肺炎Transposon Library of Klebsiella pneumoniae

Transposon Library of Klebsiella pneumoniae

1. Klebsiella pneumoniae

Klebsiella pneumoniae is a clinically common opportunistic pathogen belonging to the family Enterobacteriaceae. It widely resides in the human respiratory tract, intestinal tract and natural environments, and has important research value in the fields of clinical etiology, drug resistance mechanism research, microbial metabolic engineering and synthetic biology. Its strong environmental adaptability, capsular synthesis characteristics, multidrug resistance evolutionary capacity and diverse metabolic pathways make it a core model strain for the research of nosocomial infection prevention and control, drug resistance gene transmission and carbon source metabolic modification. Precise and mature gene editing technology is a key tool for elucidating its pathogenic mechanism and drug resistance regulation, as well as constructing metabolically engineered modified strains.

(1) Gram staining characteristics: Klebsiella pneumoniae is a Gram-negative bacterium, and Klebsiella pneumoniae subsp. pneumoniae is the most extensively studied representative strain in clinical and scientific research.

(2) Physical characteristics: It is non-spore-forming and non-flagellated, with a thick lipopolysaccharide outer membrane and a viscous capsular structure. It has a relatively stable genomic GC content and complex and diverse metabolic pathways, and can produce capsular polysaccharides, virulence factors, β-lactamases and various carbohydrate metabolic enzymes.

(3) Industrial significance: As a typical opportunistic pathogen and dominant carbon-metabolizing strain, it serves as an ideal model for studying nosocomial infection, virulence regulation, drug resistance evolution and quorum sensing mechanisms. It can be applied to research on biocatalysis, synthesis of bulk organic acids such as succinic acid, degradation of environmental pollutants, and modification of microbial carbon source utilization.

(4) Genetic transformation: It has multiple genetic introduction methods including electroporation, chemical competent transformation and conjugative transfer. Relying on broad-host-range shuttle plasmids, regulatable promoters and the CRISPR-Cas gene editing system, its genetic manipulation system for gene knockout and overexpression modification is highly mature, which is suitable for the analysis of pathogenic mechanisms and the construction of metabolically engineered strains.

2. Construction of Klebsiella pneumoniae Transposon Library

Shutong Biotechnology has achieved efficient and random insertion of resistance genes in Klebsiella pneumoniae 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 Klebsiella pneumoniae 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 Klebsiella pneumoniae 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 Klebsiella pneumoniae 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