A high-throughput, restriction-free cloning and screening strategy based on ccd B-gene replacement

Microbial Cell Factories

Technical Notes

Hanna-Kirsti Schrøder Leiros¹  Bjarte Aarmo Lund¹  GroElin Kjæreng Bjerga² 

[1] NorStruct, Department of Chemistry, Faculty of Science and Technology, UiT - The Arctic University of Norway, N-9037, Tromsø, Norway;NorStruct, Department of Chemistry, Faculty of Science and Technology, UiT - The Arctic University of Norway, N-9037, Tromsø, Norway;Uni Research AS, Centre for Applied Biotechnology, High Technology Centre, Thormøhlensgt. 55, N-5008, Bergen, Norway;

关键词: Restriction-free cloning;    Exponential megapriming PCR;    High-throughput;    Counterselection;    Recombinant DNA technology;    Parallel cloning;   

DOI  :  10.1186/1475-2859-13-38

 received in 2014-02-11, accepted in 2014-03-05,  发布年份 2014

来源: Springer

【 摘 要 】

BackgroundIn high-throughput demanding fields, such as biotechnology and structural biology, molecular cloning is an essential tool in obtaining high yields of recombinant protein. Here, we address recently developed restriction-free methods in cloning, and present a more cost-efficient protocol that has been optimized to improve both cloning and clone screening.ResultsIn our case study, three homologous β-lactamase genes were successfully cloned using these restriction-free protocols. To clone the genes, we chose a gene replacement strategy, where the recombinant genes contained overhangs that targeted a region of the expression vector including a cytotoxin-encoding ccd B-gene.ConclusionWe provide further evidence that gene replacement can be applied with high-throughput cloning protocols. Targeting a replacement of the ccd B- gene was found to be very successful for counterselection using these protocols. This eliminated the need for treatment with the restriction enzyme Dpn I that has so far been the preferred clone selection approach. We thus present an optimized cloning protocol using a restriction-free ccd B-gene replacement strategy, which allows for parallel cloning at a high-throughput level.

【 授权许可】

Unknown   
© Lund et al.; licensee BioMed Central Ltd. 2014. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.