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Bacterial Transposon Library

1. Background Introduction of Transposons

1.1 Brief Introduction to Transposons

Transposons are segments of DNA sequences that can move within the genome and insert into new positions, also known as "jumping genes" due to their jumping characteristics. Scientists have engineered transposons into powerful tools for studying gene function by leveraging this unique property.

1.2 Composition of Transposons

(1) Transposase gene: Encodes an enzyme capable of recognizing and cleaving specific DNA sequences.

(2) Inverted terminal repeats (ITR): Recognition sites for transposase.

(3) Resistance marker gene: Facilitates the screening of bacteria containing transposons.

Figure 1 Transposon Plasmid Map of GeneRulor

1.3 Classification of Transposons

Class I transposons (Retrotransposons, copy-and-paste type): These transposons move via a "copy-and-paste" mechanism. They are first transcribed into RNA, and then DNA copies are synthesized by reverse transcriptase and inserted into new genomic positions, which results in an increase in the copy number within the genome.

Class II transposons (DNA transposons, cut-and-paste type): These transposons move directly in the form of DNA, being excised from the original position and inserted into a new one through a "cut-and-paste" mechanism, which generally does not cause an increase in copy number.

Figure 2 Classification of Transposons

2. Background Introduction of Transposon Libraries

A transposon library is a technology that creates a large number of mutants by random insertion of transposons into the genome, on the basis of which screening and analysis are performed to investigate gene functions and gene-gene interactions.

2.1 Functions of Transposon Libraries

During the construction of a transposon library, transposons are typically inserted into different genomic loci, leading to alterations in gene expression or the disruption of specific gene functions. By conducting phenotypic screening of these mutants, researchers can identify genes associated with specific traits, thereby elucidating gene functions.

2.2 Mechanisms of Transposon Libraries

Tn5 is derived from bacteria, featuring a highly efficient mechanism and being widely used in the construction of libraries for high-throughput sequencing.

(1) Assembly and binding: Transposase forms a dimer, which binds to the specific recognition sequences at both ends of the transposon respectively to form a transpososome complex.

(2) Double strand cleavage and "strand transfer": Acting as a "molecular scissor", transposase simultaneously cleaves the ends of both strands of the transposon, generating a double-strand break. Immediately after, transposase catalyzes the direct attack of the 3'-hydroxyl ends on the phosphate backbone of the target DNA, inserting both ends of the transposon into the target site simultaneously. This process is referred to as strand transfer.

(3) Gap formation and repair: After strand transfer, 9-base single-strand gaps are left on the DNA strands flanking the target site. The intracellular DNA repair machinery fills these gaps, consequently generating a 9 bp direct repeat sequence on both sides of the insertion site—a hallmark of Class II transposons.

Figure 3 Schematic of the Tn5 Transposition System

Transposons of the Mariner family are extremely widely distributed, existing in the genomes from insects to humans.

(1) Recognition and binding: Monomeric transposase recognizes and binds to the inverted terminal repeats at both ends of the transposon.

(2) Cleavage ("cutting"): Transposase cleaves the 3'-ends of the transposon DNA, generating a 3'-OH end at each end of the transposon. At this point, the transposon is "cut out" from its original position.

(3) Strand transfer ("pasting"): The complex formed by the excised transposon and transposase searches for new target sites in the genome. The target site of Mariner is typically a random "TA" dinucleotide. Transposase performs staggered cleavage on the two strands of the target site (the nicks on the two strands are offset by several bases), and then ligates the 3'-OH ends of the excised transposon to the 5'-phosphate ends of the target DNA.

(4) Repair and repeat formation: The cellular repair machinery fills the single-strand gaps left after strand transfer, ultimately generating a 2 bp direct repeat sequence on both sides of the insertion site.

Figure 4 Schematic of the Mariner Transposition System

3. Workflow of Transposon Library Construction

The workflow of transposon library construction is essentially a process that combines DNA fragmentation and adapter ligation into a single step using transposase.

(1) DNA extraction and quality control: First, high-quality, non-degraded genomic DNA is obtained.

(2) Transposase-mediated fragmentation and adapter ligation: DNA, transposase and transpososomes pre-loaded with sequencing adapters are mixed. Transposase randomly cleaves DNA and simultaneously ligates adapter sequences to both ends of the DNA fragments generated by cleavage. This reaction is completed in a single tube in one step.

(3) PCR amplification: Limited-cycle PCR is performed using primers matching the adapter sequences.

(4) Library purification and quality control: PCR products are purified using magnetic beads to remove impurities such as enzymes and primer dimers. Quality detection is then carried out using a bioanalyzer to confirm whether the fragment size distribution and concentration of the library meet the expected standards.

(5) Sequencing on the machine: Qualified different libraries are mixed in proportion for high-throughput sequencing.

The greatest advantage of this technology is that it simplifies the traditional multi-step operations (fragmentation → end repair → A-tailing → adapter ligation) into a single-tube reaction, which drastically shortens the operation time (from several hours to a few minutes), and reduces sample loss and human error. It has become the mainstream method for constructing libraries for high-throughput sequencing at present.

Figure 4 Workflow of Transposon Insertion Sequencing

a | Construction of a high-density transposon insertion library to ensure multiple insertion mutations at each non-essential gene locus. b | Alignment of sequencing reads to the genome, and identification of genomic loci significantly depleted under selective growth conditions through statistical analysis of read counts at each insertion site.

Tn-seq (Transposon insertion sequencing) technology uses the inverted terminal repeats (ITR) at both ends of transposons as specific molecular tags to achieve precise localization and quantitative analysis of insertion sites via high-throughput sequencing, thus systematically elucidating gene functions.

Figure 5 Schematic of the Tn-seq Detection Principle

4. Services Provided by GeneRulor

GeneRulor offers one-stop transposon library services, fully covering the entire workflow from plasmid transformation into bacteria, transposition by transposase, library strain construction, Tn-seq sequencing to data analysis, empowering customers to efficiently carry out functional genomics research. By utilizing highly efficient transposon systems, we can construct high-quality random mutant libraries in a variety of bacterial species, ensure uniform mutation coverage across the entire genome, and accurately evaluate mutation distribution through high-throughput sequencing.

GeneRulor has successfully supported transposon library construction and TnSeq analysis in a variety of industrial and environmental microbes, providing a mature one-stop service. The covered strains include Escherichia coli, Corynebacterium glutamicum, Bacillus sp. and Pseudomonas putida, etc.

(1) One-stop library construction: We integrate the complete process from bacterial transformation to mutant library construction, adopting advanced transposon technology to ensure the constructed libraries have high coverage and uniformity to meet the needs of multiple samples.

(2) Seamless Tn-seq sequencing: Based on library construction, we provide one-stop Tn-seq sequencing services to efficiently identify insertion sites of mutant strains, quickly establish correlation analysis between phenotypic screening and genetic background, and reveal the functional impacts of specific mutations.

(3) Comprehensive genomic coverage: Our transposon library system covers the entire bacterial genome, ensuring the diversity of mutants and laying a solid foundation for in-depth studies on gene functions and gene-gene interactions.

(4) Strict quality control: Strict quality control steps are implemented from library construction to sequencing, including fragment size analysis and concentration verification, to ensure that each step meets the high standards of high-throughput sequencing.

(5) Customized solutions: According to your specific needs, we can customize transposon insertion sites and sequencing strategies, which can be flexibly adapted to both model organisms and complex bacterial systems.

(6) In-depth data analysis: As an important part of the one-stop service, we not only provide library construction and sequencing, but also offer supporting professional data analysis services to help customers deeply interpret Tn-seq results and explore the association between gene functions and phenotypes.

With GeneRulor's one-stop transposon library services, you can enjoy a worry-free process from bacterial transformation, library construction and Tnseq sequencing to data analysis, accelerating the progress of functional genomics research.