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Seamless insert-plasmid assembly at sub-terminal homologous sequences.
Plasmid ( IF 1.8 ) Pub Date : 2019-10-24 , DOI: 10.1016/j.plasmid.2019.102445
Anna-Sophia Krebs 1 , Tobias Bierig 2 , Gabriella Collu 2 , Roger M Benoit 1
Affiliation  

The engineering of fusion proteins for structural biology and protein nanotechnology often requires seamless DNA assembly with slight variations in the domain boundaries. To improve the molecular biology workflow for such projects, we evaluated the use of sub-terminal homologous sequences (HS) for co-transformation cloning and for T5 exonuclease / Phusion DNA polymerase mediated in vitro assembly. To quantify the effects of different HS-to-ends distances on cloning efficiency, we designed a blue-white-pink screening system that allowed us to easily identify positive clones (blue colonies), negative clones resulting from circular template plasmid (pink colonies) and negative colonies originating from linearized plasmids that have recircularized without an insert (white colonies). Our experiments show that both methods are feasible with HS-to-ends distances up to at least 10 base pairs. Using a combination of co-transformation cloning at sub-terminal HS and nucleotide insertions in non-annealing primer 5'-overhangs, we integrated a fusion protein into the third intracellular loop (ICL) of a G-protein-coupled receptor (GPCR) with nine different linker boundaries, using only a single plasmid linearization reaction. This molecular cloning approach is an invaluable tool for protein engineering, protein nanotechnology and synthetic biology that extends the range of applications of DNA assembly strategies.

中文翻译:

在亚末端同源序列上的无缝插入质粒组装。

用于结构生物学和蛋白质纳米技术的融合蛋白工程通常需要无缝的DNA组装,结构域边界略有变化。为了改善此类项目的分子生物学工作流程,我们评估了亚末端同源序列(HS)用于共转化克隆和T5核酸外切酶/ Phusion DNA聚合酶介导的体外组装的用途。为了量化不同的HS到末端距离对克隆效率的影响,我们设计了一个蓝白色粉红色的筛选系统,该系统使我们可以轻松地鉴定阳性克隆(蓝色菌落),环状模板质粒产生的阴性克隆(粉红色菌落)阴性菌落来自线性化质粒,没有插入而重新环化(白色菌落)。我们的实验表明,这两种方法对于至少10个碱基对的HS到末端距离都是可行的。使用在亚末端HS的共转化克隆和非退火引物5'-突出端中的核苷酸插入的组合,我们将融合蛋白整合到G蛋白偶联受体(GPCR)的第三个细胞内环(ICL)中仅使用单个质粒线性化反应,即可获得具有9个不同接头边界的序列。这种分子克隆方法是蛋白质工程,蛋白质纳米技术和合成生物学的宝贵工具,可扩展DNA组装策略的应用范围。我们仅使用一个质粒线性化反应,即可将融合蛋白整合到具有9个不同接头边界的G蛋白偶联受体(GPCR)的第三个细胞内环(ICL)中。这种分子克隆方法是蛋白质工程,蛋白质纳米技术和合成生物学的宝贵工具,可扩展DNA组装策略的应用范围。我们仅使用一个质粒线性化反应,即可将融合蛋白整合到具有9个不同接头边界的G蛋白偶联受体(GPCR)的第三个细胞内环(ICL)中。这种分子克隆方法是蛋白质工程,蛋白质纳米技术和合成生物学的宝贵工具,可扩展DNA组装策略的应用范围。
更新日期:2019-11-01
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