ORACLE: A great tool for generating many variants of phages

Issue 227 | June 9, 2023
13 min read
Capsid and Tail


This week we have a guest post from Dylan Woolsey, a University of Georgia undergraduate student, who introduces us to ORACLE, a tool for generating phage variants.


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What’s New

The UK phage biotech company, Fixed Phage, has appointed Dr Neil Clelland as its new CEO, along with a new COO and Executive Chairman. The company is looking to build on its position in the phage industry and provide innovative solutions for human and animal health.

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Biotech news

Sabrina Green and colleagues published a retrospective observational study of 12 cases of expanded access customized phage therapy. They found that customized phage production and therapy was safe and yielded favorable clinical or microbiological outcomes in 2/3 of cases. They suggest that a center dedicated to tailoring the phages against a patient’s specific infection may be a viable option.

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AMRResearch paperPhage therapy

Due to technical limitations, most gut microbiome studies have focused on prokaryotes, overlooking viruses. Yishay Pinto and colleagues have published a new paper on Phanta: a new method for quickly and simply identifying and quantifying phages in metagenomic samples.

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Bioinformatics toolGut microbiomeGut phageome

Agnès Oromí-Bosch and colleagues published a new report on phage resistance-related challenges associated with phage therapy. They review two main strategies for mitigating resistance in phage therapy: 1) smartly identify/design the phages that are administered; 2) use phages that drive evolutionary trade-offs (in bacteria) that are biomedically desirable. Twitter thread by coauthor Jyot Antani here

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Emma George and colleagues have discovered a highly complex community of endosymbionts and a phage within a single cryptomonad cell, which is a model for organelle evolution. The Cryptomonas cell is an endosymbiotic conglomeration with seven distinct evolving genomes that all show evidence of inter-lineage conflict but nevertheless remain stable, even after more than 4,000 generations in culture.

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Organelle evolutionEndosymbiosis

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CRISPR-CasPost DocMicrobiome editing
The Innovative Genomics Institute at UC Berkeley is seeking a Postdoc for in situ microbiome editing to work with a highly collaborative team of researchers in the Doudna and Cress labs to develop and use novel CRISPR-Cas editing systems, capture and engineer wild plasmids and phages, and employ cutting-edge tools for in situ control of edited organisms.
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The Bollyky Lab at Stanford is seeking a postdoctoral scholar to contribute to research developing innovative strategies for phage engineering and delivery.
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The Hill lab at APC Microbiome/University College Cork is seeking a research assistant to aid in a project investigating the role of phages in the gut microbiome, and specifically their influence on microbial community structure, function, and assembly.
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MINIMUM Bio is seeking a Research Scientist with expertise in phage display and bioinformatics to join their team in Sydney. The role involves developing innovative biopanning solutions for drug target discovery.
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ICON is recruiting for a Bacteriophage Therapeutics - Research Technician II to support Walter Reed Army Institute of Research Wound Infection Department by performing phage research toward development of therapeutics and diagnostics for microbial pathogens of military relevance.
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Jichi Medical University is seeking postdoctoral researchers passionate about advancing the field of vaccine development for infectious disease pathogens, with a specific focus on pathogenic bacteria. The role involves R&D of innovative vaccines using phage-based technology.
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The University of Ottawa is seeking a Teaching Assistant/Demonstrator/Lab Monitor for a Biomedical Research Laboratory course. The TA will assist in preparing a phage discovery lab and must have experience in phage purification and amplification, genome isolation, safe-handling of phenol, preparation of TEM samples, and familiarity with the SEA-PHAGES program.

Community Board

Anyone can post a message to the phage community — and it could be anything from collaboration requests, post-doc searches, sequencing help — just ask!

Attention all phage researchers! You’re invited to participate in an exciting initiative to help establish draft phage bank management guidelines for the community.

Please register your interest in participating here.

The process will unfold in stages:

Stage 1 (online survey, stay tuned — coming soon): Help define what topics should be included in the guidelines

Stage 2 (online survey): Collect detailed input on each of the topics identified in Stage 1

Stage 3 (in-person meetings): Please note that we have now added a second meeting in July, in addition to the first meeting in August - feel free to attend either or both

  1. July 2nd (day before VoM 2023 in Georgia): Discuss compiled responses to online surveys, especially on topics for which there are differing opinions. Virtual options will be available for remote attendees.
  2. August 6th (day before Evergreen in USA): This will be a second iteration of the July 2nd meeting, taking into account group opinions that have evolved. Virtual options will be available for remote attendees.

A limited number of scholarships will be available for those from lower- and middle-income countries who wish to attend this meeting. Please email Ria Kaelin ([email protected]) and Tobi Nagel ([email protected]) if you would like to apply for a scholarship.

Stage 4 (virtual discussions): Engage in virtual discussions to draft and finalize guidelines. Leaders will develop drafts for each topic and hold virtual meetings. Compiled draft will be shared with the group, and a final version will be published in a peer-reviewed journal and presented at VoM 2024.

To get started, please register your interest by completing this pre-questionnaire.

For questions or suggestions, please email:

Tobi Nagel: [email protected]
Francesca Hodges: [email protected]

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This week Phagecast published a new episode featuring an interview with Martin Loessner! You can hear very valuable insights in what is important to have a successful scientific career together with some phage science: how L-forms become resistant to phages & endolysins, and the implications that this has in phage therapy and endolysin research. Enjoy!

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The Evergreen International Phage Meeting is happening Aug 6-11, 2023, live in Olympia, Washington, USA! This is an in-person meeting, better known as Phage Camp! Come present your phage work, climb mountains with other phage scientists, play phage-related board games, see a special showing of the Salt in My Soul documentary, and much more! This is the 50th anniversary meeting, hosted by Betty Kutter, Queen of Phage.

Oral abstracts due June 9!

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The Evergreen Phage Meeting organizing committee invite you to take part in a photo contest and get a chance to win awesome phage swag! Send us your favorite photo featuring or taken with Betty Kutter, and let’s shower her with love at the 25th Biennial Evergreen International Phage Meeting, where your photo will be showcased in a special slide show to honor the Queen of Phage. Dig through your vintage photo album or browse your digital files - we’re excited to see the amazing moments you’ve captured! Good luck! Submit photos to [email protected].

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ORACLE: A great tool for generating many variants of phages

Profile Image
Undergraduate StudentUndergraduate Researcher
Maor Bar-Peled, University of Georgia, Athens, GA, United States

Molecular Biology, Undergraduate research, Biotechnology, Microbiology, Genomics, Microscopy, cloning / bacterial screens / lenti / TC, Plasmid prep, DNA isolation

This post is based on a paper by Phil Huss and colleagues (2021) entitle ‘Mapping the functional landscape of the receptor binding domain of T7 bacteriophage by deep mutational scanning’, published in eLife Mar 9, 2021.

How ORACLE works

For the development of phage therapy, what makes a bacteriophage able to infect a bacterium is an important question. The study of the receptor binding protein (RBP) on the phage helps to increase the understanding of how certain phages are effectively infecting certain strains of bacteria and not others. Huss et al. have developed an effective process of creating many different RBP variants to determine each residue’s function and importance while developing a greater understanding of the sequence-function relationship. This process, known as Optimized Recombination, Accumulation, and Library Expression (ORACLE), uses created T7 acceptor phages that have the tail fiber locus modified by adding CRE recombinase sites which flank the region.

Optimized recombination has the phages infect E. coli which have plasmids that constitutively produce CRE as well as having a tail fiber locus variant flanked by CRE recombination sites. Once the phage genome is in the cell, site-specific recombination occurs, and the tail fiber locus variant is inserted into the phage genome. This occurs many times with each E. coli plasmid with a tail fiber locus variant being slightly different with single amino acid substitutions of all 19 non-synonymous and 1 nonsense substitution at each codon spanning residue position.

These recombined phages are accumulated by enriching them through the elimination of non-recombined phage genomes. This occurs when progeny phages infect E. coli with a Cas9 and gRNA that targets the fixed sequence introduced into the original acceptor phages. The Cas9 gRNA cuts the non-recombined acceptor phage sequence ensuring only the recombined phages are able to replicate and accumulate.

These phages are then free to replicate on E. coli and create a full library of different variants where the genomes can be sequenced and the distribution of these sequences analyzed for use in studying important variants on the RBP on the tail fiber. The library of phages infected the 3 E. coli hosts BL21, BW25113, and 10G. After 4 infection cycles, variants were given scores based on the variants’ relative abundance after selection to before infection. Variant effectiveness was calculated using the formula shown:

Formula for showing variant effectiveness
Figure 1: Formula for calculating FN for determining whether the substitution was intolerant, tolerant, or functional. Source: Dylan Woolsey

WT are wildtype T7 phages that the variants phages are based on. If FN was less than 0.1 (less than 10% as effective as wildtype) the variant was considered to be depleted. If FN was between 0.1 and 2 (10% to twice as effective as wildtype) the variant was considered to be tolerated. If FN was greater than 2 (twice as effective as wildtype) the variant was considered to be enriched. The results of the variant effectiveness can be viewed on the heat map where darker reds represent amino acid variants at a position on the tail fiber RBP that are more enriched.

Structure of the tail fiber region and heat map
Figure 2A: The structure of the tail fiber region. 2B: Heat map showing FN values of amino acid substitutions at each position along the tail fiber. Edited from original graphic. Source:

The uses of the results created by ORACLE

Important deductions can be made from these results. For instance, there is a tradeoff between effectiveness on a host and host range. This is further supported by the top enriched variants for each host being very different from one another.

Other structural information about what contributes to the ability to bind to bacteria can be gained from these results, such as there was depletion of hydrophobic amino acids and enrichment of large and hydrophilic amino acids on variants infecting 10G.

Besides broader information about variants, more specific information can be found from the results. For example, exterior loops in the tail tip have a greater proportion of functional substitutions (ones that are depleted in 1 variant and not in another) seen in the figure below where also shown are intolerant variants (being depleted in all hosts) and tolerant variants (being tolerated in all hosts).

Intolerant, tolerant, and functional substitutions
Figure 3: Proportion of each position on the tail fiber having intolerant, tolerant, and functional substitutions. Source:

In addition to the addition of structural knowledge gained through ORACLE, the use of the library and subsequent ability to characterize each variant has been proven to be valuable tools in combatting resistant bacteria. When the researchers deleted LPS regions of bacteria, they were able to use the library to find variants able to infect these strains. As well, with a UTI E. coli strain known to develop phage insensitivity rapidly, using the library the team observed insensitivity was greatly delayed and bacterial load was decreased compared to wildtype.

From these tests, specific effective variants were also found. Since these are known variants, more information about effective tail tip RBPs can be learned by studying what is unique structurally about these variants.

Overall, the use of the ORACLE method provides the ability to learn more about phage variants structurally through amino acid changes as well as helping develop a better understanding of what makes effective bacteriophages for phage therapy.

Further Reading

Phil Huss, Anthony Meger, Megan Leander, Kyle Nishikawa, Srivatsan Raman (2021), Mapping the functional landscape of the receptor binding domain of T7 bacteriophage by deep mutational scanning, eLife 10:e63775.

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