T7 Phage Genome Editing Service

T7 Phage Genome Editing Service

1. Research Background

T7 phage is a lytic double-stranded DNA virus with high research value and extensive applications in the field of molecular biology. Owing to its compact genomic structure (approximately 40 kb), extremely rapid replication, and well-characterized genetic background, T7 and its derived T7 promoter system have become the foundation of protein expression and synthetic biology. Precise and efficient genome editing technologies serve as key tools for modifying the host range of phages, developing novel phage therapies, and constructing complex logic gates for synthetic biology. Against this backdrop, GeneRulor launches T7 phage genome modification services based on the CRISPR/Cas9 selection pressure system, which can customarily achieve gene knockout, large-fragment deletion, site-directed mutagenesis, functional gene integration and other modifications for target genes, and deliver purified and identified positive mutant strains.

2. Phage Characteristics and Biological Background

（1）Structural Properties: T7 phage belongs to the Autographiviridae family of self-replicating short-tailed phages, with a typical icosahedral head and short-tailed structure. Its genome is a linear double-stranded DNA with a length of approximately 39,937 bp.

（2）Molecular Biological Significance: T7 RNA polymerase exhibits extremely high specificity and transcription efficiency, and is widely used in prokaryotic expression systems based on the T7 promoter.

（3）Research Model: As an ideal model for studying DNA replication, transcriptional regulation and viral assembly, it is one of the most thoroughly characterized chassis viruses in current synthetic biology research.

（4）Clinical and Application Potential: In phage therapy, T7 is used for the targeted elimination of pathogenic bacteria such as Escherichia coli, and its genetic editing modification can be applied to broaden the host range or reduce immunogenicity.

Figure 1 T7 Phage

3. Reported Editing Strategies

In light of the characteristics of rapid replication and lysis of the T7 phage genome in host cells, the current mainstream editing strategies include:

3.1 CRISPR/Cas9-assisted selection system (core protocol):

（1）Principle: The Cas9 protein, guided by sgRNA, is used to specifically cleave the wild-type phage genome.

（2）Advantages: Since the cleaved wild-type genome is unable to replicate, phages carrying mutations are enriched through "negative selection", which greatly improves the extremely low mutation rate of traditional homologous recombination.

3.2 Homologous Recombination:

Phages infect host cells containing donor plasmids (carrying mutant sequences and homologous arms), and sequence exchange occurs during the infection process.

3.3 In Vitro Genome Assembly and Rebooting:

The T7 genome is split into multiple fragments, subjected to site-directed mutagenesis or integration in vitro, and then introduced into host cells via electroporation for "rebooting".

4. Core Application Fields

（1）Functional Genomics: Precisely delete or modify non-essential genes (e.g., early genes) to investigate their effects on phage replication efficiency and lytic cycle.

（2）Phage Therapy Modification: Integrate depolymerases or fluorescent proteins into the genome to enhance its biofilm degradation capacity or enable real-time monitoring of infection dynamics.

（3）Host Range Expansion: Modify the genes encoding tail fiber proteins to achieve precise recognition and elimination of specific drug-resistant bacterial strains.

（4）Synthetic Biology: Construct engineered phages with specific regulatory elements for the development of biosensors or novel alternatives to antibiotics.

5. Project Process and Validation

We offer one-stop services from design to delivery to ensure the purity and accuracy of phage modification:

（1）Protocol design and vector construction: Design donor plasmids containing mutant sequences and CRISPR screening plasmids targeting the wild-type strain.

（2）Host bacterium transformation: Transform the plasmids into recipient Escherichia coli (e.g., BL21) strains.

（3）Phage infection and screening: Wild-type T7 infects host bacteria, the Cas9 system cleaves the wild-type genome, and mutant phages are enriched.

（4）Plaque purification and validation: Perform single plaque purification via the double-layer agar plate method for multiple times, and conduct parallel PCR identification and sequencing.

6. Introduction to Genome Editing Projects

6.1 Our core services include:

（1）Gene knockout/large-fragment deletion: Remove specific genes for studying viral assembly or reducing genomic load.

（2）Exogenous gene integration: Insert reporter genes or functional enzyme genes into the non-essential regions of the T7 genome.

（3）Precise site-directed mutagenesis: Perform base substitution on the active sites of tail fiber proteins or polymerases.

6.2 Technical Advantages

（1）High success rate: Optimize the Cas9 selection pressure to ensure the accurate isolation of mutant strains from a large number of wild-type phages.

（2）Rapid delivery: Utilize the extremely short replication cycle of T7 to complete a round of editing and purification in as fast as 2-3 weeks.

（3）Full-process validation: Provide a complete validation report ranging from single plaque screening to whole-genome sequencing.

Figure 2. Schematic Diagram of Project Workflow

7. Case Introduction

We have successfully provided services for numerous top universities, research institutions and biotech companies at home and abroad. A representative case is presented below:

Case: T7 Phage Genome Modification

Project Content: T7 phage genome modification and phage reboot via the yeast recombination system.

Figure 3 Rebooted T7 Phage Infecting Host Cells

8. References

[1] Studier, F. W., & Moffatt, B. A. (1986). Use of T7 RNA polymerase to direct selective high-level expression of cloned genes. Journal of Molecular Biology.

[2] Bari, S. M., et al. (2017). Orthogonal Cas9 proteins for RNA-guided gene regulation and editing in mammalian cells. Nature Methods. (Adapted for Phage CRISPR).

[3] Kiro, R., et al. (2014). Efficient gene editing of effector-encoding genes in phages using CRISPR-Cas systems. RNA Biology.

[4] Molineux, I. J. (2006). The T7 group. The Bacteriophages.

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By choosing us, you will gain an experienced and technically proficient partner in genome editing. We commit to accelerating your research or projects with professional technologies, rigorous processes and efficient communication.

Consult us now to obtain your customized editing scheme and quotation!