Bacteriophages, viruses that infect bacteria, have been used to treat bacterial infections for over 100 years. Interest in these viruses is rising again as antibiotic resistance becomes a growing global health threat. Despite this renewed attention, most phage-based research has remained focused on naturally occurring viruses, primarily because traditional phage modification methods are slow, complex, and difficult to scale.
In the new one PNAS scientists at New England Biolabs (NEB®) and Yale University report the first fully synthetic system for engineering bacteriophages that target Pseudomonas aeruginosahighly antibiotic-resistant bacteria that poses a serious global risk. The approach relies on NEB’s High-Complexity Golden Gate Assembly (HC-GGA) platform, which allows researchers to design and build phages using digital DNA sequence data instead of relying on existing virus samples.
Using this system, the team constructed a P. aeruginosa phage from 28 synthetic DNA fragments. They then programmed the virus with new capabilities by introducing point mutations as well as DNA insertions and deletions. These changes allowed the researchers to swap out the genes of the tail filaments to alter the bacteria the phage could infect and add fluorescent markers that made infections visible in real time.
“Even in the best cases, bacteriophage engineering has been extremely labor-intensive. Researchers have spent their entire careers developing processes for constructing specific model bacteriophages in host bacteria,” reflects Andy Sikkema, co-first author of the paper and NEB research scientist. “This synthetic method offers technological leaps in simplicity, safety and speed, paving the way for biological discovery and therapeutic development.”
Building phases from digital DNA
With NEB’s Golden Gate Assembly platform, scientists can assemble an entire phage genome outside the cell using synthetic DNA and incorporate any planned genetic changes during construction. Once assembled, the genome is introduced into a safe laboratory strain, where it becomes an active bacteriophage.
This strategy avoids many of the long-standing obstacles in phage research. Traditional approaches depend on maintaining physical phage samples and using specialized host bacteria, which can be particularly challenging when working with viruses that infect dangerous human pathogens. The new method also eliminates the need for repeated rounds of screening or stepwise genetic modifications inside living cells.
Why the Golden Gate build makes a difference
Unlike other DNA assembly techniques that combine fewer but longer fragments, Golden Gate Assembly uses shorter segments of DNA. These shorter pieces are easier to produce, less toxic to host cells, and less likely to contain errors. The method also works well with phage genomes that contain repetitive sequences or extreme GC content, both of which often complicate DNA assembly.
By simplifying the process and expanding what is technically possible, this approach significantly expands the potential for developing bacteriophages as targeted therapies against antibiotic-resistant infections.
Collaboration turns tools into therapies
The development of this rapid synthetic phage engineering system grew out of a close collaboration between NEB scientists and bacteriophage researchers at Yale University. NEB researchers have spent years perfecting the Golden Gate Assembly so that it can reliably handle large DNA targets made of many fragments. Recognizing that these tools could unlock new possibilities in phage biology, the Yale researchers reached out to explore more ambitious applications.
NEB scientists first optimized the method using a well-studied model virus, Escherichia coli phage T7. From there, the collaborating teams extended the technique to non-model phages that target some of the most antibiotic-resistant bacteria known.
A related study using the same Golden Gate approach to creating high-GC content Mycobacterium of phages was published in PNAS in November 2025 in collaboration with the Hatfull Lab at the University of Pittsburgh and Ansa Biotechnologies. In another example, Cornell University researchers collaborated with NEB to create synthetically engineered T7 phages that function as biosensors to detect E. coli in drinking water, described in a December 2025 ACS study.
“My lab builds ‘weird hammers’ and then looks for the right nails,” said Greg Lohman, NEB principal investigator and co-author of the study. “In this case, the phage therapy community said to us, ‘This is exactly the hammer we’ve been waiting for.’
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