Valenzuela-Ortega Marcos, French Christopher
Centre for Systems and Synthetic Biology, School of Biological Sciences, University of Edinburgh, Roger Land Building, Alexander Crum Brown Road, Edinburgh EH9 3FF, UK.
Zhejiang University-University of Edinburgh Joint Research Centre for Engineering Biology, International Campus, Zhejiang University, Haining, Zhejiang 314400, China.
Synth Biol (Oxf). 2021 Feb 2;6(1):ysab003. doi: 10.1093/synbio/ysab003. eCollection 2021.
Generation of new DNA constructs is an essential process in modern life science and biotechnology. Modular cloning systems based on Golden Gate cloning, using Type IIS restriction endonucleases, allow assembly of complex multipart constructs from reusable basic DNA parts in a rapid, reliable and automation-friendly way. Many such toolkits are available, with varying degrees of compatibility, most of which are aimed at specific host organisms. Here, we present a vector design which allows simple vector modification by using modular cloning to assemble and add new functions in secondary sites flanking the main insertion site (used for conventional modular cloning). Assembly in all sites is compatible with the PhytoBricks standard, and vectors are compatible with the Standard European Vector Architecture (SEVA) as well as BioBricks. We demonstrate that this facilitates the construction of vectors with tailored functions and simplifies the workflow for generating libraries of constructs with common elements. We have made available a collection of vectors with 10 different microbial replication origins, varying in copy number and host range, and allowing chromosomal integration, as well as a selection of commonly used basic parts. This design expands the range of hosts which can be easily modified by modular cloning and acts as a toolkit which can be used to facilitate the generation of new toolkits with specific functions required for targeting further hosts.
新DNA构建体的生成是现代生命科学和生物技术中的一个重要过程。基于金门克隆的模块化克隆系统,使用IIS型限制性内切酶,能够以快速、可靠且便于自动化的方式,从可重复使用的基本DNA部件组装复杂的多部分构建体。有许多这样的工具包可供使用,它们的兼容性程度各不相同,其中大多数针对特定的宿主生物体。在这里,我们展示了一种载体设计,该设计允许通过模块化克隆在主要插入位点(用于传统模块化克隆)两侧的二级位点进行简单的载体修饰,以组装并添加新功能。所有位点的组装都与植物砖标准兼容,并且载体与标准欧洲载体架构(SEVA)以及生物砖兼容。我们证明,这有助于构建具有定制功能的载体,并简化了生成具有共同元件的构建体文库的工作流程。我们提供了一组载体,它们具有10种不同的微生物复制起点,拷贝数和宿主范围各不相同,并且允许染色体整合,以及一些常用的基本部件。这种设计扩展了可以通过模块化克隆轻松修饰的宿主范围,并充当一个工具包,可用于促进生成具有针对进一步宿主所需特定功能的新工具包。