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首页> 外文期刊>Journal of insect biotechnology and sericology >Design and prototyping of a Bombyx mori nucleopolyhedrovirus-based genome assembly from PCR-amplified DNA fragments
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Design and prototyping of a Bombyx mori nucleopolyhedrovirus-based genome assembly from PCR-amplified DNA fragments

机译：PCR扩增DNA片段基于PCR扩增的DNA碎片基因组组装的设计与原型

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摘要

Recent technical advances on assembling DNA fragments has enabled generation of large, circular DNA of which length reached several hundred-kilobases. Adaptation of such technologies to baculoviral genomics would allow for flexible manipulation of baculoviral genomes and thus generation of designed viral genomes for both basic and applied biology. Here we report that a BmNPV genome-size DNA could be assembled from a set of PCR-amplified DNA fragments and was infectious to Bombyx mori ovary-derived BmN cell line. The DNA fragments were designed (1) to be excised mainly at intergenic regions and (2) with no homologous sequences on the terminals among the fragments for assembly purpose, and (3) with their lengths and Tm values of priming sequences adjusted for simultaneous amplification. Although these fragments were initially designed for assembling with Gibson assembly and in yeast in the first and subsequent steps, respectively, the bacmid propagated in yeast during a feasibility test showed a deletion during in-yeast assembly and/or propagation. Therefore, we assembled the fragments using only Gibson assembly throughout the assembling steps. An assembled clone was chosen and confirmed to carry the inverted vector sequence, a watermark to distinguish the assembled clone from the parental clone, and to be infectious to BmN upon transfection. The design and assembly strategy of DNA fragments and both amplified and assembled fragments become the foundation for further development of BmNPV-based, designed genomes.
机译：最近关于组装DNA片段的技术进步已经启用了大，圆形DNA的产生，长度达到几百千碱基。对杆状病毒基因组学的这种技术的适应将允许灵活地操纵杆状病毒基因组，从而为基本和应用生物学产生设计的病毒基因组。在这里，我们报告说，可以从一组PCR扩增的DNA片段组装BMNPV基因组大小DNA，并对Bombyx Mori卵巢衍生的BMN细胞系感染。设计了DNA片段（1），以主要在代亚基地区和（2）上切除，在组装目的的片段中没有在末端上的同源序列，并且（3）其长度和TM值调节的引发序列进行同时放大。尽管最初设计用于与吉布森组件和酵母组装的第一和随后的步骤中的组装，但是在可行性试验期间在酵母中传播的Bacmid在酵母组合物和/或繁殖期间缺失。因此，我们在整个组装步骤中仅使用Gibson组装组装片段。选择组装克隆并确认以携带倒载体序列，水印以将组装克隆与亲本克隆区分开，并在转染时感染到BMN。 DNA片段的设计和组装策略和扩增和组装片段的策略成为进一步发展BMNPV的设计基因组的基础。

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    Naoto Harada; Daiki Ishibashi; Shin-ichiro Asano; Masanao Sato; Hisanori Bando;

  • 作者单位

    Laboratory of Applied Molecular Entomology Division of Applied Bioscience Graduate School of Agriculture Hokkaido University Sapporo Japan;

    Laboratory of Applied Molecular Entomology Division of Applied Bioscience Graduate School of Agriculture Hokkaido University Sapporo Japan;

    Laboratory of Applied Molecular Entomology Division of Applied Bioscience Graduate School of Agriculture Hokkaido University Sapporo Japan;

    Laboratory of Applied Molecular Entomology Division of Applied Bioscience Graduate School of Agriculture Hokkaido University Sapporo Japan;

    Laboratory of Applied Molecular Entomology Division of Applied Bioscience Graduate School of Agriculture Hokkaido University Sapporo Japan;

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  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 蚕桑;
  • 关键词

    Bombyx mori nucleopolyhedrovirus; DNA assembly; Synthetic biology;

    机译：Bombyx mori核心核细胞核病毒;DNA组装;合成生物学;

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