ZeBRα a universal, multi-fragment DNA-assembly-system with minimal hands-on time requirement
| Autor: | David Richter, Katharina Bayer, Thomas Toesko, Stefan Schuster |
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| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: | |
| Zdroj: | Scientific Reports Scientific Reports, Vol 9, Iss 1, Pp 1-16 (2019) |
| DOI: | 10.15495/epub_ubt_00004650 |
| Popis: | The recently evolved field of synthetic biology has revolutionized the way we think of biology as an “engineerable” discipline. The newly sprouted branch is constantly in need of simple, cost-effective and automatable DNA-assembly methods. We have developed a reliable DNA-assembly system, ZeBRα (Zero-Background Redα), for cloning multiple DNA-fragments seamlessly with very high efficiency. The hallmarks of ZeBRα are the greatly reduced hands-on time and costs and yet excellent efficiency and flexibility. ZeBRα combines a “zero-background vector” with a highly efficient in vitro recombination method. The suicide-gene in the vector acts as placeholder, and is replaced by the fragments-of-interest, ensuring the exclusive survival of the successful recombinants. Thereby the background from uncut or re-ligated vector is absent and screening for recombinant colonies is unnecessary. Multiple fragments-of-interest can be assembled into the empty vector by a recombinogenic E. coli-lysate (SLiCE) with a total time requirement of less than 48 h. We have significantly simplified the preparation of the high recombination-competent E. coli-lysate compared to the original protocol. ZeBRα is the least labor intensive among comparable state-of-the-art assembly/cloning methods without a trade-off in efficiency. |
| Databáze: | OpenAIRE |
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