相关实验视频

Updated: Jul 6, 2025

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

Published on: March 25, 2020

基于蛋白质的热调节巨核酶用于制遗传物质

Gary W Foo¹, Christopher D Leichthammer¹, Ibrahim M Saita¹

¹Department of Biochemistry, Schulich School of Medicine and Dentistry, London, Ontario N6A 5C1, Canada.

Nucleic acids research

|January 5, 2024

概括

一个新的生物控制系统使用温度敏感的大核酶 (TSM) 在18°C下降解工程DNA,防止其从肠道传播. 这种温度调节的方法有效地消除了哺乳动物肠道环境中的大肠杆菌等离子体.

科学领域:

合成生物学合成生物学
分子生物学分子生物学
微生物遗传学微生物遗传学

背景情况:

工程遗传材料的生物控制,特别是塑和益生菌,对于防止意外传播和生态破坏至关重要.
工程微生物及其遗传有效载荷可以逃离哺乳动物的胃肠道,对自然微生物群体构成风险.
目前的生物控制策略需要改进,以便在肠道等复杂环境中得到强大的应用.

研究的目的:

为工程DNA开发一种简单,轻量级和翻译后激活的生物控制系统.
创建温度敏感巨核酶 (TSM),在允许的温度下专门降解DNA,在更高的温度下不活跃.
在哺乳动物肠道模型中评估TSM在消除大肠杆菌等离子体方面的有效性.

主要方法:

通过将一个温度敏感的整蛋白集成到LAGLIDADG归宿性内核酶基因中,构建直角温度敏感大核酶 (TSM).
证明TSM在翻译后会激活取决于整蛋白拼接和内核酶活性的特定位点DNA内核酶.
在通过小鼠肠道模型后,测试了TSM在从实验室大肠杆菌菌株和益生菌大肠杆菌Nissle 1917中消除等离子体的疗效.

主要成果:

在18°C的温度下,TSM成功地消除了含有大肠杆菌相关点的等离子体,但在更高的温度 (37°C) 时没有.
已证实,等离子体清除依赖于TSM内核酶活性和整蛋白拼接机制.
在允许的18°C化时,TSM在小鼠肠道样本中有效消除了来自大肠杆菌Nissle 1917的等离子体,但不是在37°C时.

结论:

热调节的大核酶为工程塑体和益生菌提供了一个有前途的生物控制策略.
这个系统提供了对遗传物质的有条件控制,将其持久性限制在哺乳动物肠道内的特定温度条件下.
开发的TSM系统显示了合成生物学在与肠道相关的治疗和研究中安全和可控应用的巨大潜力.

更多相关视频

Homemade Site Directed Mutagenesis of Whole Plasmids

Homemade Site Directed Mutagenesis of Whole Plasmids

Published on: May 11, 2009

Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution

Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution

Published on: February 26, 2019

相关概念视频

Conservative Site-specific Recombination and Phase Variation

Conservative Site-specific Recombination and Phase Variation

Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...

Restriction Enzymes

Restriction Enzymes

Restriction enzymes are bacterial enzymes used to cut DNA in a sequence-specific manner. To cleave DNA, they bind to specific palindromic sequences called restriction sites. Such palindromic DNA sequences or inverted repeats are commonly found in regions of functional significance, such as the origin of replication, gene operator sites, and regions containing transcription termination signals.
The host bacteria protect their own genomic DNA from these enzymes by methylating these sites. Some...

In-vitro Mutagenesis

In-vitro Mutagenesis

To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.