ZeBR? a universal, multi-fragment DNA-assembly-system with minimal hands-on time requirement.

ABSTRACT: 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, ZeBR? (Zero-Background Red?), for cloning multiple DNA-fragments seamlessly with very high efficiency. The hallmarks of ZeBR? are the greatly reduced hands-on time and costs and yet excellent efficiency and flexibility. ZeBR? 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. ZeBR? is the least labor intensive among comparable state-of-the-art assembly/cloning methods without a trade-off in efficiency.

SUBMITTER: Richter D 

PROVIDER: S-EPMC6393441 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature