With more than 800 sequenced mycobacteriophages, we are keen to widen our net to include phages isolated on other hosts within the phylum Actinobacteria. This includes phages to hosts such as Arthrobacter, Gordonia, and Rhodococcus.
As means towards this exciting goal, SEA-PHAGES teams will pilot new Actinobacteria hosts in 3 phases, with each phase informing the next towards the ultimate goal of establishing best practices to assist phage-hunting efforts across the SEA.
SEA-PHAGES faculty can volunteer to participate at any or all steps of this endeavor. The phases are described below.
Click here to see hosts currently in the SEA-PHAGES pipeline.
Phase 1: Identification
The Identification phase is the first step in adding hosts to our collection. The goal of this phase is to determine whether a host is generally suitable for a classroom phage-hunting environment. This phase may be carried out by the Hatfull lab at the University of Pittsburgh, the HHMI SEA-phages group, or any institution within the SEA-PHAGES program.
You can participate in this phase by choosing an Actinobacterial host to work on, and determining the following:
- Growth parameters (media, temperature, incubation time, colony morphology)
- Frequency of phage discovery using enrichment
Be aware that hosts in the Identification phase may have only been preliminarily tested (i.e. colonies and lawns can be grown on solid media). This phase of testing may therefore be best suited for independent lab projects. If you are interested in choosing an Actinobacteria host other than those being piloted, we recommend that you work with hosts that meet the following criteria.
- Is a BSL1 Actinobacteria
- Has growth conditions compatible with a classroom schedule
- Grows easily as a lawn
- Can be distributed to, or is available to, the SEA-PHAGES community
Phase 2: Piloting
Hosts may move into the Piloting phase when they have been sufficiently characterized that basic guidelines for obtaining plaques have been determined. The goal of this phase is to test the host in a number of different implementation environments, and gather extensive data on using the host in SEA-PHAGES classrooms. This phase is generally coordinated by SEA-PHAGES leadership and involves a number of institutions working on the same host during the same academic year.
If you choose to participate in the Piloting phase, you will be testing following parameters:
- Phage Infection (enrichment, plaque formation/morphology, buffers, titers, storage)
- Molecular Biology (DNA extraction, restriction analyses, sequencing, electron microscopy)
Phase 3: Validated
Validated hosts are now ready for widespread use in SEA-PHAGES classrooms. Standardized protocols based on the Piloting data are available to supplement the Resource Guide, and protocols are posted on PhagesDB.
Guidelines for host selection
New SEA-PHAGES Faculty We strongly encourage all faculty members that are new to the SEA-PHAGES program to have one year of successful course implementation using Mycobacterium smegmatis mc2155 prior to working with new hosts. A successful year will entail isolation of phages through submission of at least one annotated genome to GenBank.
Current SEA-PHAGES Faculty Faculty members that have previously implemented the SEA-PHAGES program are welcome to participate in all phases of developing new hosts. If you decide to test a new host, please email the SEA-PHAGES team at email@example.com with the host(s) of choice and a brief description of how you intend to implement the pilot (e.g. in the lab, or in the classroom).
Working with new hosts
As you begin working with new hosts, we recommend that you follow the instructions for Mycobacterium smegmatis mc2155 as described in the Resource Guide or in the protocols available at PhagesDB, with the following key exception.
- Begin by growing new Actinobacteria in PYCa media at 30°C. The protocol for preparing PYCa media can be found at PhagesDB. Once you are proficient in working with the new host, you may test alternative growth media and temperatures.
Other considerations for working with new hosts can be found here on PhagesDB.
We ask that faculty members working on new hosts keep fastidious notes so that we can establish best practices quickly and efficiently. Data from working with new hosts will be collected using an online Data Collection Form sent from the SEA-PHAGES team. You can review the Data Collection Form here to become familiar with types of information that will be requested.
Ordering information can be found here on PhagesDB.
Mycobacterium smegmatis mc²155
Validated | ATCC: 700084
Rhodococcus erythropolis RIA 643
Piloting | ATCC: 15903
Originally listed on ATCC as Rhodococcus globerulus.