枯草Transposon Library of Bacillus subtilis

Transposon Library of Bacillus subtilis

1. Bacillus subtilis

Bacillus subtilis is a plant growth-promoting rhizobacterium classified as GRAS (Generally Recognized As Safe) by the U.S. FDA, possessing extremely high application value in the fields of agricultural biocontrol, food fermentation, industrial enzyme production and biomedicine. Its strong stress-resistant spore-forming capacity, broad-spectrum antibacterial activity and abundant secondary metabolite synthesis capability make it a classic model strain for research in microbial industry, biopesticides and synthetic biology. A well-established and sophisticated gene editing and genetic modification system serves as the core means to explore its metabolic potential and construct high-yield and functionally modified engineered strains.

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

(2) Physical characteristics: It has a thick peptidoglycan cell wall and can form endospores resistant to high temperature, drought and extreme environments. It features a stable genome structure and complete metabolic pathways, and can efficiently secrete amylase, protease, cellulase and various antibacterial lipopeptide active substances.

(3) Industrial significance: As a typical model strain of plant growth-promoting rhizobacteria, it is an ideal carrier for studying the mechanisms of biological disease prevention, crop growth promotion and microecological preparations. Industrially, it is widely used in the mass production of enzyme preparations, food fermentation and preservation, biopesticide preparation and the development of microecological regulators for aquaculture and livestock breeding.

(4) Genetic transformation: It has multiple well-developed genetic introduction methods including electroporation, natural competent transformation and protoplast transformation. Relying on shuttle vectors, strong constitutive/inducible promoters and the CRISPR-Cas editing system, its genetic manipulation and strain modification system are highly mature, suitable for metabolic engineering and the construction of engineered strains for heterologous expression.

2. Construction of Bacillus subtilis Transposon Library

Shutong Biotechnology has achieved efficient and random insertion of resistance genes in Bacillus subtilis using the Mariner transposon system, and established a high-quality genome-wide transposon mutant library. This system offers the following prominent advantages:

Outstanding transposition efficiency: Rigorous tests confirm that the transposition efficiency of the Mariner transposon in Bacillus subtilis is stably above 80%, ensuring broad and random insertion events and providing a reliable basis for subsequent functional screening.

Large library size and high coverage: The constructed transposon library contains more than 5×10⁵ mutants, achieving high-density coverage of non-essential genomic regions. This scale is sufficient for systematic screening of key genes related to specific traits such as stress tolerance and metabolic enhancement.

Standardization and reproducibility: Standardized workflows for transposition, screening and validation have been established. Custom library construction is available for different Bacillus subtilis strain backgrounds, covering industrial scenarios including stress resistance improvement and product synthesis optimization.

Figure 1 PCR detection: Transposition validation and resistance gene insertion verification

Figure 2 PCR detection: Plasmid residual verification

3. Example Tn-Seq Report for Bacillus subtilis Transposon Library

The Tn-Seq report first presents statistics of raw sequencing data and quality-controlled filtered data.

Figure 3 Schematic diagram of sample data volume statistics

To ensure accurate identification of integration sites, all initially detected sites are strictly filtered. Only sites supported by at least 3 unique molecular identifiers (UMIs) are retained for subsequent statistical analysis.

Figure 4 Schematic diagram of insertion site statistics

A Circos plot displays the distribution of transposon insertion sites across the host genome; each line points to a specific integration locus.

Figure 5 Schematic diagram of integration site distribution on the host genome

Genome-wide coverage and gene insertion density are two core indicators for evaluating the quality and reliability of transposon insertion mutation screening. Genome-wide coverage reflects the saturation and screening breadth of the mutant library, helping exclude false-positive essential genes caused by incomplete coverage. Gene insertion density directly quantifies the tolerance of individual genes to insertion mutations and serves as a key basis for systematic identification of essential genes.

Figure 6 Schematic diagram of genome-wide coverage

To explore the functional impacts of essential genes, the report performs KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis. The KEGG database characterizes gene interaction networks in metabolic and signaling pathways.

Figure 7 Schematic diagram of KEGG pathway enrichment

To comprehensively understand essential gene functions, the report further conducts GO (Gene Ontology) functional classification analysis covering three categories: Biological Process (BP), Cellular Component (CC) and Molecular Function (MF).

Figure 8 Schematic diagram of GO term enrichment

4. Services Provided by Shutong Biotechnology

You only need to provide the glycerol stock of the target strain and relevant information, and we will provide a full-process service for you.

Table 1 Service Content and Cycle

Table 2 Deliverables and Quality Control Standards