SEA-PHAGES Comprehensive Study Notes

Chapter 1: Welcome

  • SEA-PHAGES stands for Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science.
  • Partners and participants include student researchers, faculty, and support staff from more than 100 colleges and universities; global collaboration.
  • Funded by the Howard Hughes Medical Institute; lead scientist is Professor Graham Hatfull (University of Pittsburgh).
  • Role of SEA-PHAGES student researchers: discover new bacteriophages and analyze their genomes; contribute to understanding phage diversity, evolution, and genetics.
  • Program outputs: peer‑reviewed publications with student co‑authors; aim to increase undergraduate interest and retention in biology via authentic research.
  • Bacteriophages (phages): viruses that infect bacteria; practical applications include therapeutic agents against antibiotic‑resistant bacteria and uses in veterinary and food safety contexts.
  • Overview of the scientific workflow: two primary components
    • Phage discovery (isolation, purification, amplification, visualization by electron microscopy, DNA extraction, genome sequencing).
    • Genome analysis (bioinformatic identification of genes, regulatory elements, and genomic features).
  • The Phage Discovery Guide provides detailed, modular protocols for de novo isolation and characterization, plus explanations of experimental rationale.
  • Expanding scientific knowledge: the phage population is vast, dynamic, and ancient; estimated phage particles in the biosphere: 103110^{31}; fewer than roughly 5,0005{,}000 have been characterized genomically.
  • Authentic scientific research hallmarks: scientists do not know the answer before experiments; you formulate hypotheses, design experiments, and interpret data.
  • Resources available to students: databases, websites, scientific articles, experienced scientists, and a large SEA‑PHAGES community.

Chapter 1: Overview of the Guide and Workflow

  • This guide is organized as chapters focused on stages of phage isolation and characterization; each chapter contains step‑by‑step protocols, reagents, and troubleshooting.
  • Protocols are modules; you may choose among modules for your task; some protocols are used multiple times.
  • Chapter 12 (Toolbox) contains additional optional protocols.
  • When using online format, protocols are interconnected via hyperlinks and flowcharts.
  • Recommendation: read an entire protocol before starting; tips and troubleshooting are at protocol ends.

Chapter 1: Expanding Scientific Knowledge and the Authenticity of Research

  • Phage discovery is vast and diverse; authentic research includes occasional failed experiments and the need for problem solving.
  • You will encounter times when experiments do not work on the first try; persistence and adaptation are part of the process.

Chapter 2: Lab Basics

  • An overview of performing scientific research in a safe laboratory environment.
  • Key topic: fundamental lab safety rules and lab etiquette critical to safety and success.
  • Core universal lab safety guidelines include:
    • No eating or drinking; no open‑toed shoes; never work alone; tie back long hair; avoid baggy clothing; never leave a lit Bunsen burner unattended; keep workspace clean; know location of safety equipment (fire extinguishers, eyewash, showers).
    • Proper waste disposal; do not pour all waste down the drain; no mouth pipetting; wear safety glasses at all times; wear gloves when handling bacteria; wash hands before leaving lab.
  • Working with Actinobacteria (the primary course organisms) involves specific safety considerations:
    • Actinobacteria strains used in the course are generally safe for individuals with healthy immune systems; immunocompromised or pregnant individuals should consult a physician.
    • Primary hazards arise from exposure via mucous membranes, broken skin, or ingestion.
    • PPE: safety glasses, gloves; avoid generating aerosols; wash hands; seek instructor guidance for health concerns.
  • Record keeping and autonomy in replication: the principle of autonomous replication requires a detailed lab notebook that allows others to repeat procedures and obtain the same results.
  • Laboratory notebook outline (minimum information): 1) Date; 2) Title; 3) Aims and purpose; 4) Procedures and protocols (detailed, bulleted, including any changes); 5) Results (primary data, negative results, figures with legends); 6) Analysis and interpretation (explicit statements of interpretation); 7) Future plans; 8) Organization and storage of data; 9) The institution retains the notebook for at least seven years.
  • Labeling and traceability: all samples (Petri plates, tubes, reagents) should be labeled with name/initials, date, contents, and relevant dates.
  • Beyond notebooks: aseptic technique, contamination prevention, and bench organization.

Chapter 2: Aseptic Technique and Contamination Prevention

  • Sterility concepts: sterilization (autoclaving) and aseptic technique to avoid contamination.
  • Common sterilization methods:
    • Autoclaving: steam at 121°C and 18 psi; materials are marked post‑autoclave (white/striped tape).
    • Filtration for heat‑sensitive liquids (filter sterilization).
  • Disinfectants for surfaces: 70% ethanol, bleach (10% solution), and phenolics (e.g., CiDecon™).
  • Cleaning rationale: many phages are resistant to 70% ethanol; bleach/phenolics are preferred for decontaminating phage‑contaminated materials.
  • Aseptic technique essentials:
    • Organize the bench; disinfect surfaces; never pass hands over open vessels; keep lids closed; keep microcentrifuge tubes closed when not in use.
    • Never reuse pipette tips; always flame the neck of glass containers when opening/closing to sterilize the opening air flow.
  • Bunsen burner use and updraft concept:
    • Flame creates an updraft that helps keep the work area aseptic by pulling contaminants away from the work surface.
    • Work inside the updraft cone; keep supplies close to the flame but avoid leaving flames unattended.
  • Specific aseptic technique exercises (Protocol 2.1):
    • Steps include disinfecting surfaces with CiDecon and 70% ethanol, igniting the Bunsen burner, transferring liquids aseptically, and maintaining an aseptic field.
    • Important cautions: gloves may melt if exposed to flame; handle open flames with care; do not leave flame unattended.
  • Bench top management and disposal:
    • Keep essential items within the aseptic zone; wipe spills immediately; dispose of all waste according to protocol and instructor guidance; disinfect bench before leaving.

Chapter 3: Phage Basics

  • What is a phage?
    • A phage is a virus that infects bacteria and cannot replicate without a bacterial host; host range is typically narrow, sometimes spanning a few strains or species, but rarely across distant taxa.
  • Bacteriophages in the biosphere:
    • Phages are the most abundant biological entities in the biosphere; global phage population is vast ($10^{31}$) and genetically diverse; only a small fraction have been genomically characterized.
  • Historical context:
    • Discovery around 1915–1917 by Twort and D’Herrelle; plaque assay introduced by D’Herrelle that visualizes phage activity as plaques on a bacterial lawn (clear zones where bacteria have been lysed).
  • Phage structure and genome basics:
    • Most phages are tailed dsDNA phages (order Caudovirales) with a head (capsid) and tail; genome sizes range typically from ~50 kb to several hundred kb; many proteins per virion—around 20 different protein types in a single virion.
    • Some phages have ssDNA or RNA genomes; others may be enveloped or have multiple chromosomes or plasmids.
  • Lytic vs temperate lifestyles:
    • Lytic (virulent) phages undergo the lytic growth cycle ending in host lysis and progeny release.
    • Temperate phages can choose between lytic growth and lysogeny, wherein the phage genome integrates into the host genome (prophage) or exists as a plasmid; lysogens are resistant to superinfection by related phages due to a genetic switch and immunity repressors.
  • Phage lifecycle details:
    • Lytic cycle phases: adsorption, DNA injection, genome circularization, replication, production of heads and tails, assembly, and host lysis with burst size often ≥100 phage particles per cell.
    • Early genes vs late genes: early genes include replication/transcription factors; late genes encode structural proteins for virions.
  • Plaque morphology as an initial indicator:
    • Clear plaques typically indicate lytic activity; turbid/ cloudy plaques can indicate temperate phages or other factors affecting plaque appearance; plaque morphology depends on media, cell density, incubation time/temperature, etc.
  • Host range and infection determinants:
    • Host range influenced by receptor availability, compatibility with host transcription/translation systems, and the propensity for host range mutations in phage genomes.
    • Defense systems in bacteria include restriction enzymes, AB toxins, CRISPR, abortive infection systems; phages counter these with anti‑restriction and anti‑CRISPR genes.
  • The “Great War” analogy:
    • Phage–bacteria dynamics are rapid and continuously evolving due to selective pressure; this drives diversity and coevolution in phage populations.

Chapter 3: Phage Host Range and Immunity Mechanisms

  • Immunity, exclusion, and resistance concepts:
    • Immunity: lysogen expresses repressor proteins that prevent infection by related phages (superinfection immunity).
    • Exclusion: prophage alters host surface to prevent adsorption of other phages.
    • Resistance: bacterial defense systems (e.g., restriction, CRISPR, abortive infection) that prevent phage infection.
  • Superinfection immunity mechanics:
    • Immunity repressor binds to operator sequences and represses lytic gene expression; can also bind invading phage DNA to block lytic genes.
  • Prophage integration and maintenance:
    • Prophage can integrate into host chromosome via homologous recombination or exist as an autonomous extrachromosomal plasmid with partitioning systems.
  • Lysogeny characteristics:
    • Lysogens are cells carrying a prophage; prophage can be induced to enter lytic cycle under stress; lysogens typically show immunity to related phages.

Chapter 4: Host Basics

  • Bacteria overview:
    • Bacteria are prokaryotes, single cells, with a cytoplasmic membrane and a cell wall; some have outer lipid envelopes and capsules; some have pili or flagella.
    • Genome: typically 2–10 Mbp; transcription and translation are coupled in real time in bacteria.
  • Bacterial growth concepts:
    • Reproduction by binary fission; growth in liquid media shows lag, log, stationary, and death phases; doubling time varies by species and conditions.
    • Colony formation on solid media arises from a single bacterium, producing clonal populations.
  • rRNA and phylogeny:
    • Ribosomal RNA sequences are used for phylogenetic relationships among bacteria; conserved regions enable phylogenetic trees.

Chapter 4: Host Profiles (Host Bacteria) – Examples and Growth Parameters

  • Arthrobacter globiformis (host for some SEA‑PHAGES projects)
    • Growth media: PYCa; growth temp: 30–37°C; cycloheximide (10 μg/mL) to suppress fungi; typical colony color white to tan; capsule‑rich growth produces rapid lawns; ~3 days for a colony from a single cell; ~30% isolation success via soil enrichment with Arthrobacter globiformis.
    • Restriction enzymes: BamHI, HaeIII, MseI, NspI, SacII; viable phage yields when enriching soil samples.
  • Arthrobacter sp. KY3901 (host for isolation)
    • Growth temp: 22–28°C; not growth at 37°C; colonies tan to yellow; similar enrichment yields; ~40% success rate for phage isolation from soil.
  • Gordonia rubripertincta and Gordonia terrae; Microbacterium foliorum; Microbacterium testaceum; Mycobacterium smegmatis mc2155
    • Each host has specific growth media (often PYCa), temperatures, antimicrobials (cycloheximide; spectinomycin where applicable), and phage buffer formulations; growth patterns, colony colors, and times to saturation are provided for planning experiments.
  • Mycobacterium smegmatis mc2155 (common SEA‑PHAGES host)
    • Growth media: 7H9 (with AD) and Tween80; temperatures: 37°C (fast growth) or 30°C (slower); colony morphology: tan‑white, dry and wrinkled; suspension culture can take 3–5 days for saturated growth.
    • Phage enrichment success rate from soil: approximately 60% (varies by host).

Chapter 5: Isolation (Overview) and Protocols

  • Overview: isolation of phages from environmental samples can be direct or enriched isolation; direct isolation detects phages present in a sample; enriched isolation amplifies phages by incubating with host bacteria in growth media.
  • Direct Isolation (Protocol 5.2):
    • Mix environmental sample with host bacteria and top agar to detect plaques; use plaque assay (Protocol 5.3) to visualize plaques on lawns.
  • Enriched Isolation (Protocol 5.5):
    • Mix environmental sample with host bacteria in liquid culture; incubate to amplify phages; filter to remove bacteria; assay supernatant with spot test (Protocol 5.6) for plaques.
  • Plaque Assay (Protocol 5.3):
    • Combine host bacteria with phage sample in soft top agar; pour onto solid agar; incubate 24–48 hours; plaques indicate phage presence; each plaque originates from a single phage particle.
  • Picking a Plaque (Protocol 5.4):
    • Circle selected plaques on the bottom of the plate; transfer plaque contents to phage buffer for subsequent use; this is the starting point for purification.
  • Spot Test (Protocol 5.6):
    • Spot dilutions or lysates on a young lawn of host bacteria to observe clearing zones; helps screen multiple samples efficiently; a positive spot test indicates presence of phage.
  • Workflow summary (Fig. 5.0‑1): Collect environmental samples → Direct isolation +/− Plaque assay → Purification steps (plaque picking, spot test) → Purification confirmation and downstream work (phage purification for sequencing, TEM, DNA extraction).

Chapter 5: Collecting Environmental Samples (Protocol 5.1)

  • Sample collection:
    • Solid samples: use a cleaned bag, collect soil, and place in bag; seal and preserve.
    • Liquid samples: collect and label; fill to appropriate volume.
    • Record GPS coordinates and descriptive physical characteristics.
  • Documentation:
    • Record collection site, GPS coordinates, and environmental characteristics in the lab notebook and program database.
  • Practical notes:
    • Fresh samples yield higher phage recovery; keep samples cool; soil depth and moisture influence aerobic bacterial abundance.

Chapter 5: Enrichment and Direct Isolation Details (Protocols 5.2–5.6)

  • Direct Isolation (5.2):
    • Prepare 15 mL conical tubes with the solid sample in liquid media; shake to release phages; allow settling of solids; filter if necessary; proceed to Plaque Assay (5.3).
  • Enriched Isolation (5.5):
    • Use solid soil samples; enrich by incubating with host bacteria in liquid media; after incubation, filter lysate through a 0.22 μm filter; proceed to Spot Test (5.6).
  • Plaque Assay (5.3):
    • Inoculate host bacteria with phage samples; add molten top agar; overlay on agar plates; incubate 24–48 h; count plaques; identify morphology; prepare for purification.
  • Spot Test (5.6):
    • Plate host lawn; apply spotted lysates; incubate; zones of clearing indicate phage presence; if multiple hits or unclear spots, test further (plaque assay) to confirm.

Chapter 6: Phage Purification

  • Goal: obtain a homogeneous, clonal phage population from a mixed plaque collection.
  • Rationale: a plaque arises from a single phage particle; purify by diluting such that plaques are well separated, then verify consistency across dilutions.
  • Number of rounds: generally up to 3 rounds of purification to minimize mutation accumulation; some phages may retain multiple morphologies across rounds; in such cases, pick plaques from different morphologies and test separately.
  • Flow for purification (summary): Direct isolation plaque or enriched lysate → Plaque assay → Pick a single well‑isolated plaque → Serial dilutions → Plaque assay → If morphologies are consistent across plates, proceed to Collecting Plate Lysates; otherwise repeat purification.
  • Important caveats:
    • Phage diffusion can cause neighboring plaques to contaminate the picked plaque; pick from well‑isolated plaques (≥1.5 cm apart).
    • If multiple morphologies persist, you may have multiple phages; treat them as separate samples and test separately.
  • Outputs: a high titer, clonal lysate suitable for DNA extraction and TEM.

Chapter 6: Plaque Assay for Purification (Protocol 6.1) and Serial Dilutions (Protocol 6.2)

  • Plaque assay for purification (6.1):
    • Combine diluted phage with host bacteria and top agar; plate and incubate; identify well‑isolated plaques; pick plaques for purification.
  • Serial dilutions (6.2):
    • Prepare tenfold serial dilutions (10^-1, 10^-2, …, 10^-8); use 90 μL buffer per tube; transfer 10 μL per step; safe pipetting to ensure accurate dilutions.
  • Spot titer (6.4) and Full plate titer (6.5):
    • Spot titer uses a single plate to estimate titer across multiple dilutions; full plate titer uses multiple plates for accurate plaque counts.
    • Titer formula (used in 6.4/6.5):
      ext{Titer (pfu/ml)} = rac{N}{V} imes 10^{3} imes D
      where N = plaques counted, V = volume plated in μL, D = dilution factor (reciprocal of dilution).
  • Webbed plates and lysate collection (6.3):
    • Webbed plates produce highly concentrated lysates; flood plates (8 mL phage buffer per plate) to collect lysate; filter to remove debris; store lysates at 4°C; plan to archive later.
  • Output quality indicators:
    • High titer (>5×10^9 pfu/mL is desirable for downstream DNA extraction and TEM).
    • Purified phage should yield uniform plaque morphology across dilutions after purification rounds.

Chapter 7: Phage Amplification and Archiving

  • Making webbed plates (Protocol 7.1):
    • Use titered lysate to plate at appropriate dilution to yield densely packed, conuent plaques (webbed plates).
    • Typically yields around 4 mL of highly concentrated lysate per webbed plate; plan to harvest 8–10 mL total by using 2–3 webbed plates.
    • Volume calculations rely on prior plaque counts and lysate titer; bracket experimental conditions by testing multiple phage counts (e.g., 5× bracket around estimated optimum).
  • Entering data into PhagesDB (Protocol 7.2):
    • Create a phage entry with GPS coordinates (decimal degrees), discovery location, host, and isolation type (direct vs enriched).
    • Include all key data: plaque morphology, genome details after sequencing, TEM images, and other supporting data when available.
  • Archiving your phage sample (Protocol 7.3):
    • Archive at University of Pittsburgh and at your institution using barcoded tubes and DMSO as cryoprotectant; recommended titer ≥ 5×10^9 pfu/mL for archiving.
    • Prepare DMSO/phage lysate mixtures (e.g., 4 mL lysate + 280 μL DMSO) and aliquot into barcoded tubes; store at −80°C; maintain 4°C working stocks for ongoing studies.

Chapter 8: Electron Microscopy (TEM)

  • Purpose: visualize phage morphology at high resolution to classify phage families (e.g., Myoviridae, Siphoviridae, Podoviridae).
  • TEM basics:
    • Sample preparation involves concentrating phage, purifying away debris, placing on copper grids coated with carbon/formvar, and staining with a negative stain (uranyl acetate) or lanthanide stains.
    • The grid must not dry out during staining; use staining steps carefully to preserve capsid integrity.
    • Scale bars on micrographs; measure capsid diameter and tail length; compare to standard sizes and to other phages.
  • Protocols:
    • Protocol 8.1a: Mounting and staining with Uranyl Acetate (traditional method).
    • Protocol 8.1b: Mounting with Pelco tabs (alternative mounting method).
    • Protocol 8.1c: Lamplight (paraffin drop) method (surface tension and floatation technique).
  • Key measurements:
    • Determine capsid diameter and tail length using a known scale bar (e.g., 100 nm) and calculate the scaled tail length using a simple ratio:
      rac{( ext{size bar}){ ext{measured}}}{ ext{scale bar}} = rac{( ext{tail length}){ ext{actual}}}{( ext{tail length})_{ ext{measured}}}
    • Image quality matters: avoid grid drying, ensure proper glow discharge for hydrophilicity, and maintain grid integrity.
  • Outputs: TEM images that support morphotype assignment and genome interpretation; log images in PhagesDB with appropriate metadata.

Chapter 9: Phage DNA Extraction

  • Objective: extract high‑quality phage DNA suitable for restriction digest and sequencing.
  • Rationale:
    • Lysates contain bacterial DNA/RNA; nucleases are used to degrade contaminating nucleic acids while phage DNA is protected within the capsid.
    • DNA is released by denaturing agents and then purified with a resin/column system (e.g., Wizard DNA Clean‑Up kit) to obtain clean phage DNA.
  • Two main PEG/ZnCl2 pathways (for concentration priors):
    • PEG precipitation (Protocol 9.1 and 9.2a): concentrate phage by precipitating with PEG; resuspend in ddH2O; lysate is processed through DNA resin and column cleanup.
    • Zinc chloride precipitation (Protocol 9.2b): concentrate phage with ZnCl2 precipitation; follow dissolution and DNA extraction similar to PEG method.
  • Nuclease treatment (RNase/DNase): degrade host nucleic acids; optional EDTA to inactivate nucleases; optional Proteinase K and SDS to degrade residual proteins; incubation conditions vary by protocol.
  • Key steps in DNA extraction (Protocol 9.1):
    • Prepare high‑titer lysate (≥5×10^9 pfu/mL).
    • Add nuclease mix; incubate; optional EDTA; optional Proteinase K and SDS; incubate.
    • Denature the phage capsid using DNA resin (guanidinium thiocyanate) to release DNA.
    • Bind DNA to resin and load onto Wizard columns; wash with multiple 80% isopropanol washes; elute with hot water (≈90°C).
    • Measure DNA concentration and store at 4°C (short term) or −20°C (long term).
  • DNA integrity and troubleshooting tips:
    • Avoid nuclease contamination; use dedicated work areas and pipettes for nuclease steps.
    • Ensure fresh isopropanol; residual guanidinium must be removed to prevent interference with downstream quantification and gel loading.
    • Heavy emphasis on avoiding column damage when pipetting; use reinforced tubes for high‑g centrifugation steps.
  • Alternative DNA extraction (Protocol 9.2b): ZnCl2 pathway with Pelco tabs or other grid support; ensure no disruption of the column and maintain proper vacuum on syringe handling.
  • Outputs: High‑quality phage genomic DNA suitable for restriction digestion and sequencing; quantify using spectrophotometry or fluorometry; store appropriately.

Chapter 10: Restriction Enzyme Digestion and Gel Electrophoresis

  • Purpose: generate a genetic fingerprint of the phage genome by digesting with restriction enzymes and separating fragments by gel electrophoresis.
  • Restriction enzymes and buffers:
    • A panel of enzymes (e.g., BamHI, HaeIII, MseI, NspI, SacII, ClaI, EcoRI, HindIII, etc.) with specific buffers; isoschizomers may substitute for listed enzymes.
  • Key concepts:
    • A restriction digest cuts the phage genome at specific 4–6 bp sequences; number and size of fragments depend on the genome’s restriction sites.
    • A single enzyme digest yields a set of DNA fragments; multiple enzymes yield a fingerprint useful for comparing phages.
  • Gel electrophoresis (Protocol 10.3):
    • Cast 0.8–2% agarose gels; mix samples with 6× loading dye; heat to dissolve; load ladder and samples into wells; run at a constant voltage (e.g., 100 V) until separation is sufficient.
    • Include a DNA ladder (size markers) to estimate fragment sizes; a standard curve can be generated to convert migration distances to bp.
  • Data interpretation (Protocol 10.4):
    • Determine whether each enzyme produced fragments; compare fragment sizes to known phages; infer genome features; assess completeness of digestion and potential nuclease contamination.
  • Common considerations:
    • Load dyes, such as bromophenol blue, help monitor sample migration.
    • Ethidium bromide (EtBr) vs safer alternatives (e.g., SYBR Safe) for DNA visualization; handle mutagenic dyes with gloves.
    • Use a standard curve to estimate fragment lengths; maintain consistent gel conditions for comparability.
  • Outputs: Restriction fingerprint data and gel images; data to support genome annotation and comparison in PhagesDB.

Chapter 11: Lysogens, Efficacy of Lysogeny, and Related Assays

  • Lysogeny and the biology of temperate phages:
    • Lysogens carry a prophage; prophage maintenance can involve integration into the host chromosome or stable extrachromosomal plasmids; immunity repressors prevent superinfection by related phages.
  • Protocol 11.1–11.6 overview:
    • 11.1: Creating Lysogens by Streaking from a High Titer Spot; isolate lysogens via streak purification after spotting a high‑titer phage sample;
    • 11.2: Creating Lysogens & Determining Efficacy of Lysogeny using Phage‑Seeded Plates; quantify the efficiency of lysogeny (EOL) using phage‑seeded plates and dilution series;
    • 11.3: Verification of Potential Lysogens via Patch Assay; verify lysogens by patching onto a host lawn and observing phage release; patch plates include an lysogen‑only plate and an experimental plate with host lawn to detect lysis caused by phage release;
    • 11.4: Verification via Liquid Phage Release Assay; detect phage release from lysogens in liquid culture; use spot titers to quantify phage present in supernatant;
    • 11.5: Sensitivity Assay; test lysogen susceptibility to a panel of phages; calculate plating efficiency and EOP (efficiency of plating);
    • 11.6: Host Range Assay; test phage infection across alternative hosts; compute EOP as titer on test species divided by titer on the isolation host.
  • Key concepts:
    • EOL (Efficiency of Lysogeny): ratio of CFU on phage‑seeded plates to CFU on control plates, expressed as a percentage; ranges from 0.1% to 100% depending on temperate phage behavior.
    • EOP (Efficiency of Plating): ratio of phage titer on a test host to the titer on the isolation host; used to characterize host range and infection efficiency across hosts.
    • Kiling from without: high phage concentration can lyse cells without productive infection; distinguish from true lysogeny by observing plaques and lysogen formation on multiple dilutions.
  • PCR confirmation: a gold standard for confirming prophage integration if attB/attP junctions are identified; alternative confirmation via repeated purification and sequencing is possible.

Chapter 12: Toolbox

  • Protocol 12.1 Making Top Agar (1X Middlebrook Top Agar) for Mycobacteria (M. smegmatis)
  • Protocol 12.2 Plaque Streak Plates
  • Protocol 12.3 Taking Plaque Pictures
  • The toolbox provides practical alternatives and supplementary techniques to support the main protocols, especially when working with Mycobacteria.

Chapter 13: Bioinformatics, Genomics, and Genome Interpretation

  • Bioinformatics overview:
    • Bioinformatics uses computer tools to interpret biological data; in SEA‑PHAGES, used to locate genes, predicted promoters, and regulatory elements; to understand genome organization and function.
  • The genome and its organization:
    • Phage genomes are primarily dsDNA; most actinobacteriophages have a single chromosome; genomes range from ~15 kb to ~250 kb; typical phage genes number from tens to a few hundred.
  • The Central Dogma recap:
    • DNA -> RNA -> Protein; transcription and translation are coupled in bacteria and phages; transcription is mediated by RNA polymerase and promoters; translation is carried out by ribosomes with ribosome binding sites (RBS/ Shine‑Dalgarno sequences).
  • Promoters and transcriptional control:
    • Promoters often contain -10 and -35 motifs, differing across promoters; stronger promoters have sequences closer to consensus.
    • RNA polymerase holoenzyme with sigma factors recognizes promoter types; phage promoters can differ from bacterial promoters.
  • Open reading frames (ORFs):
    • ORFs encode proteins; start codons (AUG, and less commonly GUG/UUG in bacteria/phages); RBS (Shine‑Dalgarno sequence) enables initiation; stop codons mark termination (UAA/UAG/UGA in RNA; TAA/TAG/TGA in DNA).
  • Operons and transcriptional organization:
    • Phage genomes are often organized into operons with a single promoter and terminator controlling multiple ORFs.
  • Reading frames and frameshifts:
    • There are six reading frames in dsDNA (three frames on each strand); translation begins at start codons with the reading frame determined by the ribosome; frames determine the amino acid sequence.
  • Genome annotation and comparative genomics:
    • Annotation predicts gene locations and functions; comparative genomics compares gene content and genome architecture across phages to infer gene function and evolution; bioinformatics helps identify similarities to known proteins and operons.
  • Phage genomics databases:
    • PhagesDB, SEA‑PHAGES repositories, and GenBank submission are used for data sharing; GPS coordinates and metadata are required for GenBank submissions and sequencing reporting.

Chapter 14: Practical Tools and Data Management

  • Data handling and databases:
    • PhagesDB: phage data entry, genome submission, and thumbnail images; morphological, genetic, and metadata records are stored.
    • GPS coordinates: conversion from degrees/minutes/seconds to decimal degrees; example: 40°26′46″ N, 79°57′11″ W → 40.446111, -79.953056 (decimal degrees).
  • Reagents and recipe cards:
    • A wide catalog of common reagents (CaCl2, MgSO4, EDTA, Tris buffers, PEG 8000, ZnCl2, SDS, glycerol) and their prepared stock solutions with final concentrations; specifics for preparing 7H9 media, PYCa media, and top agar.
  • Safety data sheets and hazard awareness:
    • Uranyl acetate is a highly toxic stain; handle with care; alternative stains (lanthanides) available; always use PPE and follow institutional guidelines.
  • Data workflows and notes:
    • Document titer calculations, plaque morphologies, and TEM measurements; log plate counts, serial dilutions, and standard curve data; keep a detailed, well‑organized lab notebook.

Chapter 13: The Central Dogma, Genes, and Basic Molecular Biology in Phages

  • Genomic fundamentals:
    • DNA structure and base pairing: A–T, G–C; double helix; nucleotides with sugar‑phosphate backbone.
  • The central dogma in phages:
    • DNA -> RNA -> Protein; transcription via RNA polymerase; translation via ribosomes; promoters and terminators govern transcription initiation and termination.
  • Gene structure basics:
    • Promoters, terminators, and ORFs; Shine-Dalgarno site enables ribosome binding; codons encode amino acids via the genetic code.
  • Reading frames and transcriptional initiation:
    • Three reading frames per strand; two strands yield six possible reading frames; initiation codons and start tRNA (fMet) ensure correct initiation of translation.
  • Operons and polycistronic transcription:
    • Bacterial and phage genes often organized into operons; single transcripts containing multiple ORFs; regulatory coordination via a single promoter/terminator.
  • Practical applications of molecular biology in SEA‑PHAGES:
    • Use of restriction enzymes for genomic fingerprinting; PCR for prophage confirmation; sequencing for genome annotation; bioinformatics for function prediction and comparative genomics.

Chapter 14: Bioinformatics Tools, Data Interpretation, and Sequencing Prep

  • Bioinformatics tools and approach:
    • Use multiple tools to obtain consistent predictions; interpret results as predictions, not absolute facts; data from public databases should be evaluated critically.
  • Sequencing and genome annotation:
    • Genome sequencing provides the complete phage genome; annotation predicts gene locations and functions; experimental validation (mutants, expression studies) strengthens annotation.
  • Data interpretation and reporting:
    • Integrate genomic data with phenotypic data (plaque morphology, host range, lysogeny); document uncertainties and limits of predictions.
  • Practical data management:
    • Maintain traceability of samples across workflows; link TEM images, plaque pictures, restriction digest patterns, and sequencing results to a single phage entry in PhagesDB/SEA‑PHAGES databases.

Chapter 15: Appendices and Practical References

  • Recipes and stock solutions: detailed formulations for AD supplement, PYCa media, CaCl2 stocks, MgCl2, ZnCl2, EDTA, glycerol, isopropanol, PEG 8000, uranyl acetate (and alternatives), Tween 80, and other reagents.

  • Autoclave settings and safety notes: common cycles (liquid, wrapped, and unwrapped); typical cycle times and temperatures; safety cautions for autoclave use.

  • Lab safety and waste disposal cross‑references: instructions for proper disposal of biological waste and decontamination, with emphasis on phage handling and disinfection strategies.

  • LAB NOTES: The guide repeatedly emphasizes comprehensive documentation, careful labeling, and adherence to safety practices, with detailed protocols for each stage of phage discovery, purification, analysis, and archiving.

  • References to numerical references and formulas used throughout the guide:

    • Phage particle population estimates: 103110^{31} particles worldwide; genomic characterization to date: ~5,000 genomes; etc.
    • Plaque assay titer formula (for dilutions and plating volumes):
      ext{Titer (pfu/ml)} = rac{N}{V} imes 10^{3} imes D
      where N is plaques counted, V is volume plated in μL, and D is the dilution factor (reciprocal of the dilution).
    • Lysate titers and webbed plate calculations: bracket around target plaque counts (e.g., ~thousands of plaques) and substitute with measured plaque counts from prior plaques; use titer to determine required lysate volumes and dilutions for webbed plates.
    • GPS coordinate conversion example: 40° 26′ 46″ N, 79° 57′ 11″ W → 40.446111, -79.953056 (decimal degrees).
    • Colony counts and growth phases: lag, log, stationary, and death phases; doubling times range widely depending on host and conditions; typical colony counts and culture densities for lawn formation are discussed in host chapters.

Chapter 16: Summary of Key Concepts and Practical Guidelines

  • SEA-PHAGES integrates discovery with genomics to explore phage diversity, evolution, and genetics, with emphasis on undergraduate participation and career development.
  • The phage discovery workflow consists of phage isolation (direct or enriched), purification (via plaque purification), DNA extraction, genome sequencing, and comparative analyses.
  • TEM, phage DNA extraction, and restriction digest gel analysis provide phenotypic and genotypic data that inform genome annotation and functional prediction.
  • Host ranges and lysogeny dynamics reveal phage–bacteria interactions, including immunity and resistance mechanisms.
  • Protocols emphasize aseptic technique, careful documentation, standardized lab notebooks, and proper data management in public databases (PhagesDB, SEA‑PHAGES databases).
  • The program provides a robust framework for students to participate in authentic research, develop problem‑solving skills, and contribute to the broader phage biology field.