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

合成生物学领域的关键需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。目前缺乏这样的设计载体，但对于捕获由于其大小和/或 ** 重复性质而难以克隆的DNA序列来说，特别需要 **。为了优化、测试和改进载体开发，我们需要注释良好的大基因和/或具有重复性质的基因。我们来自西方的团队率先对叶螨进行了基因组测序，** 两种斑点叶螨（TSSM）-二斑叶螨和二斑叶螨具有小而紧凑的基因组（90 MBp），代表了最好的注释节肢动物基因组之一。这些螨纺出的丝是由高分子量蛋白质（丝心蛋白）组成的纤维，具有高度重复的序列。由于丝心蛋白基因的长度及其序列的极端重复性，即使应用目前最先进的测序技术 **（例如Oxford Nanopore），丝心蛋白基因的测序和克隆也极具挑战性。在本申请中，我们建议在我们的实验室和 ** 公司Designermicrobes（DESIGNER）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）我们将分离和注释蜘蛛螨基因组中的丝素基因，定义将被克隆 ** 的重复基因 **2）我们将优化和改进DESIGNER载体，并克隆和测序克隆在 ** DESIGNER载体中的蜘蛛螨丝素基因 ** 来自叶螨的丝素基因是用于改进DESIGNER载体的优秀DNA片段 ** 和优化DESIGNER技术的平台。捕获、克隆和测序长而重复的DNA片段的能力将是改善和扩大生物学能力的重要下一步，并允许合成生物学领域的产品扩展。

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.**