相关实验视频

Updated: Jul 16, 2025

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

Published on: January 26, 2017

针对RhtA转运体in vivo进化的目标C-to-T和A-to-G双变异发生系统

Zhandong Wei^1,2, Dongdong Zhao², Jie Wang^1,2

¹School of Biological Engineering, Dalian Polytechnic University, Dalian, China.

Applied and environmental microbiology

|September 20, 2023

概括

一个新的T7-DualMuta系统能够在体内同时进行C-to-T和A-to-GDNA突变. 这种双重功能系统加速了定向蛋白质进化,并扩展了合成生物学和酶学研究的基编辑工具箱.

科学领域:

合成生物学合成生物学
分子生物学分子生物学
酶学是一种酶学.

背景情况:

现有的T7RNA聚合酶 (T7RNAP) 融合使单基转化 (C-to-T或A-to-G) 成为有针对性的蛋白质进化.
当前系统的有限的基础转换类型限制了它们的应用范围.
需要能够同时进行多个基数转换的系统,以实现增强的定向进化.

研究的目的:

开发一种新的双功能系统,T7-DualMuta,用于同时进行C-to-T和A-to-G的体内突变.
通过T7-DualMuta系统评估突变发生的效率和分布.
为了证明T7-DualMuta在指导进化中改善生物功能的实用性.

主要方法:

结合T7RNAP与细胞氨酸脱氨酶 (PmCDA1) 和腺氨酸脱氨酶 (TadA-8e),形成T7-双Muta系统.
在24小时培养后,C-to-T和A-to-G突变发生频率的量化.
应用T7-DualMuta在体内对L-同类素载体RhtA.的定向进化.

主要成果:

T7-DualMuta实现了C-to-T和A-to-G突变发生频率分别为4.02 × 10−3和1.20 × 10−2.
突变在目标基因中均分布,显著增强了定向进化.
演化后的RhtA变异在高L-同素度 (8g/L) 中显著增加宿主生长率.

结论:

在体内,T7-DualMuta系统有效地实现了同时C/G-T/A和A/T-G/C的突变发生.
该系统代表了基础编辑工具箱的显著扩展,促进了酶学和合成生物学中的先进应用.
突变的高频率 (比自发突变高4.02×107倍) 证明了其强大的体内进化能力.

关键词:

在RhtA传送器上.在T7RNA聚合酶中,氨酸脱氨酸酶的使用.赛蒂丁脱胺酶的作用在生物体内进化的进化.

更多相关视频

Genetic Manipulation in Δku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Genetic Manipulation in Δku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Published on: July 12, 2013

Engineering Oncogenic Heterozygous Gain-of-Function Mutations in Human Hematopoietic Stem and Progenitor Cells

Engineering Oncogenic Heterozygous Gain-of-Function Mutations in Human Hematopoietic Stem and Progenitor Cells

Published on: March 10, 2023

相关概念视频

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.

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...