大肠杆菌转座文库英文

Transposon Library of Escherichia coli

1. Escherichia coli

Escherichia coli is the most classic model organism in life science research and the core chassis cell for synthetic biology and biotechnology industries. Despite its wellcharacterized genetic background, precise and highefficiency gene editing tools are still required for complex metabolic pathway engineering, sequential multiplex editing, and manipulation of specific clinical isolates such as hypervirulent or multidrugresistant strains.

(1) Gramstaining property: Escherichia coli is a Gramnegative bacterium. It has a thin peptidoglycan layer surrounded by a complex outer membrane containing lipopolysaccharide (LPS), which is distinct from Grampositive bacteria.

(2) Clinical and research significance: It is part of the normal intestinal flora and also includes pathogenic strains such as EHEC and ETEC. In basic research, it is the preferred model for cloning, expression, metabolic engineering, and fundamental genetic studies.

(3) Metabolism and applications: It exhibits strong environmental adaptability and rapid growth, serving as an important platform for biofilm research, heterologous protein expression, and industrial production of small biological molecules.

2. Construction of Escherichia coli Transposon Library

GeneRulor has achieved efficient and random insertion of antibiotic resistance genes in Escherichia coli using the Mariner and Tn5 transposon systems, and established a highquality genomewide transposon mutant library. This system offers the following prominent advantages:

Exceptional transposition efficiency: Rigorous testing confirms that transposition efficiency in Escherichia coli is stably above 95%, ensuring broad and random insertion events and providing a reliable foundation for downstream functional screening.

Large library size and high coverage: The constructed transposon library contains more than 1×10⁶ mutants, achieving highdensity coverage of nonessential genomic regions. This scale enables systematic identification 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 various Escherichia coli strain backgrounds, covering industrial applications 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 TnSeq Report for Escherichia coli Transposon Library

The TnSeq report first presents statistics for raw sequencing data and qualityfiltered data.

Figure 3 Schematic diagram of sample data volume statistics

To ensure accurate identification of integration sites, all initially detected sites undergo strict filtering. Only sites supported by at least 3 unique molecular identifiers (UMIs) are retained for subsequent 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 represents a specific integration site.

Figure 5 Schematic diagram of integration site distribution on the host genome

Genomewide coverage and gene insertion density are two core indicators for evaluating the quality and reliability of transposon insertion screening. Genomewide coverage reflects library saturation and screening breadth, helping eliminate falsepositive essential genes caused by incomplete coverage. Gene insertion density directly quantifies the tolerance of individual genes to insertion mutations and is critical for systematic identification of essential genes.

Figure 6 Schematic diagram of genomewide coverage

To reveal functional impacts of essential genes, the report includes 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

For comprehensive functional annotation of essential genes, the report further provides GO (Gene Ontology) classification 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