利用GoldenBraid克隆策略设计和构建用于植物生物技术的多基因构建体。

Design and construction of multigenic constructs for plant biotechnology using the GoldenBraid cloning strategy.

作者信息

Sarrion-Perdigones Alejandro, Palaci Jorge, Granell Antonio, Orzaez Diego

机构信息

Instituto de Biología Molecular y Celular de Plantas, Consejo Superior de Investigaciones Científicas (CSIC), Universidad Politécnica de Valencia, Valencia, Spain.

出版信息

Methods Mol Biol. 2014;1116:133-51. doi: 10.1007/978-1-62703-764-8_10.

DOI:10.1007/978-1-62703-764-8_10

PMID:24395362

Abstract

GoldenBraid (GB) is an iterative and standardized DNA assembling system specially designed for Multigene Engineering in Plant Synthetic Biology. GB is based on restriction-ligation reactions using type IIS restriction enzymes. GB comprises a collection of standard DNA pieces named "GB parts" and a set of destination plasmids (pDGBs) that incorporate the multipartite assembly of standardized DNA parts. GB reactions are extremely efficient: two transcriptional units (TUs) can be assembled from several basic GBparts in one T-DNA less than 24 h. Moreover, larger assemblies comprising 4-5 TUs are routinely built in less than 2 working weeks. Here we provide a detailed view of the GB methodology. As a practical example, a Bimolecular Fluorescence Complementation construct comprising four TUs in a 12 kb DNA fragment is presented.

摘要

GoldenBraid（GB）是一种专门为植物合成生物学中的多基因工程设计的迭代式标准化DNA组装系统。GB基于使用IIS型限制性内切酶的限制性连接反应。GB包括一组名为“GB元件”的标准DNA片段和一组目的质粒（pDGB），这些质粒整合了标准化DNA元件的多片段组装。GB反应极其高效：两个转录单元（TU）可以在不到24小时的时间内从几个基本GB元件在一个T-DNA中组装而成。此外，包含4-5个TU的更大组装体通常在不到两个工作日内就能构建完成。在这里，我们详细介绍GB方法。作为一个实际例子，展示了一个在12 kb DNA片段中包含四个TU的双分子荧光互补构建体。