希瓦Transposon Library of Shewanella

Transposon Library of Shewanella

1. Shewanella

Shewanella is a group of facultative anaerobic Gram-negative bacteria widely distributed in oceans, freshwater and sediments, with broad application prospects in the fields of environmental remediation, bioelectrochemistry, heavy metal reduction, marine microbial resource development and synthetic biology. Its unique extracellular electron transfer capability, broad-spectrum metal reduction property, excellent environmental tolerance and diverse metabolic potential enable it to occupy an important position in contaminated environmental treatment, microbial fuel cells and the research and development of functional engineered strains. Precise and efficient gene editing technology is a key tool for exploring its electron transfer mechanisms, metabolic regulation rules and constructing high-performance engineered strains.

(1) Gram staining characteristics: Shewanella belongs to Gram-negative bacteria.

(2) Physical characteristics: It does not form endospores; most strains have polar flagella with strong motility, possessing a complete outer membrane structure and a well-developed electron transport system. It has a moderate genomic GC content and flexible and diverse metabolic pathways, with strong capacities for heavy metal reduction, organic pollutant degradation and extracellular electron secretion.

(3) Industrial significance: As a model genus of dissimilatory metal-reducing bacteria, it serves as an ideal model for studying extracellular electron transfer, anaerobic respiration and heavy metal detoxification mechanisms. Industrially, it is widely applied in heavy metal pollution remediation, microbial fuel cell construction, treatment of refractory organic pollutants, and development of functional enzymes from marine extreme environments.

(4) Genetic transformation: It possesses multiple mature transformation approaches including electroporation, chemical competent cell transformation and conjugative transfer. With the continuous optimization of compatible shuttle plasmids, constitutive and inducible promoters, as well as CRISPR gene editing tools, its genetic manipulation and strain modification system has become increasingly sophisticated.

2. Construction of Shewanella Transposon Library

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