T7 Phage Gene Editing
1. Research Background
The T7 phage is a virulent double-stranded DNA virus with highresearch value and wide application in the field of molecular biology. Due toits compact genome structure (approximately 40 kb), extremely fast replicationrate, and clear genetic background, it has become a fundamental model forstudying viral replication mechanisms, gene regulation, and synthetic biologyapplications. Against this backdrop, Shutong Bio has launched a customized T7phage genome modification service, which can realize targeted editing of the T7phage genome, providing powerful technical support for related scientificresearch and industrial application promotion.
2. Phage Characteristics and Biological Background
(1) Structural Properties: The T7 phage belongs to theAutographiviridae family, featuring a typical icosahedral head and a short tailstructure. Its genome is a linear double-stranded DNA with a length ofapproximately 39,937 bp, which is compact and easy to manipulate.
(2) Molecular Biological Significance: The T7 RNA polymerase hasextremely high specificity and transcription efficiency, making it widely usedin prokaryotic expression systems. It serves as an important tool for studyinggene expression regulation and protein function.
(3) Research Model Value: As an ideal model for studying DNAreplication, transcription regulation, and viral assembly, it is one of themost thoroughly studied chassis viruses in synthetic biology.
(4) Clinical and Application Potential: In phage therapy, the T7phage can be engineered to target and clear pathogenic bacteria such asEscherichia coli. Its gene-edited derivatives can further expand the host rangeand improve antibacterial efficiency, providing a new technical approach forthe development of microbial therapy.
3. Editing Strategies Reported in Literature
Given the characteristics of the T7 phage genome’s rapidreplication and lysis in host cells, the current mainstream editing strategiesinclude:
3.1 CRISPR/Cas9-Assisted Selection System (Core Strategy)
(1) Principle: The Cas9 protein specifically cleaves thewild-type phage genome under the guidance of sgRNA.
(2) Advantages: Since the wild-type genome cannot replicateafter being cleaved, the phages with mutations are enriched through"negative selection", which greatly improves the extremely lowmutation rate of traditional homologous recombination.
3.2 Homologous Recombination
The phage infects host cells containing donor plasmids (with mutantsequences and homologous arms), and sequence exchange occurs during theinfection process.
3.3 In Vitro Genome Assembly and Rebooting
The T7 genome is split into multiple fragments, which are subjectedto point mutations or integration in vitro, then transferred into host cellsvia electroporation for "rebooting".
4. Core Application Areas
(1) Functional Genomics: Precisely delete or modifynon-essential genes (such as early genes) to study their impact on phagereplication efficiency and lysis cycle.
(2) Phage Therapy Modification: Integrate depolymerases orfluorescent proteins into the genome to enhance the ability to degrade biofilmsor monitor infection dynamics in real time.
(3) Host Range Expansion: Modify the genes encoding tailfiber proteins to enable specific recognition and killing of drug-resistantstrains.
(4) Synthetic Biology: Construct engineered phages withspecific regulatory elements for the development of biosensors or newantibiotic alternatives.
5. Project Workflow and Validation
We provide one-stop services from design to delivery to ensure thepurity and accuracy of phage modification:
(1) Design and Vector Construction: Design donor plasmidscontaining mutant sequences and CRISPR screening plasmids targeting thewild-type phage.
(2) Host Strain Transformation: Transform the plasmids intorecipient Escherichia coli (e.g., BL21) competent cells.
(3) Phage Infection and Screening: Wild-type T7 phageinfects the engineered host cells, and the Cas9 system cleaves the wild-typegenome to enrich mutant phages.
(4) Plaque Purification and Validation: Performsingle-plaque purification through multiple double-layer agar plate assays,followed by PCR identification and sequencing for confirmation.
6. Introduction to Gene Editing Projects
6.1 Core Services
(1) Gene Knockout/Large Fragment Deletion: Precisely removespecific genes to study viral assembly mechanisms or reduce genome load,facilitating research on viral replication and regulatory mechanisms.
(2) Exogenous Gene Integration: Insert reporter genes or functionalenzyme genes into non-essential regions of the T7 phage genome to expandapplication scenarios.
(3) Precise Point Mutation: Conduct site-directed modification ofkey amino acids in tail fibers or polymerases to adjust phage host recognitionspecificity and improve binding efficiency.
6.2 Technical Advantages
(1) High Success Rate: Optimized CRISPR-Cas9 selection pressureensures accurate screening of positive mutants from a large number of wild-typephages, significantly improving editing efficiency.
(2) Rapid Delivery: Leveraging the extremely short replicationcycle of T7 phage, we can complete one round of editing, purification andverification in 2-3 weeks at the earliest.
(3) Full-Process Verification: Provide a complete set ofverification services from single-plaque screening to whole-genome sequencingto ensure the accuracy and stability of modified strains.
7. Case Introduction
We have successfully provided professional gene editing servicesfor many top universities, research institutions and biotechnology companies athome and abroad. The following is a typical case:
Case: T7 Bacteriophage Genetic Modification
Project Content: T7 phage genome modification and phagereactivation via yeast recombination system
We used yeast recombination system to modify the T7 phage genome,completed the targeted editing of key genes, and realized the efficientreactivation of the modified phage, providing technical support for relatedscientific research and industrial application.
Supplementary Notes
All professional terms (such as CRISPR-Cas9, yeast recombination system, plaque screening, etc.) are consistent with your previous web page translation to ensure the overall style is unified.
The translation avoids redundant expressions, conforms to the concise and professional characteristics of web content, and is convenient for visitors to read quickly
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