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Related Experiment Video

Updated: Jan 24, 2026

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

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Published on: July 11, 2016

High-Throughput Allelic Replacement Screening in Bacillus subtilis.

Marie-Laure Diebold-Durand¹, Frank Bürmann², Stephan Gruber³

¹Department of Fundamental Microbiology, University of Lausanne, Lausanne, Switzerland.

Methods in Molecular Biology (Clifton, N.J.)

|June 1, 2019

Summary

This summary is machine-generated.

We developed a high-throughput gene targeting method for Bacillus subtilis using Golden Gate cloning. This efficient system allows rapid introduction of mutations for functional screening and genetic analysis.

Keywords:

Bacillus subtilis Cysteine scanning Gene targeting Golden Gate assembly High-throughput screening

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Area of Science:

Molecular Biology
Microbiology
Genetics

Background:

Site-directed mutagenesis is crucial for understanding biological processes.
Existing methods for gene modification can be inefficient and time-consuming.

Purpose of the Study:

To establish an efficient and systematic gene targeting strategy for Bacillus subtilis.
To enable high-throughput introduction of genetic modifications.

Main Methods:

Utilized Golden Gate cloning methodology for gene targeting.
Developed a 96-well microtiter plate format for a streamlined workflow.
Applied the system for introducing single/multiple point mutations and engineered alleles.

Main Results:

Successfully introduced genetic modifications into the endogenous gene locus in a single step.
Applied the system for high-throughput functional screening of Structural Maintenance of Chromosome (Smc) protein variants.
Demonstrated feasibility for exhaustive cysteine cross-linking mutagenesis.

Conclusions:

The developed Golden Gate-based system provides an efficient and systematic approach for gene targeting in Bacillus subtilis.
The method facilitates high-throughput genetic analysis and protein engineering.
The strategy is adaptable for applications in other bacterial species and yeast.