Lambda Phage (λ Phage) Genome Editing Service

Lambda Phage (λ Phage) Genome Editing Service

1. Research Background

Lambda phage is the most thoroughly studied temperate phage in the history of molecular biology and also the cornerstone of prokaryotic gene regulation, site-directed recombination and transduction technologies. Its genome (approximately 48.5 kb) features a unique cos site and a sophisticated lytic-lysogenic switch mechanism, making it an ideal model for constructing genetic circuits, vector systems and investigating virus-host interactions in synthetic biology.

Against this backdrop, GeneRulor launches customized genome modification services for Lambda phage. By combining the CRISPR/Cas9-assisted selection system with the classic BRED (Bacteriophage Recombineering of Electroporated DNA) technology, we can achieve efficient gene knockout, large-fragment insertion, site-directed mutagenesis and functional element integration in the λ genome, and deliver rigorously purified mutant phage strains. We are committed to providing professional and reliable Lambda phage genome editing solutions for scientific research clients worldwide.

2. Phage Characteristics and Biological Background

（1）Host Specificity: Lambda phage exclusively infects Escherichia coli, serving as a benchmark for studying virus-host interactions in Gram-negative bacteria.

（2）Structural Features: Belonging to the Siphoviridae family of long-tailed phages, it has a typical icosahedral head and a non-contractile long tail. Its linear double-stranded DNA genome circularizes via the cos site upon entering host cells.

（3）Research Value: The Red recombination system (Exo, Beta, Gam) of λ phage has been extensively developed into a universal tool for bacterial genome editing. Modification of the phage itself can further optimize the efficiency of these molecular biology tools.

（4）Life Cycle: It exhibits two distinct life forms, lytic and lysogenic, making it a natural model for studying biological toggle switches and environmental responses.

Figure 1 Lambda Phage

3. Reported Editing Strategies

In light of the complex recombination mechanism and high frequency of spontaneous mutation of Lambda phage, the currently mainstream editing strategies include:

3.1 CRISPR/Cas9 system (primary protocol):

（1）Principle: The Cas9 protein is used to specifically cleave the unmutated wild-type genome.

（2）Advantages: As a robust negative selection pressure, it can boost the recombination efficiency from the extremely low level of traditional methods to nearly 100%, enabling rapid and scarless genome modification.

3.2 BRED technology (Bacteriophage Recombineering of Electroporated DNA):

Phage DNA and homologous DNA fragments carrying mutation templates are co-introduced into host cells via electroporation, with editing mediated by endogenous or exogenous recombinases.

3.3 In vitro Assembly & Rebooting:

The genome is split into multiple modules, mutations are introduced via methods such as Gibson assembly, and viral replication is then reactivated through in vitro packaging systems or electroporation.

4. Core Application Fields

（1）Molecular Tool Optimization: Modify genes related to the λ-Red system to enhance the recombination efficiency of Escherichia coli and other bacterial species.

（2）Synthetic Biology Chassis: Integrate complex genetic logic gates or reporter genes (e.g., lux, fluorescent proteins) into the λ genome for environmental signal monitoring.

（3）Host Range Expansion: Modify the tail fiber protein (J protein) to enable λ phage to recognize and infect non-standard Escherichia coli strains or other closely related species.

（4）Novel Vector Development: Construct phage vectors with larger capacity and higher stability for the construction and screening of large-scale gene libraries.

5. Project Process and Validation

We offer one-stop services from design to delivery to ensure the accuracy of editing results:

（1）Protocol design and vector construction: Design knockout/insertion vectors and sgRNAs targeting the loci of interest.

（2）Transformation and recombination screening: Overcome host transformation barriers using optimized electroporation conditions.

（3）DNA targeted cleavage and repair: Enrich positive clones by cleaving the wild-type background with Cas9.

（4）Multiplex validation: Verify the purity of positive mutant strains via single-plaque PCR identification and Sanger sequencing.

Figure 2 Schematic of the Project Process

6. Introduction to Genome Editing Projects

6.1 Our core services include:

（1）Gene knockout/inactivation: Precisely delete redundant genes or regulatory elements for investigating viral life cycle control.

（2）Gene knock-in/overexpression: Insert exogenous functional fragments into non-coding regions or enhance the strength of endogenous promoters.

（3）Site-directed mutagenesis/scarless modification: Achieve precise single-base substitution to study the thermostability or binding affinity of key proteins (e.g., CI repressor).

（4）Continuous multi-gene editing: Realize simultaneous or stepwise modification of multiple loci in the Lambda genome to construct highly customized engineered phages.

6.2 Technical Advantages

（1）Fast delivery cycle: Rely on standardized processes to ensure the delivery of verified mutant phages in the shortest possible time.

（2）High-efficiency screening: The optimized CRISPR selection system can increase the screening efficiency to over 80%.

7. Case Introduction

Case: In Situ Targeted Base Editing of Murine Gut Bacteria

Project Content: Achieve efficient and precise genome editing of gut bacteria by delivering base editors via engineered λ phage particles.

(1) Research Background

Microbiome studies have shown that the specific gene expression of gut bacteria directly impacts host health (e.g., influencing immunotherapy efficacy, driving neurodegenerative diseases or metabolizing drugs). However, although existing CRISPR tools enable efficient editing of human cells, there is a lack of technologies for in situ editing of gut bacteria. Traditional methods rely on editing after bacterial culture, which is difficult to simulate the complex intestinal environment and may introduce the risk of transgene spread. This study aimed to develop a non-invasive strategy for precise genetic modification of target bacteria directly in the mouse gut, providing a new tool for microbiome functional research and therapy development.

(2) Protocol Design

The research team designed an engineered delivery system based on λ phage, with core innovations including:

Vector Engineering: Modify phage tail fibers (STF) and tail tip proteins (gpJ) to construct chimeric receptors (e.g., λ-P2 STF, λ-K5 STF) for targeting different bacterial surface receptors (e.g., OmpC, capsular polysaccharides) and expanding the host range. Optimize gpJ proteins (e.g., A8 and 1A2 variants) through directed evolution to enhance the binding efficiency to highly expressed intestinal receptors (e.g., OmpC), ensuring efficient delivery in complex environments.

Editing System Design: Adopt adenine base editors (ABE8e) and cytosine base editors (CBE) to achieve precise site-directed mutagenesis without DNA double-strand breaks. Design non-replicative DNA vectors that only replicate in producer bacteria and degrade after transient expression in target bacteria, avoiding transgene residue and spread.

In Vitro and In Vivo Validation Platform: Evaluate editing efficiency through in vitro models and conduct in situ editing tests using streptomycin-treated mouse models that mimic the human intestinal environment.

(3) Experimental Conclusions

High Editing Efficiency: In vitro, the editing efficiency of a single dose of ABE on the β-lactamase gene (bla) exceeded 99.99%; the editing efficiency of CBE on pathogenic bacterial genes (e.g., clbH, fimH) reached 37%-92%. Low off-target effects were verified by sequencing, and Illumina sequencing showed that the mutation frequency at candidate off-target sites was not significantly higher than the background error.

Stable In Vivo Editing: In the mouse gut, the median editing efficiency of a single dose (4×10^10 particles) on the bla gene reached 93%, and the edited microbiota was stably maintained for at least 42 days. Multiple-dose treatment could further increase the editing ratio (e.g., from 36% to 88%), and the editing effect was positively correlated with the dose.

Broad Applicability: Successful editing of virulence factors (e.g., csgA, cnf1) in pathogenic strains (e.g., uropathogenic E. coli UTI89, Klebsiella pneumoniae ST258) demonstrated that the technology can be extended to a variety of bacterial species.

8. References

[1] Datsenko, K. A., & Wanner, B. L. (2000). One-step inactivation of chromosomal genes. PNAS.

[2] Ptashne, M. (2004). A Genetic Switch: Phage Lambda Revisited. Cold Spring Harbor Laboratory Press.

[3] Jiang, W., et al. (2013). RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature Biotechnology.

[4] Luo, M. L., et al. (2016). The CRISPR-Cas9 system as a tool for editing the genomes of Staphylococcus aureus. Applied and Environmental Microbiology.

[5] Brodel, A. K., et al. (2024). In situ targeted base editing of bacteria in the mouse gut. Nature.

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