金黄色Transposon Library of Staphylococcus aureus

Transposon Library of Staphylococcus aureus

1. Staphylococcus aureus

Staphylococcus aureus is a clinically common Gram-positive opportunistic pathogen that widely colonizes human skin, nasal cavities and natural environments. It holds important research value in the fields of clinical etiology, virulence mechanisms, drug resistance research, antimicrobial drug screening and microbial synthetic biology. Its strong environmental tolerance, abundant ability to secrete virulence factors, biofilm-forming characteristics and evolutionary potential of multidrug resistance make it a core model strain in nosocomial infection prevention and control, antibacterial target development and pathogenic mechanism analysis. Precise and efficient gene editing technology serves as a key tool to explore its pathogenic molecular mechanisms, drug resistance regulatory networks and construct genetically modified research strains.

(1) Gram staining characteristics: Staphylococcus aureus is classified as Gram-positive bacterium.

(2) Physical characteristics: Cells arrange in grape-like clusters, with no spores and no flagella, and possess a thick peptidoglycan cell wall. It has a conserved genome structure and complex metabolic pathways, and can secrete hemolysins, coagulase, enterotoxins and various extracellular hydrolases. It tends to form biofilms to enhance drug resistance and colonization ability.

(3) Industrial significance: As a typical model pathogenic staphylococcus species, it is an ideal model for studying bacterial virulence expression, quorum sensing, biofilm formation and antibiotic resistance mechanisms. It is widely applied in the screening of antibacterial active substances, efficacy evaluation of disinfectants, food microbial safety detection and basic research on pathogenic mechanisms.

(4) Genetic transformation: It has multiple genetic manipulation approaches including protoplast transformation, electroporation, phage transduction and conjugative transfer. With the development of specific shuttle plasmids, inducible promoters and CRISPR-Cas editing systems, its genetic manipulation system for gene knockout, gene complementation and heterologous expression has become increasingly mature.

2. Construction of Staphylococcus aureus Transposon Library

Shutong Biotechnology has achieved efficient and random insertion of resistance genes in Staphylococcus aureus 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 Staphylococcus aureus 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 Staphylococcus aureus 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 Staphylococcus aureus 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