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Using Coculture to Detect Chemically Mediated Interspecies Interactions

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Fast genome editing in Bacillus subtilis.

Guo Wu¹, Erin Drufva¹, Kang Wu¹

¹Department of Chemical Engineering University of New Hampshire Durham New Hampshire USA.

Engineering in Life Sciences

|July 7, 2020

Summary

Researchers developed a faster method for genome editing in Bacillus subtilis. This new approach uses linear DNA from Gibson assembly, eliminating the need for E. coli cloning and reducing the process time significantly.

Keywords:

DNA assembly gene knockout/knockin genome editing mutant libraries transformation efficiency

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CRISPR-Cas9-Mediated Genome Editing in the Filamentous Ascomycete Huntiella omanensis

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Area of Science:

Microbiology
Molecular Biology
Synthetic Biology

Background:

Bacillus subtilis is a crucial model organism for Gram-positive bacteria research.
It serves as a vital industrial host for producing proteins and chemicals.
Current genome editing methods for B. subtilis are time-consuming, often requiring cloning in Escherichia coli.

Purpose of the Study:

To investigate the direct transformation and integration of linear DNA into Bacillus subtilis.
To bypass the conventional cloning step in Escherichia coli for B. subtilis genome editing.
To establish a streamlined and efficient genome editing protocol for B. subtilis.

Main Methods:

Utilized Gibson assembly to generate linear deoxyribonucleic acid (DNA).
Directly transformed Bacillus subtilis with linear DNA fragments (8-10 kb).
Evaluated transformation efficiency compared to traditional methods.

Main Results:

Achieved high transformation efficiency using linear DNA from Gibson assembly.
Efficiency was comparable to using linearized plasmids constructed in E. coli.
Successfully integrated multiple genes in a single step.

Conclusions:

Direct transformation with Gibson assembly-generated linear DNA is feasible for B. subtilis genome editing.
This method significantly reduces the genome editing process time from one week to one day.
Offers a simplified, rapid, and efficient approach for genetic manipulation of B. subtilis.