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, 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.
Highlights
Seamless multi-fragment DNA assemblies have recently been added to the toolkit of molecular biology
In search of a method that unites the advantages of previous methods for robust and high-throughput cloning, we have developed a novel strategy that combines a multi-fragment seamless assembly method with positive selection for the desired cloning products
Since different authors used commercially available highly competent cells (2 × 108 cfu/μg pUC19 DNA13), or electrocompetent DH10B5, we investigated if the recombinogenic PPY-extract would efficiently assemble three-fragments with “home-brewed” chemically competent New England Biolabs (NEB) 5-alpha prepared by the Inoue-method[21]
Summary
Seamless multi-fragment DNA assemblies have recently been added to the toolkit of molecular biology. Only a few non-commercial methods like LIC3, SLIC4, SLiCE5, Hot-Fusion[6], Golden-Gate[7,8] to name a few, reached wider acceptance outside synthetic-biology. This is probably attributable to the specific requirements of different cloning projects, the lack of universality of some of the aforementioned methods, and the effort needed to evaluate and establish novel methods. We exploit the recently developed, highly recombinogenic cell-extracts of E. coli (PPY-strain[5], NEB 5-alpha) to overcome the limitation of cloning a single DNA-fragment at a time (Fig. 1a). For a typical number of 10 to 100 cloning experiments the laborious final step needs to be carried out up to 2000 times, making it plain that time and resources can significantly be saved by any improvement in the verification step
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
