Oligo- and dsDNA-mediated genome editing using a tetA dual selection system in Escherichia coli
| Autor: | Kyungchul Kim, Young Shin Ryu, Sung Kuk Lee, Sathesh-Prabu Chandran |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
lcsh:Medicine medicine.disease_cause Genome Biochemistry Genome Engineering Antiporters Synthetic biology Negative selection Plasmid Genome editing Nickel Antibiotics Medicine and Health Sciences lcsh:Science Homologous Recombination Genetics Gene Editing Multidisciplinary Mammalian Genomics Bacterial Genomics Antimicrobials Microbial Genetics Drugs Genomics Chromosomes Bacterial Nucleic acids Chemistry Metabolic Engineering Tetracyclines Physical Sciences Engineering and Technology Synthetic Biology Transformation Associated Recombination Research Article Chemical Elements Biotechnology DNA recombination 030106 microbiology DNA Single-Stranded Bioengineering Microbial Genomics Biology Microbiology Genome engineering 03 medical and health sciences Bacterial Proteins Microbial Control medicine Escherichia coli Bacterial Genetics Gene Pharmacology Pentosephosphates Biology and life sciences Base Sequence lcsh:R Bacteriology DNA 030104 developmental biology Animal Genomics Synthetic Bioengineering lcsh:Q |
| Zdroj: | PLoS ONE PLoS ONE, Vol 12, Iss 7, p e0181501 (2017) |
| ISSN: | 1932-6203 |
| Popis: | The ability to precisely and seamlessly modify a target genome is needed for metabolic engineering and synthetic biology techniques aimed at creating potent biosystems. Herein, we report on a promising method in Escherichia coli that relies on the insertion of an optimized tetA dual selection cassette followed by replacement of the same cassette with short, single-stranded DNA (oligos) or long, double-stranded DNA and the isolation of recombinant strains by negative selection using NiCl2. This method could be rapidly and successfully used for genome engineering, including deletions, insertions, replacements, and point mutations, without inactivation of the methyl-directed mismatch repair (MMR) system and plasmid cloning. The method we describe here facilitates positive genome-edited recombinants with selection efficiencies ranging from 57 to 92%. Using our method, we increased lycopene production (3.4-fold) by replacing the ribosome binding site (RBS) of the rate-limiting gene (dxs) in the 1-deoxy-D-xylulose-5-phosphate (DXP) biosynthesis pathway with a strong RBS. Thus, this method could be used to achieve scarless, proficient, and targeted genome editing for engineering E. coli strains. |
| Databáze: | OpenAIRE |
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