Team:Calgary/Experiments

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Our Experiments

General Protocols

Experimental Details and Rationale

Registry DNA was rehydrated for completion of the Interlab Study. Also, Part:BBa_K934001 (phaC1-A-B1) was rehydrated and transformed into our chassis so that PHB was produced and preliminary secretion assays could be performed before the Synthesis subgroup had completed their cloning.

Materials

iGEM 2017 distribution kit

ddH₂O

Protocol

  1. Add 10μL of ddH₂O to the desired well.
  2. Pipette up and down 3-5 times.
  3. Incubate at room temperature for 10 minutes.
  4. Transform cells with 1μL of rehydrated DNA. Store the remaining amount at -20°C.

Experimental Details and Rationale

Our genetic parts were ordered from IDT and arrived as a dry, lyophilized powder. They were resuspended in aqueous solution for cloning into pSB1C3 or pET29B vectors and to ligate multiple parts together.

Materials

Synthesized DNA from IDT (gBlocks)

ddH₂O

Protocol

  1. Centrifuge tube containing the synthesized DNA for 3-5 seconds at 3000g to ensure that all material is at the bottom of the tube.
  2. Add ddH₂O to reach a final concentration of 50 ng/μL.
  3. Vortex.
  4. Incubate at 50°C for 20 minutes.
  5. Briefly vortex and centrifuge . Store at -80°C.

Experimental Details and Rationale

Antibiotics were added to agar to select for successful E.coli transformants. The vector pSB1C3 was selected for with chloramphenicol, pET29B was selected for with kanamycin, and pSB1A3 was selected for with ampicillin.

Materials

Luria-Bertani broth with agar:

  • 10% (w/v) tryptone

  • 5% (w/v) NaCl

  • 10% (w/v) yeast extract

  • 15% (w/v) agar

Appropriate antibiotic:

  • kanamycin (final concentration of 50 μg/mL)

  • chloramphenicol (final concentration of 30 μg/mL)

  • ampicillin (final concentration of 100 μg/mL)

dH2O

1500-mL Erlenmeyer flask

Stir bar

Aluminum foil

Protocol

  1. In a 1500-mL Erlenmeyer flask add 10g tryptone, 5g yeast extract, 10g NaCl and 15g agar. Dissolve solids in 1000mL dH2O and add a stir bar.
  2. Cover flask loosely with aluminum foil, secure with autoclave tape and sterilize by autoclaving for 20 minutes.
  3. Remove agar from autoclave using oven mitts. Allow agar to cool until warm to the touch before adding appropriate antibiotic.
  4. Stir on hot plate and magnetic stirrer for 30 seconds.
  5. Pour agar into plates using aseptic technique.

Experimental Details and Rationale

Culture broth was plated on agar to isolate single colonies of E.coli.

Materials

Luria-Bertani agar plate with appropriate antibiotic (if required)

Overnight culture of desired bacteria

70% ethanol

Spreading rod

Bunsen burner

Protocol

  1. Using aseptic technique pipette 50-100μL of bacterial culture onto agar plate.
  2. Dip spreading rod in 70% ethanol, pass over flame, and allow for excess liquid to burn off. Cool rod on agar, avoiding bacterial culture.
  3. Use rod to spread bacterial culture over entire plate, spinning the plate at the same time.
  4. Dip spreading rod in 70% ethanol, pass over flame, and allow excess liquid to burn off.
  5. Incubate plates at 37°C overnight or until growth is observed.

Experimental Details and Rationale

Culture broth was streaked on agar to isolate single colonies of E.coli.

Materials

Luria-Bertani agar plate with appropriate antibiotic (if required)

Overnight culture of desired bacteria or single isolated colony on agar plate

Inoculation loop

Bunsen burner

Protocol

  1. Using aseptic technique, flame inoculation loop until red hot. Allow it to cool for 10 seconds or touch it to agar.
  2. Dip the inoculation loop in bacterial culture or touch a single colony and streak the loop on ¼ of the surface of agar in a zigzag motion.
  3. Flame the inoculation loop until red hot. Allow it to cool for 10 seconds or touch it to agar.
  4. Run the cooled inoculation loop through one of the previous streaks ONCE, then streak 1/4 of the surface of the agar.
  5. Repeat Steps 3 and 4 two more times.
  6. Flame the inoculation loop until red hot. Allow it to cool for 10 seconds.
  7. Incubate plates at 37°C overnight or until growth is observed.

Experimental Details and Rationale

E. coli DH5ɑ and BL21(DE3) were lysed and the pSB1C3 or pET29B vectors were isolated to be used in the cloning of our genetic constructs. Bacterial clones were lysed for analysis (eg: confirmation restriction digest, genetic sequencing).

Materials

>2mL overnight culture of bacteria in Luria-Bertani broth with appropriate buffer in 16x125mm culture tube

Resuspension buffer (stored at 4°C):

  • 50mM Tris-HCl, pH 8

  • 10mM EDTA

  • 100μg/mL RNase A

Lysis buffer:

  • 200mM NaOH

  • 1% (v/v) SDS

Precipitation buffer:

  • 3M CH₃CO₂K, pH 5.5

Isopropanol

70% ethanol

Table-top centrifuge

Vacuum Centrifuge

Ice bucket

2-mL microcentrifuge tubes

1.5-mL microcentrifuge tubes

ddH₂O

Protocol

  1. Transfer 2mL of the overnight culture to a 2-mL microcentrifuge tube and pellet the cells by spinning at 3500 g for 1 minute. Discard supernatant.
  2. Resuspend pellet in 300μL Resuspension buffer.
  3. Add 300μL Lysis buffer. Invert gently and incubate at room temperature for 3-5 minutes.
  4. Add 300μL Precipitation buffer. Invert gently. A white precipitate should form.
  5. Centrifuge at 14,000g for 10 minutes at room temperature.
  6. Retain supernatant in a clean 1.5-mL microcentrifuge tube.
  7. Add 650μL isopropanol. Gently invert and incubate at room temperature for 10 minutes.
  8. Centrifuge at 14,000g for 10 minutes at 4°C. Discard supernatant.
  9. Wash pellet with 500μL cold 70% ethanol. Add to microcentrifuge tube. Do not resuspend.
  10. Centrifuge at 14,000g for 5 minutes at 4°C. Discard supernatant.
  11. Dry pellet in speed vac for 15-30 minutes, or until no more liquid remains visible in the tube.
  12. Resuspend pellet in ddH₂O and store at - 20°C.

Experimental Details and Rationale

Our genetic parts and vectors were digested with restriction enzymes before they were ligated. Plasmids isolated from transformants (through Plasmid Miniprep) were also digested for confirmation of ligation and transformation.

Materials

DNA (eg: from plasmid miniprep)

Restriction enzymes

10X appropriate buffer

ddH₂O

100X Bovine Serum Albumin (BSA) (if using PstI)

0.2-mL PCR tubes or 1.5-mL microcentrifuge tubes

Protocol

  1. Into a 0.2-mL PCR tube or 1.5-mL microcentrifuge tube add the following:

    • 1μg DNA

    • 1μL restriction enzyme 1

    • 1μL restriction enzyme 2

    • 2μL 10X appropriate buffer

    • 1μL 100X BSA, if using

    • ddH₂O to a final volume of 20 μL

  2. Incubate at 37°C for one hour.
  3. Deactivate restriction enzymes via heat shock by incubating tube at 80°C for 20 minutes.

Experimental Details and Rationale

DNA was precipitated between steps during sequential digestions in order to isolate the DNA from excess buffer and enzymes, allowing us to start “from scratch” for the subsequent digest. This protocol has been adapted from www.openwetware.org.

Materials

DNA sample that has already been digested once with the desired restriction enzyme(s)

3M Sodium acetate, pH 5.2

100% ethanol

Table-top centrifuge

Vacuum Centrifuge

ddH2O

Protocol

  1. Add the following to your sample, in this order:

    • 1/10 volume of 3M sodium acetate

    • 2-3 volumes of 100% ethanol

  2. Mix and freeze overnight at -20°C or at -80°C for 30-60 minutes.
  3. Spin at 3000g for 30 minutes at 4°C.
  4. Discard supernatant.
  5. Dry the pellet in speed vac for 15-30 minutes.
  6. Resuspend in ddH2O and store at -20°C or proceed with subsequent digest.

Experimental Details and Rationale

Fragments of DNA are separated by size on the gel. This was used to visualize the results of restriction digests, particularly those done to confirm ligation or transformation.

Materials

TAE buffer:

  • 40mM Tris, pH 7.6
  • 20mM CH₃COOH
  • 1mM EDTA

Agarose

250-mL Erlenmeyer flask

RedSafe Nucleic Acid Staining Solution

Gel casting tray and comb

Microwave

6X loading dye

DNA sample

Protocol

  1. For a 1% gel (standard), add 0.3 g agarose to 30 mL TAE buffer in a 250-mL Erlenmeyer flask and microwave until agarose is fully dissolved (avoid boiling for too long).
  2. Allow flask to cool in fumehood until warm to the touch before adding 1.5μL RedSafe Nucleic Acid Staining Solution. Gently swirl to mix.
  3. Pour agarose into assembled gel casting tray. Remove any bubbles with a pipette tip and place comb in gel.
  4. Allow gel to solidify and transfer to a gel running apparatus filled with TAE buffer.
  5. Load samples of 10 μL DNA containing 2μL loading dye.
  6. Run gel at 100 V for 30 minutes or until loading dye is 1/2 way down the gel.

Experimental Details and Rationale

Fragments of DNA are separated by size on the gel, as with agarose gel electrophoresis, but DNA bands can be excised from low-melting-point agarose gel for improved ligation efficiency. With this method, specific inserts or linearized backbones can be cut out and ligated together. This decreases the chances of insert ligating back to its old backbone instead of the new backbone that we want it to be ligated to.

Materials

TAE buffer:

  • 40mM Tris, pH 7.6
  • 20mM CH₃COOH
  • 1mM EDTA

Low-melting-point agarose

250-mL Erlenmeyer flask

RedSafe Nucleic Acid Staining Solution

Gel casting tray and comb

Microwave

6X loading dye

DNA sample

Razor blade

UV-safe mask and shield

1.5-mL microcentrifuge tubes

Protocol

  1. For a 1% gel, add 0.3 g low-melting-point agarose to 30 mL TAE buffer in a 250-mL Erlenmeyer flask and microwave until agarose is fully dissolved (avoid boiling for too long).
  2. Allow flask to cool in fumehood until warm to the touch before adding 1.5μL RedSafe Nucleic Acid Staining Solution. Gently swirl to mix.
  3. Pour agarose into assembled gel casting tray. Remove any bubbles with a pipette tip and place comb in gel.
  4. Allow gel to solidify and transfer to a gel running apparatus filled with TAE buffer.
  5. Load samples of 10 μL DNA containing 2μL loading dye.
  6. Run gel at 80 V for 30 minutes or until loading dye is 1/2 way down the gel.
  7. Remove the gel from casting tray and place it on a UV-light viewer. While using a UV-protecting face mask and shield, turn on the UV light and use razor blade to excise out the desired DNA bands. Try to cut the bands as quickly as possible as UV-light damages the DNA. Place each band in its own 1.5-mL microcentrifuge tube
  8. Melt the gel at 65°C for 5 minutes for ligation.

Experimental Details and Rationale

Digested registry DNA or digested genetic parts from IDT were ligated to either pSB1C3 or pET29B for propagation in E.coli DH5ɑ or protein expression in E.coli BL21(DE3). Later, our parts were ligated to pSB1C3 for submission to the iGEM registry.

Materials

Digested vector DNA

Digested insert DNA

10X T4 DNA ligase buffer (from New England BIolabs)

T4 DNA ligase (1 U/μL) (from New England Biolabs)

ddH2O

1.5-mL microcentrifuge tubes

Protocol

  1. To a 1.5-mL microcentrifuge tube add:

    • 50ng digested vector DNA

    • Appropriate amount of digested insert DNA to give a 3:1 molar ratio of insert:vector

    • 1μL T4 DNA ligase

    • 2μL 10X T4 DNA ligase buffer

    • ddH2O to a total volume of 20μL

  2. Incubate tube at room temperature overnight.
  3. Use 10μL to transform cells, store at -20°C.

Experimental Details and Rationale

Chemically competent DH5 Alpha and BL21(DE3) E. coli cells were prepared, which enabled them to be transformed with recombinant DNA.

Materials

Luria-Bertani broth:

  • 10% (w/v) tryptone

  • 5% (w/v) NaCl

  • 10% (w/v) yeast extract

Stock MgSO4

Stock KCl

250-mL Erlenmeyer flask

16x125 mm culture tubes

Spectrophotometer

Centrifuge

50-mL Falcon tubes

100mM CaCl₂

100mM CaCl₂ + 10% glycerol

Chilled 1.5-mL microcentrifuge tubes

Chilled pipette tips

Protocol

  1. Subculture strain (1:50) into 50ml LB. Add Stock MgSO4 and KCl to a final concentration of 10mM MgSO4 and 1mM KCl.
  2. Shake at 28°C until OD600 = 0.3 - 0.4 is reached.
  3. Chill on ice at least 10 minutes.
  4. Put into 50 ml pre-chilled tube, centrifuge at 2500g for 8 minutes, at 4°C.
  5. Re-suspend in 10ml ice-cold 100 mM CaCl₂, gently mix on ice, and incubate on ice for 10 minutes.
  6. Centrifuge at 2500g for 8 minutes, at 4°C.
  7. Re-suspend in 500ul 100mM CaCl₂ + 10% glycerol by gently pipetting up and down a few times on ice.
  8. Incubate on ice for at least 10 minutes.
  9. Use large tip to separate to 1.5 ml pre-chilled tubes, with 50 ul of cells in each tube. Keep on ice while separating.

Experimental Details and Rationale

Chemically competent E.coli DH5α were transformed with pSB1C3 or pET29b containing our genetic parts in order for the vector and insert to be propagated. Chemically competent E.coli BL21(DE3) was transformed with pSB1C3 or pET29B containing our genetic parts in order for those proteins to be expressed.

Materials

Competent E.coli aliquots (50 μL)

1M CaCl₂

DNA for transformation

Luria-Bertani broth or SOC Media

Agar plate with appropriate antibiotic

Protocol

  1. Thaw 50μL aliquot of competent E.coli DH5α cells on ice just before use.
  2. Add 0.3-1μg DNA to cells, flick gently to mix. For every 9μL of DNA used, add 1μL of cold 1M CaCl₂ to maintain competency of the cells. Place on ice for 45 minutes.
  3. Heat shock for 60-75 seconds at 42°C.
  4. Place on ice for 5 minutes
  5. Add 2 mL Luria-Bertani or SOC medium to aliquot of cells.
  6. Incubate cells for 60-90 minutes at 37°C, shaking at 200 rpm for 1 hour.
  7. Plate 50-100μL of re-suspended culture on agar plate with appropriate antibiotic and spread.
  8. Incubate plates at 37°C overnight or until desired growth is observed.

Experimental Details and Rationale

Glycerol stocks of transformed E.coli were prepared for long-term storage of the cells at -80°C.

Materials

Overnight culture of transformed bacteria

Sterile 1.5-mL cryo-tubes

Sterile 50% glycerol

Protocol

  1. Using aseptic technique, pipette 0.5mL of 50% sterile glycerol into a 1.5-mL cryo-tube.
  2. Using aseptic technique add 0.5mL of overnight culture.
  3. Pipette up and down gently to mix.
  4. Store at -80°C for up to 1 year.

Experimental Details and Rationale

Proteins are isolated, denatured, and separated by size on the gel. This helps to identify the proteins created from our parts and expressed by the E.coli.

Materials

1x SDS gel loading buffer:

  • 50mM tris-Cl (pH 6.8)
  • 100mM dithiothreitol
  • 2% sodium dodecyl sulfate
  • 0.1% bromophenol blue
  • 10% glycerol

1x Tris-Glycine electrophoresis buffer:

  • 25mM tris
  • 250mM glycine
  • 0.1% (w/v) sodium dodecyl sulfate

Stacking gel:

  • dH₂O
  • 30% acrylamide mix
  • 1.0M tris (pH 6.8)
  • 10% sodium dodecyl sulfate
  • 10% ammonium persulfate
  • TEMED

10% Resolving gel:

  • dH₂O
  • 30% acrylamide mix
  • 1.5M tris (pH 8.8)
  • 10% sodium dodecyl sulfate
  • 10% ammonium persulfate
  • TEMED

250-mL Erlenmeyer Flasks

Protocol

  1. Assemble glass plates into a holding cassette.
  2. In a 250-mL Erlenmeyer flask, place all the ingredients of 10% resolving gel, mix rapidly, and pour into casting plates up to 1 cm below where the well comb would be. Wait to put TEMED and 10% APS until ready to pour gel. For 5mL gel:
    • 1.9mL dH2O
    • 1.7mL 30% acrylamide mix
    • 1.3mL 1.5M Tris
    • 0.05mL 10% SDS
    • 0.05mL 10% APS
    • 0.002mL TEMED.
  3. Place distilled water into the remaining space upto the top of the glass plates. Wait about 30 minutes, or until gel is set.
  4. After the gel sets, pour off the distilled water on top and use a kimwipe to wipe up any excess water.
  5. Mix the stacking solution in another 250-mL Erlenmeyer flask. Wait to put TEMED and 10% APS until ready to pour gel. For 2mL gel:
    • 1.4mL dH2O
    • 0.33mL 30% acrylamide mix
    • 0.25mL 1.0M Tris
    • 0.02mL 10% SDS
    • 0.02mL 10% APS
    • 0.002mL TEMED
  6. Pour the stacking gel and place Teflon comb into solution.
  7. Prepare the sample:
    • 3μL of 1X SDS gel-loading buffer
    • 12uL of protein sample
  8. Heat the sample at 100°C for 3 minutes.
  9. Mount the gel into the electrophoresis apparatus and fill the inside well with 1X Tris-glycine electrophoresis buffer. For 1000mL:
    • 3.02g Tris
    • 18.8g glycine
    • 10mL 10% SDS
    • Adjust volume to 1000mL
  10. Remove the Teflon comb gently and load up to 15μL of samples into the well. Attach the apparatus to the power supply and run for 60 minutes at 30mA per gel.
  11. The resulting gel is then dried using a paper towel and fixed using a variety of methods such as Coomassie Blue or sivler salts, fluorographed, autoradiographed or used for immunoblotting.

Experimental Details and Rationale

After running a SDS-Page gel, the resulting gel was stained with Coomassie Blue to visualize protein bands. Coomassie Blue binds to proteins and makes them appear as blue bands.

Materials

Staining solution:

  • 55 % Methanol (250 mL Methanol, 200 mL dH2O)
  • 50 mL glacial acetic acid
  • 1.25 g Coomassie blue (0.25 g/100mL of solution)

De-staining solution:

  • 55 % Methanol (250 mL Methanol, 200 mL dH2O)
  • 50 mL glacial acetic acid

Plastic container that fits your gels

Polyacrylamide gel run on a SDS-Page apparatus

Shaker

dH2O

Protocol

  1. Place the polyacrylamide SDS-Page gel into a plastic container.
  2. Pour the staining solution over top of the gel until it is just covered.
  3. Incubate at room temperature on the shaker overnight.
  4. Place the stained gels in a new plastic container .
  5. Pour the de-staining solution over top of the gel and place a Kimwipe on top.
  6. Incubate at room temperature on the shaker for 4 hours. Rinse with dH2O .



PHB Synthesis

NOTE: Media with other % glucose levels can be prepared in the same way, but with different amounts of glucose in step 1.

Experimental Details and Rationale

3% glucose media (with appropriate antibiotics if required) was inoculated with PHB-producing E. coli cells and incubated overnight. Glucose was used as a carbon source for the PHB synthesis and the antibiotics are necessary to select for cells that contain the correct plasmids for PHB production.

Materials

300 mL Luria-Bertani broth:

  • 10% (w/v) tryptone

  • 5% (w/v) NaCl

  • 10% (w/v) yeast extract

Glucose

Appropriate antibiotic if required:

  • kanamycin (final concentration of 50 μg/mL)

  • chloramphenicol (final concentration of 30 μg/mL)

  • ampicillin (final concentration of 100 μg/mL)

dH2O

1500-mL Erlenmeyer flask

Stir bar

Aluminum foil

Hot water bath

Overnight culture of PHB-producing bacteria

Protocol

  1. Make Luria-Bertani broth and after it has been autoclaved add 9g of glucose to it.
  2. Microwave for 30 seconds.
  3. Incubate in hot water bath at 80°C for 1-2 hours.
  4. Add antibiotic to the broth (if required).
  5. Inoculate the broth with 1mL of overnight culture and incubate at 37°C overnight.

Experimental Details and Rationale

Nile Red LB agar plates were used to detect the presence of PHB granules inside of E.coli cells. Nile Red is lipophilic stain that binds to the PHB granules inside the cell and fluoresces once bound. Fluorescing cells on these plates strongly suggest that PHB granules are present. This protocol was adapted from the Imperial College iGEM Team, 2013.

Materials

Luria-Bertani broth with agar:

  • 10% (w/v) tryptone

  • 5% (w/v) NaCl

  • 10% (w/v) yeast extract

  • 15% (w/v) agar

Appropriate antibiotic:

  • kanamycin (final concentration of 50 μg/mL)

  • chloramphenicol (final concentration of 30 μg/mL)

  • ampicillin (final concentration of 100 μg/mL)

dH2O

1500-mL Erlenmeyer flask

Stir bar

Aluminum foil

Nile Red stain

Protocol

  1. Make Luria-Bertani broth with agar and after it has been autoclaved add stock Nile Red solution to a final concentration of 0.5 μg/mL.
  2. Add appropriate antibiotic.
  3. Pour agar plates using aseptic technique.

Experimental Details and Rationale

Nile Red is lipophilic stain that binds to PHB granules inside the cells and fluoresces once bound. Our PHB-producing E. coli were incubated to allow for PHB production, then they were treated with Nile Red stain. The flourescence was read in a flow cytometer at an excitation wavelength of 535 nm and an emission wavelength of 605 nm.

Materials

Minimum 1-mL samples of PHB-producing cells

Nile Red (80 μg/mL dissolved in dimethyl sulfoxide (DMSO))

dH₂O

1.5-mL microcentrifuge tubes

Table-top centrifuge

96-well microplate

Protocol

  1. Centrifuge 1 mL of PHB-producing cells at 12,000xg for 5 minute. Discard supernatant and resuspend pellet in 1 mL dH₂O.
  2. Add 40 μL of Nile Red in DMSO to each solution (final concentration = 3.1 μg Nile Red per mL suspension.
  3. Incubate at room temperature for 30 minutes, covered.
  4. Centrifuge the stained suspension at 12,000xg for 5 minutes. Discard supernatant and add 1 mL dH₂O. Vigorously vortex to resuspend the pellet.
  5. Add an aliquot of each sample to a 96-well microplate.
  6. Read the fluorescence at an excitation wavelength of 535 nm and an emission wavelength of 605 nm.

Experimental Details and Rationale

Chloroform chemically lyses the bacterial cells, causing them to release PHB into their media, which can then be isolated via centrifugation. This method of extraction was carried out before the Secretion subgroup had completed their parts for the secretion pathway. This protocol has been adapted from theTokyo Tech iGEM Team, 2012. We initially used chlorform to extract PHB, however we found bleach extraction simpler, therefore we primarily used that method for PHB extraction.

Materials

50mL overnight culture of PHB-producing bacteria

Chloroform

Methanol

50-mL Falcon Tubes

Filtration apparatus

Centrifuge

Shaker

Protocol

  1. Centrifuge the 50mL overnight culture at 3275g for 10 minutes in a 50-mL Falcon Tube.
  2. Discard supernatant and weigh pellet. Resuspend pellet in chloroform (1 mL chloroform per 2 mg of dried cells).
  3. Incubate the solution in a shaker for 72 hour at 25°C
  4. Filter the chloroform solution and concentrate the solution into a pellet by evaporation in a fume hood overnight.
  5. Add methanol drop-wise to the pellet until it is just resuspended.
  6. Filter the solution in methanol and dry the polymer in a fume hood overnight.
  7. Add a little chloroform to dissolve the polymer then pour it into a sheet in the small Petri dish.
  8. Let the sheet dry at room temperature.
  9. Allow powder to dry overnight in an open tube.

Experimental Details and Rationale

Sodium hypochlorite (bleach) chemically lyses the bacterial cells, causing them to release PHB into their media, which can then be isolated via centrifugation. This method of extraction was carried out before the Secretion subgroup had completed their parts for the secretion pathway. This protocol has been adapted from the Imperial College iGEM Team, 2013. We initially used chlorform to extract PHB, however we found bleach extraction simpler, therefore we primarily used this method for PHB extraction.

Materials

50mL overnight culture of PHB-producing bacteria

Sodium hypochlorite (bleach)

1X PBS

Triton X-100

70% ethanol

50-mL Falcon Tubes

Centrifuge

Protocol

  1. Centrifuge the 50mL overnight culture at 3275g for 10 minutes in a 50-mL Falcon Tube. Discard supernatant and resuspend pellet in 5mL 1X PBS solution.
  2. Centrifuge again at 3275g for 10 minutes. Discard supernatant and resuspend pellet in 5mL 1% (v/v) Triton X-100 in PBS.
  3. Incubate for 30 minutes at room temperature.
  4. Centrifuge at 3275g for 10 minutes. Discard supernatant and resuspend pellet in 5mL 1X PBS solution.
  5. Centrifuge at 3275g for 10 minutes. Discard supernatant then resuspend in5mL of sodium hypochlorite.
  6. Incubate at 30°C for 1 hour
  7. Centrifuge at 3275g for 20 minutes. Discard supernatant and wash pellet in 5mL 70% ethanol. Repeat this wash several times, with 20 minutes of centrifugation at 3275g between each step.
  8. Allow powder to dry overnight in an open tube.

NOTE:Scale reagant volumes proportionally for higher volumes of overnight culture. All steps should be carried out with 1/10 volume of the overnight culture.

Experimental Details and Rationale

The different conditions used for this experiment are to identify whether the gene construct present in bacteria is able to utilize VFAs and glucose. Glucose is used a positive control, whereas pET29(b)+ plasmid containing no insert is negative control. Three replicates are carried out for each of the four conditions

Materials

10 ml OD600 0.4-0.8 overnight culture of PHB-producing bacteria (x9)

10 ml OD600 0.4-0.8 overnight culture of negative control bacteria (x3)

20% Glucose

MgSO4

CaCl2

M9 salts

1 M IPTG

410 uL propionic acid (x3)

118 uL acetic acid (x3)

55 uL butyric acid (x3)

Syn poo (see Syn Poo Recipe 1 in the Process Section of this page)

dH2O

125 ml Erlenmeyer flasks (x12)

10 ml culture tubes

50 ml Falcon tubes

Centrifuge

Shaker

Protocol

    (Note: final concentrations are enclosed in () and total volume in each flask was 50 ml.

  1. Label the 12 erlenmeyer flasks
  2. Obtain the three flasks for positive control and add the following to each:
    • 10 ml O/Ns in LB+Kan
    • 7 ml 20% Glucose
    • 100 uL MgSO4 (2mM)
    • 5 uL CaCl2 (0.1 mM)
    • 10 ml M9 salts (1x)
    • 5 uL 1M IPTG
    • 20 ml dH2O
  3. Obtain the three flasks for negative control and add the following to each:
    • 10 ml O/Ns in LB+Kan
    • "Syn poo" fermented supernatant (see Syn Poo Recipe 1 in the Process Section of this page)
    • 100 uL MgSO4 (2mM)
    • 5 uL CaCl2 (0.1 mM)
    • 10 ml M9 salts (1x)
    • 5 uL 1M IPTG
    • 30 ml dH2O

  4. Obtain the three flasks for Fermented "syn poo" supernatant and add the following to each:
    • 10 ml O/Ns in LB+Kan
    • 10 ml "Syn poo" fermented supernatant (see Syn Poo Recipe 1 in the Process Section of this page)
    • 100 uL MgSO4
    • 5 uL CaCl2
    • 10 ml M9 salts
    • 5 uL 1M IPTG
    • 23 ml dH2O
  5. Obtain the three flasks for Pure VFAs and add the following to each:
    • 10 ml O/Ns in LB+Kan
    • VFAs
      • 410 uL propionic acid
      • 118 uL acetic acid
      • 55 uL butyric acid
    • 100 uL MgSO4
    • 5 uL CaCl2
    • 10 ml M9 salts
    • 5 uL 1M IPTG
    • 30 ml dH2O
  6. Incubate the flasks in a shaker for 24 hours at 37°C and 100 rpm
  7. Obtain the flasks and pour solution to 50 ml Falcon tubes
  8. Follow the extraction protocol



PHB Secretion

Experimental Details and Rationale

For secretion assays, a plasmid with PHB-producing and PHB-secreting genes (pSB1C3-PhaCAB-Phasin-HlyA Tag) was transformed into E. coli BL21(DE3) and inoculated in LB with 3% glucose + chloramphenicol. After incubation for at least 24h, the samples were separated into secreted and cellular fractions for analysis of PHB secretion. CaCl2 addition causes secreted PHB to agglomerate for easier separation. This method has been adapted from Rahman et. al (2013).

Materials

50mL of PHB-producing and PHB-secreting bacterial samples that have been incubating in LB media + 3 % glucose for at least 24 hours

50-mL Falcon Tubes

1 M CaCl2

Centrifuge

Protocol

  1. Transfer the sample to a 50-mL Falcon Tube
  2. Add 1 M CaCl2 to the sample so that a final concentration of 0.01 M is reached.
  3. Incubate at room temperature for 10 minutes.
  4. Centrifuge at 50xg for 5 minutes. This pellet contains secreted PHB.
  5. Remove supernatant to a new 50-mL Falcon Tube.
  6. Centrifuge at 3270xg for 10 minutes. Discard supernatant. The pellet contains bacteria with intracellular PHB.

Experimental Details and Rationale

For secretion assays, PHB was separated into secreted and intracellular fractions. Intracellular PHB was purified with sodium hypochlorite (bleach extraction) and secreted PHB was purified from cellular debris with Triton X-100, which extracts lipids and proteins from the PHB.

Materials

Secreted fraction of PHB (pellet) from 50-mL of culture.

1 % Triton X-100 in PBS

Centrifuge

Protocol

  1. Resuspend the pellet of secreted PHB in 5 mL of 1% Triton X-100 in PBS.
  2. Incubate for 30 minutes at room temperature.
  3. Centrifuge at 3275g for 10 minutes. Discard supernatant and let pellet dry overnight in an open tube.

NOTE:Scale reagant volumes proportionally for higher volumes of culture from which the secreted fraction was obtained. All steps should be carried out with 1/10 volume of culture.

Experimental Details and Rationale

This process is also known as a Western Blot. After running a SDS-Page gel of our Phasin-HlyA Tag, the resulting gel was incubated with FLAG antibodies because our protein contains FLAG tags. Excess antibody and proteins are washed away then a second antibody that binds to the first antibody is added. The second antibody contains horseradish peroxidase (HRP) that produces a color change when bound to the first antibody and this signals the presence of our Phasin-HlyA Tag. This protocol has been adapted from Thermo Fisher Scientific.

Materials

TBST:

  • 20 mM Tris, pH 7.5
  • 150 mM NaCl
  • 0.05 % TWEEN-20

TBSTM:

  • 20 mM Tris, pH 7.5
  • 150 mM NaCl
  • 0.05 % TWEEN-20
  • 5% Non-fat dry milk

Transfer Buffer Solution

  • 15.6 mM Tris-base, pH 8.3
  • 120 mM Glycinel
  • 020 % Methanol/li>
  • dH2O to fill up to one Liter

Electrophoretic Transfer Apparatus

Polyacrylamide gel run on a SDS-Page apparatus

dH2O

Scotch-brite pads

Transfer Membrane

Filter paper

Primary Antibody:

  • Sigma Aldrich Anti-FLAG antibody produced in rabbit

Secondary Antibody:

Sigma Aldrich goat anti-rabbit igG-HRP

Blotting Solution:

Thermo Scientific 1-Step TMB-Blotting Solution

Shaker

Protocol

  1. Place the polyacrylamide SDS-Page gel into a plastic container.
  2. Prepare a membrane “sandwich” in the Electrophoretic Transfer Apparatus as shown in the diagram below, while making sure you wet each component with cold TBST as you go and removing any air bubbles:

    3. Western Blot Sandwich

  3. Transfer the proteins to the membrane at 100V for 1 hour .
  4. Block the membrane by incubating for 1 hour at room temperature or in 100 mL TBSTM on a shaker.
  5. Incubate with the primary antibody for 1 hour at room temperature at a dilution of 1:1000 in 10 mL of TBSTM on a shaker.
  6. Wash the membrane three times with 20 mL TBST for 5 minutes.
  7. Incubate for 1 hour with secondary antibody at a dilution of 1:5000 in 10 mL TBSTM on a shaker.
  8. Wash membrane three times with 20 mL TBST for 5 minutes.
  9. Drain membrane of remaining TBST and incubate with 10 mL of TMB-Blotting Solution until there is a color change (several minutes) to blue. Then wash the TMB away with dH2O.



Process Development

Experimental Details and Rationale

This recipe was used to prepare synthetic feces (syn poo) for VFA/PHB quantification experiments using HPLC, VFA fermentation experiments, and PHB production experiments by the synthesis sub-group.

Materials

Water

Yeast Extract

Microcrystalline Cellulose

Psyllium

Miso Paste

Sodium Chloride

Potassium Chloride

Calcium Chloride

Protocol

Add the following materials to prepare 1 L of syn feces, while mixing well:

  1. 800 mL of water
  2. 60 g of yeast extract
  3. 20 g of microcrystalline cellulose
  4. 35 g of psyllium
  5. 35 g of miso paste
  6. 4 g of sodium chloride
  7. 4 g of potassium chloride
  8. 2 g of calcium chloride

Experimental Details and Rationale

To test different technologies for the solid-liquid separation, feces fermentation and E. coli fermentation we had to research a methodology to create synthetic human feces, which would have chemical and physical characteristics similar to those of real feces. The following recipes were obtained from the NASA paper, and the recipe 1 in the chart was found to be the best representation of the physical properties for feces and hence was chosen for some of the experiments.

Recipe chart

Experimental Details and Rationale

The goal of these experiments was to test the VFA Fermentation step of the process at lab scale and to determine the optimal operating conditions. The two different temperatures tested were 37°C and 22°C and two fermentation durations tested were 3 and 5 days. After VFA fermentation experiments, the supernatant was collected and sterilized. PHB-producing bacteria were then cultured in the obtained supernatants and the amounts of produced PHB were then compared.

Materials

Syn poo

E. coli BL21(DE3) transformed with pET29b(+) vector without any inserts

PHB-producing E.coli strain

Flasks

0.22 micron syringe filters

syringes

Shaker

Centrifuge

Protocol

  1. Prepare syn poo according to "Syn Poo Recipe 1" protocol
  2. Prepare overnights of E. coli BL21(DE3) transformed with pET29b(+) vector without any inserts (control) and PHB-producing E. coli
  3. Transfer syn poo into flasks, label all conditions
  4. Inoculate with bacteria (1 mL of overnight culture per 100 mL of syn poo)
  5. Cover the flasks with aluminum foil
  6. Put inoculated flasks into a shaker at 80 rpm and desired temperature
  7. Ferment for desired amount of time
  8. After fermentation is complete, centrifuge fermented syn poo at 3000 rcf for 20 minutes
  9. Collect the supernatant and perform VFA measurements using titrations protocol
  10. Sterlize the supernatant using syringe-driven 0.22 micron filters
  11. Follow "PHB Synthesis using VFAs as feedstock" protocol

Experimental Details and Rationale

Originally we have considered simple, low power consumption methods for solid-liquid separation. Gravity-driven filtration was inspired by the sand pack filtration used in the early oil industry. We have also evaluated the effect of dilution on the efficiency of liquid recovery.

Materials

Funnel (x3)

Lab filter paper (x3)

Large beaker (500ml) (x3)

Synthetic feces samples

Pure Syn Feces Sample (recipe 2) (25g)

Syn Feces Sample (recipe 2) (25g) mixed with 25g of water

Syn Feces Sample (recipe 2) (25g) mixed with 50g of water

Protocol

  1. Place filter paper into the funnel and then place the funnel on top of the beaker
  2. Label the beaker with one of the three samples and then place the representative sample into the funnel
  3. Let the sample filter under the gravitational force for 24 hours.
  4. measure the weight of the liquid obtained

Experimental Details and Rationale

Originally we considered using low power requirement methods for liquid (&VFA) and solid separation method. Gravity-driven sedimentation appeared to be the simplest method, hence we decided to test it's efficiency first under the Earth gravitational field. We have also examined the effect of dilution on the water recovery efficiency. /p>

Materials

Beaker (500ml) (x3)

1ml pipet

Scale

Synthetic feces samples

Pure Syn Feces Sample (recipe 2) (25g)

Syn Feces Sample (recipe 2) (25g) mixed with 25g of water

Syn Feces Sample (recipe 2) (25g) mixed with 50g of water

Protocol

  1. Place samples into the beakers and label them representatively
  2. Leave the beakers untouched for 24 hours
  3. Pipet the middle liquid layer out of the beaker and measure the volume of collected liquid

Experimental Details and Rationale

Centrifugation is a power intensive method for solid-liquid separation; however, literature and industrial example show that it should be the most efficient method for solid-liquid extraction, hence we decided to compare the centrifugation liquid recovery efficiency to the efficiency of other methods. We have also tested the effect of dilution on the recovery efficiency.

Materials

Lab scale centrifuge (goes up to 3700rpm)

Beaker (x3)

Scale

Synthetic feces samples

Pure Syn Feces Sample (recipe 2) (25g)

Syn Feces Sample (recipe 2) (25g) mixed with 25g of water

Syn Feces Sample (recipe 2) (25g) mixed with 50g of water

Protocol

  1. Place each of three syn feces samples into the 50ml centrifugation tubes and label them accordingly
  2. Centrifuge at 3700rpm for 15 minutes
  3. Collect the supernatant from each tube
  4. Centrifuge the supernatant again at the same conditions
  5. Measure the volume of supernatant collected after the second centrifugation

Experimental Details and Rationale

Original gravity driven filtration experiments proved to recover insufficient amount of liquid and require high degree of power when a single filter paper is used due to clogging. Therefore the staged pressure filtration experiment was developed (to test how having a series of filters decreasing in ore size would affect the required power and the amount of liquid recovered)

Materials

25g of Syn Poop

AeroPress Coffee press

Strainer

paper towel

coffee filter

11 and 0.2 micron filter paper

Protocol

  1. Collect 25 g of Syn Feces sample. Place the strainer on top of the beaker and poor the sample into the strainer. Record the weight of the liquid collected in the beaker
  2. Place a paper towel filter disc into the AeroPress coffee filter device. Poor the liquid obtained in step 2 into the AeroPress.
  3. Press on the device and collect all the liquid in the beaker. Press on the device repeatedly and make sure to shake the device every time to allow all the liquid drop to fall down. Weight the obtained liquid.
  4. Repeat the steps 3-4 with coffee filter, 11micron filter, and 0.2micor filter

Experimental Details and Rationale

After successfully recovering liquids from the feces sample, we had to measure the VFA concentration of the sample. This was an important step for proving VFA presence in the recovered liquids, proving the increase in VFA concentration post bacterial fermentation. Wastewater treatment industry uses five-point titration method for the determination of average VFA concentration in the water. This method requires extensive calculus and long experimental procedure when simple titration have proven to work with low VFA concentrations. The following Simple titration method was developed by Anderson and Yang (1992)

Materials

beaker (100ml)

50ml Sample for titration

pH meter

Magnet stirring bar

Protocol

  1. Place the liquid sample into the beaker
  2. Add magnetic stirring bar to the beaker and place the beaker onto the stirring magnetic plate
  3. Place the tip of the pH meter into the solution, make sure that the pH meter was recently collaborated and that the tip is fully immersed into the solution
  4. Measure the initial pH of the sample (should be above 6pH ), dilute with a base if needed
  5. Add 1N sulphuric acid to the beaker 1ml at a time and measure the pH with every addition
  6. Measure the volume of acid added to titrate the sample to each end point: pH of 5.1 and 3.5. Adjust the volume of each consecutive sulphuric acid addition when appropriate

Post titration calculations:

A1=[VFA]*([H2]-[H1])/([H2]+k2)

A2=[VFA]*([H3]-[H1])/([H3]+k2)

where

  1. A1, A2 - molar equivalents of acid consumed to the first (5.1ph) and second (3.5pH) titration points
  2. A1=(sulphuric acid concentration)(mol/ml)*(volume of acid added)(ml)
  3. [H1]=10^(-pH)
  4. [VFA]- VFA concentration
  5. k1- conditional dissociation acid for carbonic acid = 3.2*10^(-7)
  6. k2 - combined dissociation constant of the VFAs = 2.4*10^(-5)

Rearrange the equation to obtain the expressions for VFA concentration

Experimental Details and Rationale

One of the design options we considered for the PHB extraction used the principle of chemical coagulation. The PHB particles secreted by the E.coli were expected to be in the 20-60 nm range (Rahman et al., 2013). Therefore, we hoped to demonstrate in the lab that adding a coagulant helped the nanoparticles to agglomerate into larger particles that could be more easily recovered by centrifugation.

Materials

Lab scale centrifuge

Spectrophotometer

Probe-type Sonicator

50 ml tubes

1L of 5g/L PHB suspension in distilled water

200 ml of 1M CaCl2 solution

Protocol
  1. The PHB suspension was sonicated in 250 ml batches in a probe-type sonicator to break up the particles in suspension.
  2. The suspension was then allowed to settle for 48 hours before the top phase was separated by decanting.
  3. 50ml of the suspension is transferred into 50 ml tubes three different conditions are tested.
  4. The first three samples are centrifuged at 1000 PRM
  5. The second three samples are centrifuged at 3750 PRM
  6. CaCl2 solution is added to the final three samples to make a final concentration of 10mM. The samples are then centrifuged at 3750 RPM.
  7. The absorbance of each of the samples and a sample of PHB suspension after step 2 is measured at 600nm.

Experimental Details and Rationale

We considered electrocoagulation for extracting PHB. Thus, we hoped to show that we could recover PHB from synthetic feces supernatant. PHB particles have a zeta potential of pH 3.5 (van Hee et al., 2006), therefore in a suspension in water or supernatant (pH 5.3), we would be able to coagulate the particles by supplying positively charged ions. Therefore we planned to use an iron anode (which would release Fe 2+ ions) and a steel cathode for our experiments. We plan to test a suspension of PHB in water to see if we can settle PHB via electrocoagulation. Then we plan to go on to test just synthetic poop supernatant as a negative control and then a mixture of PHB and synthetic poop supernatant.

Materials

9V Battery

Alligator clips

Iron nail

Steel rod

Mason jars

Lab scale centrifuge

Probe-type Sonicator

Zetasizer Nano for particle size measurements

50 ml tubes

1L of 5g/L PHB suspension in distilled water

1L of supernatant from the synthetic poop recipe

Protocol
  1. Supernatant from the synthetic poop recipe was prepared by centrifuging samples in 50 ml tubes at 3750 RPM for 20 minutes, removing the supernatant and then centrifuging the supernatant again at 3750 RPM for 20 minutes.
  2. The first 500 ml of the 5g/L suspension of PHB was sonicated in 250 ml batches in a probe-type sonicator to break up the particles in suspension. The suspension was then allowed to settle for 48 hours before the top phase was separated by decanting.
  3. Average size of the particles in this sample was measured on the Zetasizer Nano and found to be in the range of 1500 nm to 1000nm. This sample was referred to as the microscale PHB.
  4. The second 500 ml of the 5g/L suspension of PHB was first sonicated in 250 ml batches in a probe-type sonicator and then allowed to settle for 48 hours before the top phase was separated by decanting. The top phase was then sonicated again and centrifuged at 3750 RPM for 30 minutes.
  5. Average size of the particles in the supernatant from the centrifugation step was measured on the Zetasizer Nano and found to be in the range of 70 nm to 400 nm. This sample was referred to as the nanoscale PHB.
  6. 250 ml of both the microscale and nanoscale PHB are loaded into the electrocoagulation cells. The cells are set up using an iron anode and a steel cathode similarly to the set up used in ("Make Water - Collaborative Water Purification", 2017).
  7. The cells are run for 3 hours and the settlement is observed.
  8. 250 ml of synthetic feces supernatant was also loaded into an electrocoagulation cell which was run for 3 hours.
  9. A 250 ml 1:1 mixture of synthetic feces supernatant and PHB suspension was then loaded into a cell and run for 3 hours.

Experimental Details and Rationale

We measured acetic, propionic and butanoic acid in the synthetic feces supernatant using High Performance Liquid Chromatography.

Materials

Aminex HPX-87H ion-exclusion HPLC column with a UV detector

0.2 micron filters

Lab-scale centrifuge

50 ml tubes

2 ml tubes

Supernatant from the synthetic feces recipe

Standard solutions of acetic acid, propionic acid and butyric acid

Protocol
  1. Supernatant from the synthetic poop recipe was prepared by centrifuging samples in 50 ml tubes at 3750 RPM for 20 minutes, removing the supernatant and then centrifuging the supernatant again at 3750 RPM for 20 minutes.
  2. The supernatant was then diluted using dilution factor of 32 (to get the concentration of butyric acid within the range of the HPLC) and 256 (to get the concentration of propionic acid within the range of the HPLC)
  3. The diluted supernatant was filtered using a 0.2 micron filter into 2 ml tubes
  4. The samples were then run on the HPLC

Experimental Details and Rationale

We hoped to confirm the presence of PHB within the samples extracted from the engineered E.coli by the Synthesis group using High Performance Liquid Chromatography (HPLC). The samples are first digested in concentrated sulphuric acid to produce crotonic acid which is measured on the HPLC.

Materials

Aminex HPX-87H ion-exclusion HPLC column with a UV detector

o.2 micron filters

Lab-scale centrifuge

50 ml tubes

2 ml tubes

Concentrated sulphuric acid

Crotonic acid standards

At least 0.01g PHB-containing samples

Protocol
  1. We followed the protocol in (Karr et al., 1983) to convert PHB to crotonic acid.
  2. We digested the PHB-containing samples in 1 ml concentrated sulphuric acid at 90 degrees Celsius for 30 miinutes.
  3. After the digestion was complete we cooled the samples on ice and rapidly mixed in 4 ml of 0.014N sulphuric acid.
  4. We diluted the samples by predicting the possible concentration of crotonic acid assuming a 80% conversion and then filtered them using a 0.2 micron filter before running them on the HPLC.

Works Cited

Anderson, G., & Yang, G. (1992). Determination of Bicarbonate and Total Volatile Acid Concentration in Anaerobic Digesters Using a Simple Titration. Water Environment Research, 64(1), 53-59

Ethanol precipitation of nucleic acids. (2012, July 1). Retrieved from https://openwetware.org/wiki/Ethanol_precipitation_of_nucleic_acids.

Karr, D. B., Waters, J. K., & Emerich, D. W. (1983). Analysis of poly-β-hydroxybutyrate in Rhizobium japonicum bacteroids by ion-exclusion high-pressure liquid chromatography and UV detection. Applied and environmental microbiology, 46(6), 1339-1344

Make Water - Collaborative Water Purification. (2017). Hackaday.io. Retrieved 30 October 2017, from https://hackaday.io/project/20812-make-water-collaborative-water-purification#menu-description

Rahman, A., Linton, E., Hatch, A., Sims, R., & Miller, C. (2013). Secretion of polyhydroxybutyrate in Escherichia coli using a synthetic biological engineering approach. Journal Of Biological Engineering, 7(1), 24. http://dx.doi.org/10.1186/1754-1611-7-24

Thermo Fisher Scientific. Overview of Western Blotting. Retrieved from https://www.thermofisher.com/ca/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/overview-western-blotting.html

van Hee, P., Elumbaring, A., van der Lans, R., & Van der Wielen, L. (2006). Selective recovery of polyhydroxyalkanoate inclusion bodies from fermentation broth by dissolved-air flotation. Journal Of Colloid And Interface Science, 297(2), 595-606