Pseudomonas putida

Transposon Library of Pseudomonas putida

1. Pseudomonas putida

Pseudomonas putida is an environmental and industrial microorganism with high application potential, classified as Generally Recognized as Safe (GRAS). Its remarkable metabolic diversity, strong environmental adaptability, and excellent stress tolerance make it a core strain in bioremediation, biocatalysis, and synthetic biology. Precise and efficient gene editing technology is a key tool for exploring its metabolic potential and constructing high-performance engineered strains.

(1) Gram-staining property: Pseudomonas putida is a Gram-negative bacterium.

(2) Physical characteristics: It has a typical outer membrane structure and a thin peptidoglycan cell wall. The outer membrane acts as an effective permeability barrier, conferring intrinsic tolerance to multiple antibiotics and organic solvents. As a model species of the genus Pseudomonas, it possesses diverse metabolic pathways.

(3) Industrial significance: As a model Gram-negative bacterium, it is an ideal model for studying aromatic hydrocarbon degradation, organic solvent tolerance, and biofilm formation. It is widely used in industry for bioremediation, chiral compound synthesis, and polymer material production.

(4) Genetic transformation: It exhibits high efficiency in exogenous DNA uptake, and gene-editing tools can be introduced via electroporation or conjugation with relatively high transformation efficiency.

2. Construction of Pseudomonas putida Transposon Library

GeneRulor has achieved efficient and random insertion of resistance genes in Pseudomonas putida using the Mariner transposon system, and established a high-quality genome-wide transposon mutant library. This system has the following prominent advantages:

Exceptional transposition efficiency: Rigorous testing confirms that the transposition efficiency of the Mariner transposon in Pseudomonas putida is stably above 95%, ensuring the universality and randomness of insertion events and providing a reliable foundation for subsequent functional screening.

Large library size and high coverage: The constructed transposon library contains more than 1×10⁶ mutants, achieving high-density coverage of non-essential genomic regions. This scale is sufficient for systematic screening of key genes associated with specific traits such as stress tolerance and metabolic enhancement.

Standardization and reproducibility: We have established standardized workflows for transposition, screening, and validation. Custom library construction is available for different Pseudomonas putida 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 Pseudomonas putida Transposon Library

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

Figure 3 Schematic diagram of sample data volume statistics

To ensure the accuracy of integration site identification, all initially detected integration sites undergo strict filtering. 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 illustrates the distribution of transposon insertion sites across the host genome; each line indicates 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 and can exclude false-positive essential genes caused by incomplete experimental coverage. Gene insertion density directly quantifies the tolerance of a single gene to insertion mutations and serves as a key basis for the 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 describes gene interaction networks in metabolic and signaling pathways.

Figure 7 Schematic diagram of KEGG pathway enrichment

To comprehensively characterize the functions of essential genes, 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 by GeneRulor

You only need to provide the glycerol stock of your target strain and relevant information; we handle the entire workflow.

Table 1 Service Content and Cycle

Table 2 Deliverables and Quality Control Standards