Design and Implementation of Multi-protein Expression Constructs and Combinatorial Libraries using Start-Stop Assembly.
Design and Implementation of Multi-protein Expression Constructs and Combinatorial Libraries using Start-Stop Assembly.
复制标题
使用启停组装设计和实现多蛋白表达构建体和组合文库。
DOI：
10.1007/978-1-0716-0908-8_13
复制
发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Taylor GM
中科院分区：
文献类型：
--
作者：
Taylor GM
启停组装是一个多部分，模块化，基于金门的DNA组装系统，具有两个关键特征，使其区别于以前的DNA组装方法。首先，编码序列通过对应于起始密码子和终止密码子的突出端与上游和下游序列组装，避免在编码序列边界处组装的构建体中的不需要的“疤痕”。这些关键敏感部位的疤痕会影响mRNA结构、核糖体结合位点的活性，以及潜在的其他功能性RNA特征。因此，启停组装在功能上是无疤痕的（通常只有使用定制的、基于MEMS的组装方法才能实现的优势），并且适合于高效、无偏和组合组装（基于Golden Gate的方法的一般优势）。其次，Start-Stop Assembly具有新的流线型组装层次结构，这意味着通常只需要一个新的载体就可以为任何新的目的地背景（如新的生物体或基因组位置）组装构建体。这将有助于更快速，更方便地开发各种非模式生物的工程代谢途径，以利用其应用潜力。本章解释了设计考虑和实际程序，以实现多部分，多蛋白质表达构建体的分层组装，无论是单独或组合库，使用起止组装。
Start-Stop Assembly is a multi-part, modular, Golden Gate-based DNA assembly system with two key features which distinguish it from previous DNA assembly methods. Firstly, coding sequences are assembled with upstream and downstream sequences via overhangs corresponding to start and stop codons, avoiding unwanted ‘scars’ in assembled constructs at coding sequence boundaries. Scars at these crucial, sensitive locations can affect mRNA structure, activity of the ribosome binding site, and potentially other functional RNA features. Start-Stop Assembly is therefore both functionally scarless (an advantage usually only achieved using bespoke, overlap-based assembly methods) and suitable for efficient, unbiased and combinatorial assembly (a general advantage of Golden Gate-based methods). Secondly, Start-Stop Assembly has a new, streamlined assembly hierarchy, meaning that typically only one new vector is required in order to assemble constructs for any new destination context, such as a new organism or genomic location. This should facilitate more rapid and convenient development of engineered metabolic pathways for diverse nonmodel organisms in order to exploit their applied potential. This chapter explains both design considerations and practical procedures to implement multi-part, hierarchical assembly of multi-protein expression constructs, either individually or as combinatorial libraries, using Start-Stop Assembly.
影响因子：
14.9
作者：
Taylor, George M.;Mordaka, Pawel M.;Heap, John T.
通讯作者：
Heap, John T.