Paenibacillus

Transposon Library of Paenibacillus

1. Paenibacillus

Paenibacillus is a plant-growth-promoting rhizobacterium designated as GRAS (Generally Recognized as Safe) by the U.S. FDA, with broad application prospects in agriculture, medicine, industry and environmental bioremediation. Its prominent plant-growth-promoting activity, capacity to produce broad-spectrum antimicrobial metabolites, and excellent environmental adaptability establish its core role in biocontrol, industrial enzyme production and synthetic biology. Precise and efficient gene editing techniques are key tools for exploring its biological resource potential and constructing high-performance engineered strains.

(1) Gram-staining property: Paenibacillus is a Gram-positive bacterium. Representative species such as Paenibacillus polymyxa are the most extensively studied strains.

(2) Physical characteristics: It has a thick peptidoglycan cell wall and can form spores with strong stress resistance. Its genomic GC content ranges widely (40–55%), with diverse metabolic pathways enabling production of various antimicrobial peptides, hydrolases and polysaccharides.

(3) Industrial significance: As a model genus of plant-growth-promoting rhizobacteria (PGPR), it is an ideal model for studying biocontrol, plant growth promotion mechanisms and antimicrobial metabolite synthesis. Industrially, it is widely used for producing antimicrobial peptides, industrial enzymes and bioremediation materials.

(4) Genetic transformation: Multiple transformation approaches are available, including penicillin-mediated transformation, electroporation and magnesium-amino-acid-mediated transformation. With the development of shuttle plasmids, promoters and CRISPR tools, its genetic manipulation system has become increasingly mature.

2. Construction of Transposon Library for Paenibacillus

GeneRulor has achieved efficient and random insertion of resistance genes in Paenibacillus 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 Paenibacillus 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 Paenibacillus 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 Paenibacillus 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 GeneRulor

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