One-step generation of error-prone PCR libraries using Gateway® technology

Microbial Cell Factories

Technical Notes

Sonia Longhi¹  Antoine Gruet¹  Christophe Bignon¹ 

[1] Architecture et Fonction des Macromolécules Biologiques (AFMB), UMR 7257 CNRS and Aix-Marseille University, 163, Avenue de Luminy, Case 932, Cedex 09, 13288, Marseille, France;

关键词: Cloning;    Sub-cloning;    Gateway;    Directed evolution;    GFP;    Error-prone PCR;    epPCR;    Library;    Screening;   

DOI  :  10.1186/1475-2859-11-14

 received in 2011-11-08, accepted in 2012-01-30,  发布年份 2012

来源: Springer

【 摘 要 】

BackgroundError-prone PCR (epPCR) libraries are one of the tools used in directed evolution. The Gateway® technology allows constructing epPCR libraries virtually devoid of any background (i.e., of insert-free plasmid), but requires two steps: the BP and the LR reactions and the associated E. coli cell transformations and plasmid purifications.ResultsWe describe a method for making epPCR libraries in Gateway® plasmids using an LR reaction without intermediate BP reaction. We also describe a BP-free and LR-free sub-cloning method for in-frame transferring the coding sequence of selected clones from the plasmid used to screen the library to another one devoid of tag used for screening (such as the green fluorescent protein). We report preliminary results of a directed evolution program using this method.ConclusionsThe one-step method enables producing epPCR libraries of as high complexity and quality as does the regular, two-step, protocol for half the amount of work. In addition, it contributes to preserve the original complexity of the epPCR product.

【 授权许可】

Unknown   
© Gruet et al; licensee BioMed Central Ltd. 2012. 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/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.