Falkenberg Kristoffer Bach, Mol Viviënne, de la Maza Larrea Arrate Sainz, Pogrebnyakov Ivan, Nørholm Morten H H, Nielsen Alex Toftgaard, Jensen Sheila Ingemann
The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet Building 220, Kongens Lyngby 2800, Denmark.
ACS Synth Biol. 2021 Dec 17;10(12):3278-3289. doi: 10.1021/acssynbio.1c00130. Epub 2021 Nov 18.
Versatile DNA assembly standards and compatible, well-characterized part libraries are essential tools for creating effective designs in synthetic biology. However, to date, vector standards for Gram-positive hosts have limited flexibility. As a result, users often revert to PCR-based methods for building the desired genetic constructs. These methods are inherently prone to introducing mutations, which is problematic considering vector backbone parts are often left unsequenced in cloning workflows. To circumvent this, we present the ProUSER2.0 toolbox: a standardized vector platform for building both integrative and replicative shuttle vectors for. The ProUSER2.0 vectors consist of a ProUSER cassette for easy and efficient insertion of cargo sequences and six exchangeable modules. Furthermore, the standard is semicompatible with several previously developed standards, allowing the user to utilize the parts developed for these. To provide parts for the toolbox, seven novel integration sites and six promoters were thoroughly characterized in . Finally, the capacity of the ProUSER2.0 system was demonstrated through the construction of signal peptide libraries for two industrially relevant proteins. Altogether, the ProUSER2.0 toolbox is a powerful and flexible framework for use in .
通用的DNA组装标准以及兼容且特征明确的元件库是在合成生物学中创建有效设计的重要工具。然而，迄今为止，革兰氏阳性宿主的载体标准灵活性有限。因此，用户常常转而采用基于PCR的方法来构建所需的基因构建体。这些方法天生容易引入突变，鉴于在克隆工作流程中载体骨架部分通常不进行测序，这就成了问题。为了规避这一问题，我们推出了ProUSER2.0工具箱：一个用于构建整合型和复制型穿梭载体的标准化载体平台。ProUSER2.0载体由一个用于轻松高效插入目标序列的ProUSER盒式结构和六个可交换模块组成。此外，该标准与之前开发的几个标准半兼容，允许用户使用为这些标准开发的元件。为了为该工具箱提供元件，在……中对七个新的整合位点和六个启动子进行了全面表征。最后，通过构建两种与工业相关蛋白质的信号肽库，证明了ProUSER2.0系统的能力。总之，ProUSER2.0工具箱是一个用于……的强大且灵活的框架。
