mutants, and thus provides a counterselection stringency close to the maximum possible. We improved the efficiency of integrating long PCR-generated cassettes by exploiting the Ocr antirestriction function of T7 phage, which completely prevents degradation of unmethylated DNA by restriction endonucleases in wild-type bacteria. The employment of highly restrictive counterselection and ocr-assisted λRed recombineering allowed markerless integration of operon-sized cassettes into arbitrary genomic loci of four enterobacterial species with an efficiency of 50–100%. In the case of Escherichia coli, our strategy ensures simple combination of markerless mutations in a single strain via P1 transduction. Overall, the proposed approach can serve as a general tool for synthetic biology and metabolic engineering in a range of bacterial hosts. |
| Databáze: | OpenAIRE |
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