大肠杆菌的轨道:千基基尺度的寡核酸在高吞吐量和高效率下进行重组
Scott H Saunders1, Ayesha M Ahmed1
1Green Center for Systems Biology - Lyda Hill Department of Bioinformatics, University of Texas Southwestern Medical Center, Dallas, TX 75320, USA.
Nucleic acids research
|April 8, 2024
概括
我们开发了Oligonucleotide重组,然后是Bxb-1整合酶向 (ORBIT) 以实现高效的细菌基因组工程. 这种灵活的系统能够在大肠杆菌中高通量创建复杂的突变库.
科学领域:
- 微生物学 微生物学
- 合成生物学 合成生物学
- 细菌遗传学 细菌遗传学
背景情况:
- 反向遗传学对于微生物学和合成生物学中的细菌基因组操纵至关重要.
- 现有的基因组操纵方法往往是低效的,低吞吐量,需要广泛的分子生物学技术.
研究的目的:
- 开发一种新,高效,可扩展的系统,用于大规模的Escherichia coli基因组修饰.
- 适应和优化Oligonucleotide重组,然后对大肠杆菌进行Bxb-1整合酶向 (ORBIT) 系统.
主要方法:
- 开发了一种重新设计的等离子体工具包,用于在大肠杆菌中进行寡核酸重组.
- 利用DNA寡核酸来指导非复制性等离子体的整合以进行基因组修改.
- 用于构建单个转换中的多个突变的直角连接点.
- 使用针对性寡核酸和基因组条形码的池,用于高通量,全基因组突变库的构建.
主要成果:
- 与传统的 λ 红色重组相比,实现了显著更高的效率.
- 启用了精确和稳定的淘汰 (高达134kb) 和集成 (高达11kb).
- 在单个转换中成功构建了多个突变.
- 通过针对几乎所有转录因子和小RNA基因,生成精确的全基因组单个突变库.
- 使用基因组条形码向数千个独特成员 (>30k) 证明了可扩展性.
结论:
- 轨道是一个灵活和高效的逆转基因系统为大肠杆菌.
- 促进复杂细菌菌株的快速构建.
- 易于扩展,用于创建复杂的,高通量突变库,用于各种研究应用.
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