谷棒文库英文

Transposon Library of Corynebacterium glutamicum

1. Corynebacterium glutamicum

Corynebacterium glutamicum is a universally recognized core strain in the industrial production of amino acids and classified as a Generally Recognized as Safe (GRAS) strain. Its exceptional capacity for amino acid synthesis, well-characterized genetic background and excellent industrial production traits have established its central role in the fields of food additives, feed nutrition and biomedicine. Precise and efficient gene editing technology serves as a pivotal tool for optimizing its production performance and constructing high-efficiency cell factories.

(1) Gram-staining property: Corynebacterium glutamicum is a Gram-positive bacterium with a high-GC-content genome.

(2) Physical characteristics: It possesses a unique cell wall structure comprising arabinogalactan polymers and an outer mycolic acid layer, which forms an additional permeability barrier and endows the strain with certain hydrophobicity and environmental tolerance.

(3) Industrial significance: As a model strain for amino acid production, it is an ideal model for investigating amino acid biosynthesis, metabolic regulation and the optimization of industrial fermentation. Discovered in 1957, it has been widely used for the large-scale industrial production of amino acids such as glutamic acid and lysine across the globe.

(4) Genetic transformation: Exogenous DNA delivery can be achieved via electroporation or conjugation, yet the transformation efficiency requires optimization due to its distinctive cell wall structure. A variety of inducible promoter systems have been developed for regulated gene expression.

2. Construction of Transposon Library for Corynebacterium glutamicum

GeneRulor has successfully achieved efficient and random insertion of resistance genes in Corynebacterium glutamicum 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 Corynebacterium glutamicum is stably above 90%, 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 Corynebacterium glutamicum 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 Corynebacterium glutamicum 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 GeneRulor

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