Transposon Library of Pseudomonas chlororaphis

Transposon Library of Pseudomonas chlororaphis

1. Pseudomonas chlororaphis

Pseudomonas chlororaphis is a beneficial microorganism of great interest in agricultural biocontrol. It is regarded as an ideal strain for developing new microbial pesticides due to its excellent biocontrol activity and plant growth-promoting ability.

(1) Gram staining property: Pseudomonas chlororaphis is a Gram-negative bacterium comprising multiple subspecies.

(2) Morphological characteristics: Cells are rod-shaped and motile by a single polar flagellum. They do not form spores; some strains have a capsule. On solid media, they form round, smooth, convex colonies. Colonies show characteristic colors including pale yellow, greenish, orange-red, or orange-yellow due to production of phenazine antibiotics, carotenoids, and other pigments.

(3) Industrial/agricultural significance: Pseudomonas chlororaphis is recognized as an important biocontrol agent against plant diseases. It produces diverse secondary metabolites such as phenazine antibiotics, hydrocyanic acid, siderophores, and plant growth hormones, exhibiting broad-spectrum antagonism against many phytopathogenic fungi and bacteria. Studies show that its wettable powder controls tomato fusarium wilt better than the chemical fungicide carbendazim (50%), and effectively suppresses potato late blight, tobacco root rot, and other crop diseases. Additionally, it indirectly promotes crop growth and reduces the incidence of soil-borne diseases by regulating soil microbial communities.

(4) Genetic transformation: As a Gram-negative bacterium, its genetic manipulation system is relatively mature. Electroporation is commonly used to introduce exogenous DNA. The genetic background of this bacterium is gradually being elucidated, and several strains have been genome-sequenced and analyzed, laying a foundation for gene function research and metabolic pathway engineering. Gene editing techniques enable optimization of biosynthetic pathways for bioactive metabolites, providing effective tools to enhance biocontrol efficacy and industrial production capacity.

2. Construction of Transposon Library for Pseudomonas chlororaphis

Shutong Biotechnology has successfully achieved efficient and random insertion of resistance genes in Pseudomonas chlororaphis using the Mariner transposon system, and established a high-quality genome-wide transposon mutant library. This system features the following remarkable advantages:

Superior transposition efficiency: Rigorous tests have demonstrated that the transposition efficiency of the Mariner transposon in Pseudomonas chlororaphis is stably above 85%, ensuring the universality and randomness of insertion events and providing a reliable foundation for subsequent functional screening.

High library scale and coverage: The constructed transposon library contains more than 5×10⁵ mutants, achieving high-density coverage of the non-essential regions of the genome. This scale is sufficient for the systematic screening of key genes associated with specific traits (e.g., stress tolerance, metabolic enhancement).

Standardization and reproducibility: We have established standardized protocols for transposition, screening and verification, and can construct customized libraries for different Pseudomonas chlororaphis strain backgrounds. The services cover various industrial scenarios such as stress resistance improvement and product synthesis optimization.

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

Figure 2 PCR detection: Plasmid residual verification

3. Example Report of Tn-Seq for Pseudomonas chlororaphis Transposon Library

The Tn-Seq report first presents statistical analysis of the raw sequencing data and the data after quality control and filtering.

Figure 3 Schematic diagram of sample data volume statistics

To ensure the accuracy of integration site identification results, all initially detected integration sites are rigorously filtered, with only those supported by at least 3 Unique Molecular Identifiers (UMIs) retained for subsequent statistical analysis.

Figure 4 Schematic diagram of insertion site statistics

The Circos plot displays the associated distribution of transposon insertion sites on the host genome, with each line in the plot pointing to a specific integration site.

Figure 5 Schematic diagram of the distribution of integration sites 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 results. Genome-wide coverage reflects the saturation and screening breadth of the mutant library, and can be used to exclude false positive essential genes caused by incomplete experimental coverage. Gene insertion density directly quantifies the tolerance of a single gene to insertion mutations, serving 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 enrichment analysis of the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways. The main function of the KEGG database is to describe the interactive networks of genes in metabolic and signaling pathways.

Figure 7 Schematic diagram of KEGG pathway enrichment

For a more comprehensive understanding of the functions of essential genes, the report further conducts GO (Gene Ontology) functional classification analysis, which includes three dimensions: 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