Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

1. Preparation of Multi-phenotype Assay Plate (MAP) Substrates, Basal Media, Pre-growth Media, and Buffer

1. Make 50 ml of 1.0 – 1.25% substrate stocks.
   1. Dissolve 1.0 – 1.25% (w/v) of solid substrate in sterile water (heat if necessary). Filter sterilize solutions with a 0.22 µm filter unit and store stocks at RT. Examples of substrates used in these experiments are provided in Table 2.
2. Make 250 ml of 3x basal media for all substrate classes (carbon, nitrogen, sulfur and phosphorus). The MOPS (3-morpholinopropane-1-sulfonate) based basal media²² contains the following: 1x MOPS (40 mM MOPS + 10 mM Tricine), 0.4% glycerol*, 9.5 mM NH₄Cl*, 0.25 mM NaSO₄*, 1.0 mM MgSO₄*, 1.32 mM K₂HPO₄*, 10 mM KCl, 0.5 µM CaCl₂, 5 mM NaCl, 6 µM FeCl₃, and 0.1 % (w/v) L-arabinose** (Tables 1 and 2).
   Note: *These compounds are not included depending on the basal media. For example, 0.4% glycerol is not used in the carbon basal media and in the sulfur basal media 1.0 mM MgSO₄ is replaced by 1.0 mM MgCl₂. **L-arabinose is specific for induction of pBAD promoter vector, pEMB11, which was transformed into an ara^–E. coli K-12 strain (BW 27784)²³.
   1. Add each component of the basal media to a sterile flask and bring solution to volume. Mix well. With a 0.22 µm filter unit, filter sterilize each basal media into a sterile bottle.
3. Make 500 ml of MOPS pre-growth media. MOPS based pre-growth media²² contains the following: 1x MOPS (40 mM MOPS + 10 mM Tricine), 0.4% glycerol, 9.5 mM NH₄Cl, 0.25 mM NaSO₄, 1.0 mM MgSO₄, 1.32 mM K₂HPO₄, 10 mM KCl, 0.5 µM CaCl₂, 5 mM NaCl, and 6 µM FeCl₃.
   1. Add each component of the pre-growth media to a sterile flask and bring to volume. Filter sterilize with a 0.22 µm filter unit, then add ampicillin at a final concentration of 100 µg/ml. Store solution at 4 °C.
4. Make 500 ml of 0.5x Luria-Bertani (LB) agar plates. To a sterile flask, add LB components to make a 0.5x concentration (1.25 g yeast extract, 2.5 g NaCl, 2.5 g tryptone, 7.5 g agar). Mix well and autoclave to sterilize.
   1. Cool agar, then add 100 µg/ml of antibiotic ampicillin with mixing. Pour ~25 ml of agar into Petri dishes, allow to set, and store at 4 °C until needed.
5. Make 250 ml of 10 mM Tris (pH 7.4) plus 10 mM MgSO₄ buffer solution. Filter sterilize with a 0.22 µm filter unit, and store solution at RT.

2. Preparation of Bacterial Cell-suspension

1. Prepare fresh colonies. From a 12.5% glycerol frozen stock, plate fresh E. coli clones onto 0.5x LB agar containing 100 µg/ml ampicillin. Incubate at 37 °C for 24 hr.
2. Prepare liquid cultures:
   1. Pipette 3 ml of pre-growth media containing 100 µg/ml ampicillin into culture tubes. For each clone, use biological triplicates.
   2. Inoculate independent colonies from section 2.1 into prepared culture tubes. Incubate at 37 °C with shaking for 22 hr.
3. Pellet and wash bacterial cells:
   1. Transfer O/N cultures into 1.7 ml microfuge tubes. Pellet cells via centrifugation at maximum speed (16,900 x g) for 2 min in a microcentrifuge. Decant supernatant and wash cells by re-suspending in 500 µl of 10 mM Tris/10 mM MgSO₄ buffer. Repeat.
   2. Re-pellet cells, decant supernatant and re-suspend cells in 1 ml of 10 mM Tris/10 mM MgSO₄ buffer.
4. Measure cell density and concentrate cells (if necessary):
   1. Dilute cells from section 2.3.3 10-fold in the 10 mM Tris/10 mM MgSO₄ buffer and transfer this dilution into a cuvette. Measure optical density (OD_{600 nm}) of this dilution and record.
   2. Concentrate cells to achieve final concentration of 0.07 (OD_{600 nm}) in the MAPs (the cells will be diluted 15 fold when they are added to the MAPs, therefore cells need to be at an initial concentration of OD_{600 nm} = 1.05). Transfer concentrated cell suspension into a reservoir and save (at RT) until step 3.5 of MAPs preparation.

3. Preparation of Multi-phenotype Assay Plates (MAPs)

1. Label MAPs. Label sterile 96-well micro-titer plates with an E. coli clone identification number and MAP schematic type.
2. Aliquot sterile water into MAPs. Aseptically transfer sterile water into a liquid reservoir. Pipette 60 µl into each well of the micro-titer plate using a multi-channel pipette.
3. Aliquot basal media into MAPs. Aseptically transfer 3x basal media into a liquid reservoir. Pipette 50 µl into each well of the micro-titer plate using a multi-channel pipette. Repeat steps 3.2 and 3.3 for each basal media used in the MAP schematic.
4. Aliquot substrates into MAPs. Transfer 30 µl of each substrate into the appropriate well of the MAPs.
5. Add bacterial suspension to the MAPs. Transfer 10 µl of bacterial cells from section 2.4.2 into each well of the MAP using a multi-channel pipette. Change tips before re-introducing pipette back into the culture stock.
6. Cover MAPs with adhesive film. Cover each plate with a single adhesive plate film. Firmly press the film atop the wells of the plate and along the edges to create an even and tight seal. Remove excess film from the edges of the micro-titer plate with a sterile razor blade.
7. Measure optical density of MAPs:
   1. Place prepared MAPs in the “Input” sleeve of a multi-plate spectrophotometer plate reader. Open plate reader software and create a protocol that measures absorbance (OD_{600 nm}) every 30 min, for a total of 32 hr, with 60 sec of shaking between reads.
   2. Set temperature in the apparatus to 37 °C. Start protocol once MAPs have equilibrated to set temperature.
   3. After 32 hr, remove MAPs from the “Output” sleeve of the plate reader. Label each data text file to include: the E. coli clone identification number, date the MAP ran, and the MAP schematic type.

4. Multi-phenotype Assay Plates (MAPs) Processing and Parameterization

1. Assess growth curves using an automated QA process, e.g., PMAnalyzer²⁴.
   1. Verify that growth curves have OD_{600 nm} < 0.20 in the first 2 hr. Do not use absorbance at the initial time point (t₀) in the filter due to artifacts of condensation, which typically resolve at t₁ (30 min).
   2. Remove growth curves that violate QA filter. Save curve information (sample name, well number, and OD_{600 nm} values) in a separate output file for future reference. Continue with analysis if growth curve passes QA filter.
2. Calculate median growth curves. Using replicate data, per well, calculate the median growth curve by taking the median optical density (OD_{600 nm}) value at each time point. Record resulting median growth curves in a separate output file for future use.
3. Calculate the best-fit logistic model for each growth curve using an adaptation of the equation proposed by Zwietering²⁵. The logistic equation includes three parameters describing bacterial growth: the lag time, λ (hr), the maximum rate of growth, µ_max (OD_{600 nm} 100 ^-1), and the final biomass yield, α (OD_{600 nm}). Use data at time = 30 min (y₁) as the initial value due to artifacts of condensation.
   [1]
   1. Calculate the maximum growth rate using a direct search. Record the largest rate of change over a 90 min interval within the data.
      ​ [2]
   2. Estimate the final biomass yield by searching for the upper asymptotic value of the largest moving average over a 90 min window. Specifically, define as the asymptote of the growth curve.
      [3]
   3. Using the µ_max and A values from 4.3.1 and 4.3.2, find the λ value by trying all values λ:
      [4]
      1. Use each λ value to calculate a sum of squared error (SSE). Use the lowest SSE is the SSE (λS):
         [5]

5. Phenotype Analysis of MAPs

1. Calculate the growth level (GL) for each growth curve to assess the overall growth per clone per substrate. Define GL as the harmonic mean of the shifted logistic-fitted values:
   [6]
2. Assign a phenotype to each growth curve based on the GL values. Here, the four phenotypes used are: expected growth, no growth, gain of function, and loss of function.
   1. Determine phenotypes by comparing growth across all clones on a specific substrate in terms of standard deviations away from the mean. Use this statistic and a minimum growth threshold to designate the phenotype presented by the growth curve (Figure 1A,B). Figure 1C provides examples of phenotype assignment.
   2. Define growth as GL ≥ 0.4 (Figure 1A,B). Define Gain of Function (Figure 1C, green) as having a GL > two standard deviations above the mean and a mean > the growth threshold. Loss of Function (Figure 1C, red) is defined as having a GL < two standard deviations below the mean and a mean > the growth threshold.
3. Create visual representations of the data set based on phenotypes and growth curves.
   1. View growth dynamics portraying OD_{600 nm} values as color to quickly compare growth curves between multiple clones (Figure 2).
   2. View phenotypic distributions among all clones based on growth conditions (Figure 3).

6. Building the Continuous Culture Apparatus

1. Reactor port construction (Figure 4A,B). Drill three 1/4 in. holes, spaced 3/4 in. apart, in the cap of the continuous culture reactor.
   1. For port α: screw a female luer thread style panel mount to 1/16 in. barb from the bottom of the cap with the barb pointing up, out of the cap. Secure barb with a 1/4 in. panel mount lock nut.
   2. For port β: screw a female luer thread style panel mount to 1/16 in. barb with the barb pointing up, out of the cap. Secure barb with a 1/4 in. panel mount lock nut.
   3. For port γ: screw a female luer thread style panel mount to 1/16 in. barb from the bottom of the cap so the barb is pointing up, out of the cap. Secure barb with 1/4 in. panel mount lock nut. Screw a male luer with integral lock ring to 1/8 in. wide bore hose to the barb fitting on the inside of the cap.
   4. For the outflow port: drill a 5/16 in. hole at the 75 ml mark of the continuous culture reactor. Cut 3/4 in. off the nut end of the 1/4 in. barbed bulkhead fitting. Screw in the 1/4 in. barbed bulkhead fitting (gasket is on the outside of the bottle and the nut is on the inside, Figure 4B).
2. Feeding bottle port construction (Figure 4C). Drill two 1/4 in. holes spaced 1 in. apart in the cap of the feeding bottle.
   1. For the port δ: screw a female luer thread style panel mount to 1/8 in. barb with the barb pointing up, out of the cap. Secure barb with a 1/4 in. panel mount lock nut.
   2. For the port ε: screw a female luer thread style panel mount to 1/16 in. barb with the barb pointing up, out of the cap. Secure barb with a 1/4 in. panel mount lock nut.
      1. Screw a male luer with integral lock ring to 1/8 in. wide bore hose to the barb fitting, on the inside of the cap.
3. Feeding tubes and tube extensions:
   1. Cut two 1 in. pieces of tubing (1/8 in. outer diameter (OD) x 1/16 in. inner diameter (ID)). Fit pieces onto ports α and γ of the continuous culture reactor made in section 5.2.
   2. Cut a single 1 in. piece of tubing (1/4 in. OD x 1/8 in. ID). Fit piece onto port Β of the continuous culture reactor.
   3. Cut a single 3.5 in. piece of tubing (1/8 in. OD x 1/16 in. ID). Fit this piece to the inside of port γ, on the male luer with integral lock ring to 1/8 in. wide bore hose. Port γ is the sampling port of the continuous culture reactor.
   4. Cut a single 1 in. piece of tubing (1/4 in. OD x 1/8 in. ID). Fit this piece on the δ port of the feeding bottle.
   5. Cut a single 11 in. piece of tubing (1/16 in. OD x 1/8 in. ID). Fit this piece to the inside of port ε of the feeding bottle, on the male luer with integral lock ring to 1/8 in. wide bore hose.
   6. Cut two 18 in. pieces of tubing (1/8 in. OD x 1/16 in. ID). Fit one piece to port ε of the feeding bottle. Save the other piece for section 7.

7. Running Continuous Cultures for Metabolomics

1. Sterilize materials:
   1. Autoclave the dry materials for 30 min. Make sure to wrap cap of the feeding bottle made in section 5 in aluminum foil. Keep attached tubing straight.
      1. Wrap cap of the continuous culture reactor, made in section 5, in aluminum foil. Keep attached tubing straight. Wrap 18 in. tubing cut in section 6.3.5 and the smallest peristaltic pump tubing adaptor in aluminum foil. Keep tubing straight. Autoclave for 30 min.
   2. Make 2 L of 0.5x LB broth. In the feeding bottle, make 0.5x LB broth. Cover feeding bottle with foil. Autoclave for 1 hr.
   3. Make 70 ml of 0.5x LB broth. Pour broth into the continuous culture reactor. Place a stir bar in the continuous culture reactor. Cover reactor with foil and autoclave for 30 min.
2. Continuous culture set-up:
   1. Bacterial cell-suspension.
      1. From a 12.5% glycerol frozen stock, plate fresh E. coli clones onto 0.5x LB agar containing 100 µg/ml ampicillin. Incubate at 37 °C for 24 hr.
      2. Inoculate a single colony into 3 ml of 0.5x LB broth containing 100 µg/ml ampicillin. For each clone use biological triplicates. Incubate at 37 °C with shaking for 22 hr.
   2. Connect the parts together.
      1. Place all autoclaved materials into a biological safety cabinet and turn ultraviolet light on while media cools. Once media has cooled, add 0.1% L-arabinose and 100 µg/ml ampicillin to all 0.5x LB broth.
      2. Unwrap continuous culture reactor cap and screw onto the reactor. Avoid touching the sampling tube. Unwrap the feeding bottle cap and screw onto the feeding bottle. Avoid touching the sampling tube.
      3. Keep the 18 in. tubing attached to port ε sterile. Un-wrap the 18 in. tubing and peristaltic pump tubing adaptor.
      4. Fit a free end of the 18 in. tubing onto one end of the peristaltic pump tubing adaptor. Fit the other end of the peristaltic pump tubing adaptor to the 18 in. tubing attached to port ε of the feeding bottle cap.
      5. Screw 0.22 µm filter units onto ports β and δ of the continuous culture reactor. Screw a 0.22 µm filter unit on the adaptor of port α. To the 0.22 µm filter unit, fit the free end of the 18 in. tubing.
   3. Start the continuous culture.
      1. In the biosafety hood, inoculate the continuous culture reactor with 100 µl of O/N culture made in section 7.2.1.1.
      2. Close system before moving the continuous culture into a 37 °C incubator. In the incubator have a magnetic stir plate and mini peristaltic pump set up.
      3. Fit the peristaltic pump tubing adaptor into the peristaltic pump. Tubing from the feeding bottle starts at the “In” side and tubing leading to the reactor starts at the “Out” side.
      4. Set the peristaltic pump to: “FAST”. Start the peristaltic pump by switching to “FORWARD”. Check that media begins to flow through the tubing.
      5. Place reactor on magnetic stir plate and begin mixing. Continuous culture reactor must equilibrate for 24 hr before sampling.
   4. Sampling of the continuous culture. Screw a 5 ml luer lok syringe onto port γ and pull up 4.5 ml of culture.
      1. Use 500 µl of the culture to measure the density (OD_{600 nm}). Dilute cells to make 4 x 1 ml cultures at OD_{600 nm} = 0.35.
      2. Aliquot diluted cultures into 1.7 ml microfuge tubes. Repeat sampling every 12 hr for 4 days.
   5. Prepare samples for GC-TOFMS:
      1. Pellet cells via centrifugation at max speed (16,900 x g) for 2 min. Decant supernatant. Wash cells with 500 µl of phosphate buffered saline (PBS). Repeat this step.
      2. Decant supernatant a final time, and then submerge microfuge tubes with pelleted cells in liquid nitrogen until bubbling stops. Store samples in -80 °C freezer.

8. Running Serial Passage Batch Cultures for Metabolomics

1. Preparing bacterial cell-suspension. From a 12.5% glycerol frozen stock, plate fresh E. coli clones onto 0.5x LB agar containing 100 µg/ml ampicillin. Incubate at 37 °C for 24 hr.
   1. Inoculate individual colonies into 3 ml of 0.5x LB broth containing 100 µg/ml ampicillin. Use biological triplicates for each clone.
2. Serial passage batch cultures.
   1. Subculture O/N from 8.1.1 via a 500-fold dilution into 3 ml of LB broth containing 50 µg/ml ampicillin and 0.1% L-arabinose. Subculture should have an OD_{600 nm} < 0.005. Incubate at 37 °C with shaking.
   2. Check optical density (OD_{600 nm}) at 2 and 2.5 hr. Grow cells to achieve OD_{600 nm} = 0.35.
      1. Subculture via a 500-fold dilution into 3 ml of LB broth containing 50 µg/ml ampicillin and 0.1% L-arabinose. Subculture should have an OD_{600 nm} < 0.005.
   3. Check optical density (OD_{600 nm}) at 2 and 2.5 hr. Grow cells to achieve OD_{600 nm} = 0.35.
3. Sampling serial passage batch cultures.
   1. Using sterile forceps, place a 0.22 µm membrane filter on top of a filter manifold. Aliquot 1 ml of Phosphate Buffered Saline (PBS) onto the filter paper and then turn on vacuum pump.
   2. Repeat addition of 1 ml of PBS. Transfer 1 ml of subculture from section 8.2.3 onto the filter paper. From here, move quickly to get samples into liquid nitrogen immediately. Complete this in 1 min.
   3. Aliquot 1 ml of PBS onto filter paper to wash sample. Flush rapidly without spilling sample over sides of filter paper. Repeat.
   4. Using sterile forceps place filter into a 1.7 ml microfuge tube by carefully folding the filter. Submerge microfuge tube in liquid nitrogen until bubbling stops. Store sample in -80 °C freezer.

9. Metabolite Analysis & Processing

1. Ship 3 samples per clone on dry ice to a metabolomics core facility for sample processing, analysis and normalization of GC-TOFMS.
2. For each sample determine the mean, median, standard deviation and coefficient of variation across metabolites, using replicate data.
3. For statistical analysis, manually code a QA pipeline using conditional statements and repetitive executions²⁶ to remove metabolites that do not follow defined conditions.
   1. For Condition 1, remove metabolites in which more than 2 samples have zero abundance. Remove internal standards from the metabolomic profiles.
   2. For Condition 2, remove those metabolites that are not found in the cells of interest. Here, remove the following metabolites: indole 3 acetate, dihydroabietic acid, nonadecanoic acid, salicylic acid, salicylaldehyde, cholesterol, phenol, 1-monostearin, octadecanol, 1-monopalmitin, dodecane, dodecanol, 1-hexadecanol, 5-methoxytryptamine, benzoic acid, pentadecanoic acid, phosphoric acid, pelargonic acid, palmitic acid, capric acid, hydroxylamine, stearic acid, myristic acid, maleimide, levoglucosan, and 4-hydroxybutyric acid.
   3. For Condition 3, remove those metabolites whose coefficient of variation is greater than 1.
4. Capture global metabolomics effects by looking at metabolite relative abundances.
   1. Create a visual representation of the median metabolite abundance per clone for each metabolite (Figure 6).
5. Identify outliers by observing clone-metabolite pair frequencies.
   1. Calculate the Z score for each median value of every metabolite in a clone. Calculate z scores using the following equation:
      [7]
      ​Note: Term X_MC represents the abundance level of a specific metabolite for a specific clone. Term µ_C represents the mean of all clones for a specific metabolite. Term σ_C is the associated standard deviation.
   2. Create a visual representation of the data in which outliers are highlighted (data not provided).