Designing a simple and efficient phage biocontainment system using the amber suppressor initiator tRNA

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Multidrug-resistant infections pose a growing global health threat.
• Phage therapy is an emerging alternative and adjuvant treatment.
• Engineered phages offer enhanced efficacy but raise safety concerns regarding uncontrolled replication.

Purpose of the Study:

• To design and validate a biocontained phage system for enhanced safety in phage therapy.
• To engineer phage φX174 with conditional replication based on amber stop codon suppression.
• To ensure engineered phages cannot replicate or produce infectious particles without a specific host factor.

Main Methods:

• Engineered phage φX174 mutants with amber stop codons in essential capsid genes.
• Utilized host cells expressing amber initiator tRNA for conditional replication.
• Tested phage infectivity, replication, and particle production in vitro.
• Assessed phage host range and reversion propensity.

Main Results:

• Amber mutant phages efficiently infected host cells expressing amber initiator tRNA.
• Phages reduced bacterial populations in liquid and solid cultures.
• Infectious particle production was strictly dependent on the presence of amber initiator tRNA or complementing capsid gene.
• No growth inhibition observed in non-host E. coli strains; limited reversion observed.

Conclusions:

• The developed biocontained phage system effectively controls engineered phage replication.
• Conditional replication via amber stop codon suppression enhances phage therapy safety.
• This system offers a viable strategy for safe deployment of engineered phages in clinical and laboratory settings.