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建立一个简单的I-SceI介导的重组单质粒系统,以有效编辑P. putida KT244040的基因组

Hao Meng¹, Sebastian Köbbing¹, Lars M Blank¹

¹iAMB-Institute of Applied Microbiology, ABBt-Aachen Biology and Biotechnology, RWTH Aachen University, Aachen, Germany.

Microbial biotechnology

|July 20, 2024

概括

一个新的单等离子体系统,pEMG-RIS,可以在Pseudomonas putida中快速有效地编辑基因组. 这种工具加速了这种重要的生物制品底盘的工程,在短短三天内完成了循环.

科学领域:

合成生物学合成生物学
微生物工程微生物工程
分子生物学分子生物学

背景情况:

Pseudomonas putida 是生物制品制造的关键微生物底盘.
基因工具的进步,包括基因组编辑,对它的实用性至关重要.
现有的工具需要优化速度和效率.

研究的目的:

开发一个精简的,单质粒系统,用于Pseudomonas putida的快速基因组工程.
提高已建立的pEMG/pSW基因组编辑系统的效率和速度.
为了促进更快的生物产品开发,使用工程P. putida.

主要方法:

构建一种新型单体质粒,pEMG-RIS,集成I-SceI和sacB对选择.
使用pEMG-RIS系统在P. putida KT2440中进行针对性基因组修改.
展示表达卡塞特 (msfGFP) 和一个大DNA片段 (12.1 kb) 的快速集成.

主要成果:

通过pEMG-RIS系统,基因组编辑周期可以在3天内完成.
快速实现了 msfGFP 表达盒的成功集成.
大量的DNA片段 (高达12.1kb) 被有效地集成到P. putida基因组中.
新系统通过 sacB 反选择标记加速了等离子体固化.

结论:

由pEMG-RIS体现的更新的pEMG/pSW系统显著提高了P. putida.的基因组编辑速度和效率.
这种优化的工具加速了P. putida用于生物生产应用的工程.
开发的等离子体将通过Addgene提供,用于更广泛的研究.

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