Bacillus subtilis

Bacillus subtilis Gene Editing Services

1. Research Background

Bacillus subtilis is an extremely important industrial microorganism, classified as GRAS (Generally Recognized as Safe) by the U.S. FDA. With its strong protein secretion capacity, well-characterized genetic background and excellent growth properties, it occupies a core position in industrial enzymes, biomedicine and metabolic engineering. Precise and efficient gene editing technology is a key tool for optimizing its production performance and constructing high-efficiency cell factories.

Against this background, GeneRulor provides genome modification of Bacillus subtilis using the CRISPR/Cas9 system. Customized services including target gene knockout, large-fragment deletion, point mutation, and gene knock-in (overexpression) can be achieved in different strains, and positive mutant strains will be delivered. We are committed to providing professional and reliable customized Bacillus subtilis gene editing solutions for global research and industrial customers.

2. Strain Characteristics and Biological Background

(1) Gram-staining property: Bacillus subtilis is a Gram-positive bacterium.

(2) Physical characteristics: It has a thick peptidoglycan cell wall (about 20–80 nm) and lacks the outer membrane structure of Gram-negative bacteria. A prominent feature is that it can form highly stress-resistant endospores in the late growth stage.

(3) Industrial significance: As a model Gram-positive bacterium, it is an ideal model for studying protein secretion mechanisms, cell differentiation, and biofilm formation. It is widely used industrially for the secretory expression of various hydrolases, antibiotics, and vitamins.

(4) Genetic transformation: It possesses natural competence, but transformation efficiency still needs to be optimized in specific environments (such as industrial strains) to achieve efficient editing.

Figure 1. Growth of Bacillus subtilis on Culture Medium Plates

3. Gene-Editing Strategies Reported in the Literature

To overcome transformation challenges caused by the thick cell wall of Bacillus subtilis, mainstream editing strategies adopted in current literature and research practice include:

3.1 CRISPR/Cas9 System (Primary Solution)

(1) Principle: The Cas9 protein (containing RuvC and HNH domains) performs site-specific cleavage of target genomic DNA under the guidance of sgRNA.

(2) Advantage: It induces repair through double-strand breaks (DSBs), enabling highly efficient gene knockout, large-fragment deletion, or gene knock-in (overexpression).

3.2 Homologous Recombination

It is often used with allele exchange plasmids, and precise editing is achieved by using the powerful endogenous recombination system of Bacillus subtilis.

Transformation efficiency optimization: For industrial production strains (such as WB600 or high-enzyme-producing strains), the transformation barrier caused by the thick cell wall is overcome by optimizing culture conditions to induce natural competence or adopting electroporation.

4. Core Application Areas

(1) Functional genomics: Precisely delete key genes in metabolic pathways to study their effects on growth, sporulation, or protein secretion.

(2) Cell factory construction: Weaken byproduct synthesis and improve the yield of target proteins or metabolites through point mutations or gene knockouts.

(3) Synthetic biology engineering: Integrate strong promoters or reporter genes into the genome to construct smart sensing strains or high-efficiency expression systems.

(4) Chassis cell streamlining: Perform large-fragment deletions to remove unnecessary metabolic burden genes (such as prophage sequences) and construct industrial strains with higher stability.

5. Project Workflow and Validation

We provide a one-stop service from design to delivery to ensure the accuracy of editing results:

(1) Design and vector construction: Design knockout vectors for target sites.

(2) Bacterial transformation and screening: Introduce the editing system using natural competence or electroporation.

(3) Multi-level validation: Confirm positive mutations through PCR identification and Sanger sequencing.

Figure 2. Schematic Diagram of Project Workflow

6. Genome Editing Project Introduction

6.1 Core services include:

(1) Gene Knockout/Inactivation: Precisely delete protease genes or metabolic byproduct genes to optimize chassis performance.

(2) Gene Knock-in/Overexpression: Integrate exogenous expression cassettes at specific loci or enhance the expression of endogenous rate-limiting enzymes.

(3) Point Mutation/Modification: Perform site-directed mutagenesis of key enzymes to improve thermostability or catalytic activity.

(4) Multi-gene editing: Edit multiple genes successively to construct strains with complex metabolic network modifications.

6.2 Technical Advantages:

(1) High success rate: Extensive experience in editing industrial strains and laboratory model strains.

(2) Customized design: Develop optimal editing strategies according to production goals (such as improving enzyme activity, enhancing growth).

(3) Full-process validation: Provide a complete closed-loop report from scheme design to final genotype confirmation.

7. Case Studies

We have successfully provided services for many top universities, research institutes, and biotechnology companies worldwide. Below are selected examples:

Case: Construction of Bacillus subtilis BS168 Mutant Strain

Project Description: The target gene sequence in the BS168 genome was successfully knocked out.

Figure 3. Monoclonal Identification of Target Gene Knockout

Figure 4. Sequencing Results Confirming Successful Target Gene Knockout

8. References

[1] Zhu, B., et al. (2020). Engineering Bacillus subtilis as a versatile cell factory. Current Opinion in Biotechnology.

[2] Bruckner, R. (1997). Gene replacement in Staphylococcus aureus and Bacillus subtilis. Methods in Molecular Biology.

[3] Jiang, W., et al. (2013). RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature Biotechnology.

[4] Altenbuchner, J. (2016). Editing the Bacillus subtilis genome by cotransformation of a CRISPR-Cas9 system. Applied and Environmental Microbiology.

Cooperate with Us

By choosing us, you will gain an experienced and technically proficient partner in genome editing. We commit to accelerating your research or projects with professional technologies, rigorous processes， and efficient communication.

Consult us now to obtain your customized editing scheme and quotation!