Related Experiment Video
Updated: Dec 26, 2025
08:31
Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning
Published on: February 5, 2021
14.7K
[A high efficiency cloning approach of multi-points combinational mutagenesis]
Jie Wang1,2, Xiaoyun Lu2, Aipo Diao1
1College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China.
Summary
A new Multi-points Combinational Mutagenesis (MCM) method enhances enzyme directed evolution. This technique efficiently creates diverse mutant libraries, leading to improved enzyme function, such as a 10-fold increase in benzoylformate decarboxylase activity.
Area of Science:
- Biotechnology
- Enzyme Engineering
- Molecular Biology
Background:
- Enzyme directed evolution requires efficient mutagenesis and screening.
- High-throughput methods are crucial for generating diverse mutant libraries.
Purpose of the Study:
- To develop a high-efficiency cloning construction method for Multi-points Combinational Mutagenesis (MCM).
- To evaluate the efficiency of MCM in creating mutant libraries for enzyme directed evolution.
Main Methods:
- Developed MCM using DNA assembly, fusion PCR, and hybridization techniques.
- Optimized electroporation for high Colony Forming Units (CFUs) in E. coli Trelief™ 5α.
- Applied MCM to directed evolution of benzoylformate decarboxylase.
Main Results:
- Achieved >10⁶ CFUs/μg DNA with optimized electroporation.
- Demonstrated 90% precise assembly of targeted clones.
- Reached 88% efficiency for simultaneous mutations at 5 specific sites.
- Obtained a benzoylformate decarboxylase mutant with a 10-fold increase in kcat/Km.
Conclusions:
- The developed MCM method significantly enhances the efficiency of creating diverse mutant libraries.
- MCM promotes rapid advancements in enzyme directed evolution.
- This method is effective for engineering enzymes with improved catalytic properties.
Keywords:
benzoylformate decarboxylasedirected evolutionhigh-throughput screeningmulti-points combinatorial mutagenesisMore Related Videos
Related Concept Videos
02:53Conservative Site-specific Recombination and Phase Variation
6.5K
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...
The recognition sites for Cre recombinase called LoxP...
6.5K
01:16In-vitro Mutagenesis
15.9K
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.
15.9K