Development of novel vectors for synthetic biology industrial applications which require capture of large, difficult to clone repetitive DNA fragments

开发用于合成生物学工业应用的新型载体，这些应用需要捕获大的、难以克隆的重复 DNA 片段

• 批准号: 528291-2018
• 负责人: Grbic, Miodrag
• 金额: $ 1.82万
• 依托单位: University of Western Ontario
• 依托单位国家: 加拿大
• 项目类别: Engage Grants Program
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=652379
• 关键词: Development; novel; vectors; synthetic; biology

The key requirement in area of synthetic biology is development of vectors for cloning, sequencing and**expression of large DNA fragments for industrial applications. Such designer vectors are currently lacking, but**are particularly needed for the capturing of DNA sequences that are difficult to clone due to their size and/or**repetitive nature. To optimize, test and improve the vector development we need well annotated genes that are**large and/or have repetitive nature. Our team from Western pioneered genome sequencing of spider mites, the**Two Spotted Spider Mite (TSSM) - Tetranychus urticae and Tetranychus lintearius that have small and**compact genomes (90 MBp) and represent one of best annotated arthropod genomes. These mites spin silk that**is made of fibers consisting of high molecular weight proteins (fibroins) with highly repetitive sequences. Due**to their length and the extreme repetitive nature of their sequences, fibroin genes are extremely challenging to**sequence and clone even with the application of the most advanced sequencing technologies currently available**(e.g. Oxford Nanopore). In this application we propose to establish new collaboration between our lab and**company Designermicrobes (DMI) and to perform following experiments aimed at development of novel**vectors for synthetic biology:**1) We will isolate and annotate fibroin genes in spider mite genomes defining repetitive genes to be cloned**into DMI vectors**2) We will optimize and improve DMI vectors and clone and sequence spider mite fibroin genes cloned in**DMI vector**Fibroin genes from Tetranychid mites are excellent DNA fragments for the improvement of the DMI vectors**and platform for optimizing the DMI technology. The ability to capture, clone and sequence long and repetitive**DNA fragments will be an important next step in improving and expanding the DMI capabilities and allow the**expansion of products in the area of synthetic biology.**

合成生物学领域的关键要求是开发用于工业应用的大 DNA 片段的克隆、测序和**表达的载体。目前缺乏此类设计载体，但**特别需要用于捕获由于其大小和/或**重复性质而难以克隆的DNA序列。为了优化、测试和改进载体开发，我们需要注释良好的基因，这些基因**大和/或具有重复性。我们来自西方的团队率先对叶螨进行基因组测序，**二斑叶螨 (TSSM) - 二斑叶螨和林特叶螨具有小而**紧凑的基因组 (90 MBp)，代表了注释最好的节肢动物基因组之一。这些螨虫纺丝，丝由具有高度重复序列的高分子量蛋白质（丝心蛋白）组成的纤维制成。由于**其长度和序列的极端重复性，即使应用目前最先进的测序技术**（例如牛津纳米孔），丝心蛋白基因的测序和克隆也极具挑战性。在此应用中，我们建议在我们的实验室和**公司 Designermicrobes (DMI) 之间建立新的合作，并进行以下实验，旨在开发用于合成生物学的新型**载体：**1) 我们将分离和注释叶螨基因组中的丝心蛋白基因，定义要克隆到 DMI 载体中的重复基因**2) 我们将优化和改进 DMI 载体和 克隆和测序在**DMI载体中克隆的红蜘蛛丝心蛋白基因**来自叶螨的丝心蛋白基因是用于改进DMI载体**和优化DMI技术的平台的优秀DNA片段。捕获、克隆和测序长且重复的** DNA 片段的能力将是改进和扩展 DMI 功能的重要下一步，并允许**合成生物学领域的产品扩展。**