CIFR (克隆-整合-翻转-重复):一种用于代基因组和Gram-负细菌路径工程的工具集
Filippo Federici1, Francesco Luppino1, Clara Aguilar-Vilar1
1The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby, Denmark.
Metabolic engineering
|January 8, 2025
概括
我们开发了CIFR,这是一种用于代细菌基因组工程的小Tn5集成系统. 该系统使可重复使用的选择标记成为可能,促进了各种细菌宿主中复杂的遗传修饰.
科学领域:
- 合成生物学 合成生物学
- 细菌遗传学 细菌遗传学
- 分子生物学分子生物学
背景情况:
- 先进的基因组工程需要便携式工具集来进行精确的细菌修改.
- Tn5转子子载体对于随机DNA整合是有用的,但受选择标记物的重用性限制.
- 代基因组工程需要有效的方法来清除标记物和重新使用宿主菌株.
研究的目的:
- 为代细菌基因组工程开发一个迷你Tn5集成系统.
- 为了实现可重复使用的选择标记,以简化基因操纵.
- 为各种细菌宿主创建一个便携和多功能工具.
主要方法:
- 开发了CIFR (可定制的基因组代功能) 系统,使用具有attP/attB位点的pCIFR向量.
- 包含用于初始选择的抗生素耐药性标记物和用于标记物去除的反选择标记物.
- 利用Bxb1整合酶有效去除抗生素耐药性决定因素.
- 在Pseudomonas putida,大肠杆菌和Cupriavidus necator中验证了该系统.
主要成果:
- 具有稳定的DNA插入和没有抗生素耐药性磁带的工程菌株,使工具可重复使用.
- 在P. putida中证明了染色蛋白基因的成功组合集成,创建了色调板.
- 展示了P. putida中一种胡卜素生物合成途径的两步工程,用于途径平衡.
结论:
- 该CIFR系统为代细菌基因组工程提供了一个便携式和可重复使用的解决方案.
- 这种工具箱扩大了代谢工程和复杂的表型构造的能力.
- CIFR为细菌遗传操纵和合成生物学应用开辟了新的途径.
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