iGEM 2017 — Team Calgary

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# Team:Calgary/Notebook

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[Design](https://2017.igem.org/Team%3ACalgary/Design)
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HUMAN PRACTICES

[Silver HP](https://2017.igem.org/Team%3ACalgary/HP/Silver)
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[Public Engagement](https://2017.igem.org/Team%3ACalgary/Engagement)

AWARDS

[Applied Design](https://2017.igem.org/Team%3ACalgary/Applied_Design)
[Entrepreneurship](https://2017.igem.org/Team%3ACalgary/Entrepreneurship)
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[Software](https://2017.igem.org/Team%3ACalgary/Software)

[JUDGING FORM](https://igem.org/2017_Judging_Form?team=Calgary)

# Calgary

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

##### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

##### Inspiration

You can see what others teams have done to organize their notes:

* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)

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# Team:Calgary/Experiments

Header

## ![iGEM](https://static.igem.org/mediawiki/2017/3/32/Calgary2017_Patch.png)

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* [Home](https://2017.igem.org/Team%3ACalgary)
* Project

  + [Problem/Description](https://2017.igem.org/Team%3ACalgary/Description)
  + Synthesis
    - [Synthesis Overview](https://2017.igem.org/Team%3ACalgary/Synthesis)
    - [Beta Oxidation](https://2017.igem.org/Team%3ACalgary/BetaOxidation)
    - [Glycolysis](https://2017.igem.org/Team%3ACalgary/Glycolysis)
  + [Secretion](https://2017.igem.org/Team%3ACalgary/Secretion)
  + Process
    - [Process Overview](https://2017.igem.org/Team%3ACalgary/Process)
    - [VFA Fermentation](https://2017.igem.org/Team%3ACalgary/VFA_Fermentation)
    - [Solid-Liquid Separation](https://2017.igem.org/Team%3ACalgary/SolidLiquidSeparation)
    - [PHB Fermentation](https://2017.igem.org/Team%3ACalgary/PHB_Fermentation)
    - [Extraction](https://2017.igem.org/Team%3ACalgary/Extraction)
    - [Products](https://2017.igem.org/Team%3ACalgary/Products)
  + [Applied Design](https://2017.igem.org/Team%3ACalgary/Applied_Design)
  + [Experiments](https://2017.igem.org/Team%3ACalgary/Experiments)
  + [Results](https://2017.igem.org/Team%3ACalgary/Results)
  + [Composite Parts](https://2017.igem.org/Team%3ACalgary/Composite_Part)
  + [Modelling](https://2017.igem.org/Team%3ACalgary/Model)
* [Notebook](https://2017.igem.org/Team%3ACalgary/Journal)
* ## ![iGemLogo](https://static.igem.org/mediawiki/2017/3/32/Calgary2017_Patch.png)
* Team
  + [Team Members](https://2017.igem.org/Team%3ACalgary/Team)
  + [Attributions](https://2017.igem.org/Team%3ACalgary/Attributions)
  + [Sponsors](https://2017.igem.org/Team%3ACalgary/Sponsors)
* Human
  Practices
  + [HP Summary](https://2017.igem.org/Team%3ACalgary/Human_Practices)
  + [HP Silver](https://2017.igem.org/Team%3ACalgary/HP/Silver)
  + [HP Gold/Integrated HP](https://2017.igem.org/Team%3ACalgary/HP/Gold_Integrated)
  + [Collaborations](https://2017.igem.org/Team%3ACalgary/Collaborations)
  + [Engagement](https://2017.igem.org/Team%3ACalgary/Engagement)
  + [Safety](https://2017.igem.org/Team%3ACalgary/Safety)
* Judging
  + [Medal Requirements](https://2017.igem.org/Team%3ACalgary/MedalRequirements)
  + [Interlab](https://2017.igem.org/Team%3ACalgary/InterLab)

# Our Experiments

## General Protocols

### Rehydration of Registry DNA

|  |  |
| --- | --- |
| **Experimental Details and Rationale** | Registry DNA was rehydrated for completion of the Interlab Study. Also, [Part:BBa\_K934001](http://parts.igem.org/Part%3ABBa_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** | - Add 10μL of ddH₂O to the desired well. - Pipette up and down 3-5 times. - Incubate at room temperature for 10 minutes. - Transform cells with 1μL of rehydrated DNA. Store the remaining amount at -20°C. |

### Rehydration of IDT Synthesized DNA

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

### Preparation of Agar with Antibiotics

|  |  |
| --- | --- |
| **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** | - 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. - Cover flask loosely with aluminum foil, secure with autoclave tape and sterilize by autoclaving for 20 minutes. - Remove agar from autoclave using oven mitts. Allow agar to cool until warm to the touch before adding appropriate antibiotic. - Stir on hot plate and magnetic stirrer for 30 seconds. - Pour agar into plates using aseptic technique. |

### Plating Culture Broth on Agar Plates

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

### Streaking Agar Plates

|  |  |
| --- | --- |
| **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** | - Using aseptic technique, flame inoculation loop until red hot. Allow it to cool for 10 seconds or touch it to agar. - 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. - Flame the inoculation loop until red hot. Allow it to cool for 10 seconds or touch it to agar. - Run the cooled inoculation loop through one of the previous streaks ONCE, then streak 1/4 of the surface of the agar. - Repeat Steps 3 and 4 two more times. - Flame the inoculation loop until red hot. Allow it to cool for 10 seconds. - Incubate plates at 37°C overnight or until growth is observed. |

### Plasmid Miniprep from *Escherichia coli*

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

### Restriction Digest

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

### DNA Ethanol Precipitation

|  |  |
| --- | --- |
| **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.](http://www.openwetware.org/wiki/Ethanol_precipitation_of_nucleic_acids) |
| **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** | - Add the following to your sample, in this order:  * 1/10 volume of 3M sodium acetate * 2-3 volumes of 100% ethanol  - Mix and freeze overnight at -20°C or at -80°C for 30-60 minutes. - Spin at 3000g for 30 minutes at 4°C. - Discard supernatant. - Dry the pellet in speed vac for 15-30 minutes. - Resuspend in ddH2O and store at -20°C or proceed with subsequent digest. |

### Agarose Gel Electrophoresis

|  |  |
| --- | --- |
| **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** | - 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). - Allow flask to cool in fumehood until warm to the touch before adding 1.5μL RedSafe Nucleic Acid Staining Solution. Gently swirl to mix. - Pour agarose into assembled gel casting tray. Remove any bubbles with a pipette tip and place comb in gel. - Allow gel to solidify and transfer to a gel running apparatus filled with TAE buffer. - Load samples of 10 μL DNA containing 2μL loading dye. - Run gel at 100 V for 30 minutes or until loading dye is 1/2 way down the gel. |

### DNA Excision from Low-Melting Point Agarose 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** | - 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). - Allow flask to cool in fumehood until warm to the touch before adding 1.5μL RedSafe Nucleic Acid Staining Solution. Gently swirl to mix. - Pour agarose into assembled gel casting tray. Remove any bubbles with a pipette tip and place comb in gel. - Allow gel to solidify and transfer to a gel running apparatus filled with TAE buffer. - Load samples of 10 μL DNA containing 2μL loading dye. - Run gel at 80 V for 30 minutes or until loading dye is 1/2 way down the gel. - 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 - Melt the gel at 65°C for 5 minutes for ligation. |

### Ligation of DNA Inserts to Plasmid Backbones

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

### Preparation of Chemically Competent *Escherichia coli*

|  |  |
| --- | --- |
| **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** | - Subculture strain (1:50) into 50ml LB. Add Stock MgSO4 and KCl to a final concentration of 10mM MgSO4 and 1mM KCl. - Shake at 28°C until OD600 = 0.3 - 0.4 is reached. - Chill on ice at least 10 minutes. - Put into 50 ml pre-chilled tube, centrifuge at 2500g for 8 minutes, at 4°C. - Re-suspend in 10ml ice-cold 100 mM CaCl₂, gently mix on ice, and incubate on ice for 10 minutes. - Centrifuge at 2500g for 8 minutes, at 4°C. - Re-suspend in 500ul 100mM CaCl₂ + 10% glycerol by gently pipetting up and down a few times on ice. - Incubate on ice for at least 10 minutes. - 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. |

### Transformation of *Escherichia coli*

|  |  |
| --- | --- |
| **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** | - Thaw 50μL aliquot of competent E.coli DH5α cells on ice just before use. - 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. - Heat shock for 60-75 seconds at 42°C. - Place on ice for 5 minutes - Add 2 mL Luria-Bertani or SOC medium to aliquot of cells. - Incubate cells for 60-90 minutes at 37°C, shaking at 200 rpm for 1 hour. - Plate 50-100μL of re-suspended culture on agar plate with appropriate antibiotic and spread. - Incubate plates at 37°C overnight or until desired growth is observed. |

### Glycerol Stock Preparation of *Escherichia coli*

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

### SDS-Page Gel Electrophoresis

|  |  |
| --- | --- |
| **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** | - Assemble glass plates into a holding cassette. - 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.  - Place distilled water into the remaining space upto the top of the glass plates. Wait about 30 minutes, or until gel is set. - After the gel sets, pour off the distilled water on top and use a kimwipe to wipe up any excess water. - 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  - Pour the stacking gel and place Teflon comb into solution. - Prepare the sample:  * 3μL of 1X SDS gel-loading buffer * 12uL of protein sample  - Heat the sample at 100°C for 3 minutes. - 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  - 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. - 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. |

### Coomassie Blue Staining of SDS-Page gels

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

## PHB Synthesis

### Preparation of 3% Glucose Media for PHB Production

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

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

### Preparation of Nile Red LB Agar Plates

|  |  |
| --- | --- |
| **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.](https://2013.igem.org/Team%3AImperial_College/Protocols) |
| **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** | - 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. - Add appropriate antibiotic. - Pour agar plates using aseptic technique. |

### Flow Cytometry with Nile Red for Detection of Intracellualr PHB

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

### Chloroform Extraction of PHB From *Escherichia coli*

|  |  |
| --- | --- |
| **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 the[Tokyo Tech iGEM Team, 2012.](https://2012.igem.org/Team%3ATokyo_Tech/Experiment/PHB2) 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** | - Centrifuge the 50mL overnight culture at 3275g for 10 minutes in a 50-mL Falcon Tube. - Discard supernatant and weigh pellet. Resuspend pellet in chloroform (1 mL chloroform per 2 mg of dried cells). - Incubate the solution in a shaker for 72 hour at 25°C - Filter the chloroform solution and concentrate the solution into a pellet by evaporation in a fume hood overnight. - Add methanol drop-wise to the pellet until it is just resuspended. - Filter the solution in methanol and dry the polymer in a fume hood overnight. - Add a little chloroform to dissolve the polymer then pour it into a sheet in the small Petri dish. - Let the sheet dry at room temperature. - Allow powder to dry overnight in an open tube. |

### Sodium Hypochlorite Extraction of PHB From *Escherichia coli*

|  |  |
| --- | --- |
| **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.](https://2013.igem.org/Team%3AImperial_College/Protocols#P3HB_production_and_extraction) 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 PBSTriton X-100 70% ethanol 50-mL Falcon TubesCentrifuge |
| **Protocol** | - 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. - Centrifuge again at 3275g for 10 minutes. Discard supernatant and resuspend pellet in 5mL 1% (v/v) Triton X-100 in PBS. - Incubate for 30 minutes at room temperature. - Centrifuge at 3275g for 10 minutes. Discard supernatant and resuspend pellet in 5mL 1X PBS solution. - Centrifuge at 3275g for 10 minutes. Discard supernatant then resuspend in5mL of sodium hypochlorite. - Incubate at 30°C for 1 hour - 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. - 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. |

### PHB Synthesis using VFAs as feedstock

|  |  |
| --- | --- |
| **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.- Label the 12 erlenmeyer flasks - 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 - 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 1. 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 2. 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 3. Incubate the flasks in a shaker for 24 hours at 37°C and 100 rpm 4. Obtain the flasks and pour solution to 50 ml Falcon tubes 5. Follow the extraction protocol |

## PHB Secretion

### Separation of PHB into secreted and intracellular fractions

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

### Purification of secreted PHB pellet

|  |  |
| --- | --- |
| **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** | - Resuspend the pellet of secreted PHB in 5 mL of 1% Triton X-100 in PBS. - Incubate for 30 minutes at room temperature. - 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. |

### Immunoblot of SDS-Page gels

|  |  |
| --- | --- |
| **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](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). |
| **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** | - Place the polyacrylamide SDS-Page gel into a plastic container. - 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:  ![Western Blot Sandwich](https://static.igem.org/mediawiki/2017/e/ed/Calgary2017_WesternBlot.jpg) - Transfer the proteins to the membrane at 100V for 1 hour . - Block the membrane by incubating for 1 hour at room temperature or in 100 mL TBSTM on a shaker. - 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. - Wash the membrane three times with 20 mL TBST for 5 minutes. - Incubate for 1 hour with secondary antibody at a dilution of 1:5000 in 10 mL TBSTM on a shaker. - Wash membrane three times with 20 mL TBST for 5 minutes. - 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

### Syn Poo Recipe 1

|  |  |
| --- | --- |
| **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:    - 800 mL of water - 60 g of yeast extract - 20 g of microcrystalline cellulose - 35 g of psyllium - 35 g of miso paste - 4 g of sodium chloride - 4 g of potassium chloride - 2 g of calcium chloride |

### Syn Poo Recipe 2

|  |  |
| --- | --- |
| **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** | ![](https://static.igem.org/mediawiki/2017/4/45/IgemCalgary2017_feces_recipe_2.jpg) |

### VFA Fermentation Experiments

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

### Gravity driven filtration Experiement

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

### Gravity driven sedimentation

|  |  |
| --- | --- |
| **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** | - Place samples into the beakers and label them representatively - Leave the beakers untouched for 24 hours - Pipet the middle liquid layer out of the beaker and measure the volume of collected liquid |

### Centrifugation for solid-liquid separation

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

### Staged Pressure Filtration Experiment

|  |  |
| --- | --- |
| **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** | - 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 - Place a paper towel filter disc into the AeroPress coffee filter device. Poor the liquid obtained in step 2 into the AeroPress. - 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. - Repeat the steps 3-4 with coffee filter, 11micron filter, and 0.2micor filter |

### Titration Experiments

|  |  |
| --- | --- |
| **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** | - Place the liquid sample into the beaker - Add magnetic stirring bar to the beaker and place the beaker onto the stirring magnetic plate - 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 - Measure the initial pH of the sample (should be above 6pH ), dilute with a base if needed - Add 1N sulphuric acid to the beaker 1ml at a time and measure the pH with every addition - 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- A1=(sulphuric acid concentration)(mol/ml)\*(volume of acid added)(ml)- [H1]=10^(-pH)- [VFA]- VFA concentration- k1- conditional dissociation acid for carbonic acid = 3.2\*10^(-7)- k2 - combined dissociation constant of the VFAs = 2.4\*10^(-5)   Rearrange the equation to obtain the expressions for VFA concentration |

### Chemical Coagulation

|  |  |
| --- | --- |
| **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. |

### Electrocoagulation

|  |  |
| --- | --- |
| **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. |

### High Performance Liquid Chromatography Measurement of Volatile Fatty Acid

|  |  |
| --- | --- |
| **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 |

### High Performance Liquid Chromatography Measurement of PHB

|  |  |
| --- | --- |
| **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

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  + [Problem/Description](https://2017.igem.org/Team%3ACalgary/Description)
  + Synthesis
    - [Synthesis Overview](https://2017.igem.org/Team%3ACalgary/Synthesis)
    - [Beta Oxidation](https://2017.igem.org/Team%3ACalgary/BetaOxidation)
    - [Glycolysis](https://2017.igem.org/Team%3ACalgary/Glycolysis)
  + [Secretion](https://2017.igem.org/Team%3ACalgary/Secretion)
  + Process
    - [Process Overview](https://2017.igem.org/Team%3ACalgary/Process)
    - [VFA Fermentation](https://2017.igem.org/Team%3ACalgary/VFA_Fermentation)
    - [Solid-Liquid Separation](https://2017.igem.org/Team%3ACalgary/SolidLiquidSeparation)
    - [PHB Fermentation](https://2017.igem.org/Team%3ACalgary/PHB_Fermentation)
    - [Extraction](https://2017.igem.org/Team%3ACalgary/Extraction)
    - [Products](https://2017.igem.org/Team%3ACalgary/Products)
  + [Applied Design](https://2017.igem.org/Team%3ACalgary/Applied_Design)
  + [Experiments](https://2017.igem.org/Team%3ACalgary/Experiments)
  + [Results](https://2017.igem.org/Team%3ACalgary/Results)
  + [Composite Parts](https://2017.igem.org/Team%3ACalgary/Composite_Part)
  + [Modelling](https://2017.igem.org/Team%3ACalgary/Model)
* [Notebook](https://2017.igem.org/Team%3ACalgary/Journal)
* ## ![iGemLogo](https://static.igem.org/mediawiki/2017/3/32/Calgary2017_Patch.png)
* Team
  + [Team Members](https://2017.igem.org/Team%3ACalgary/Team)
  + [Attributions](https://2017.igem.org/Team%3ACalgary/Attributions)
  + [Sponsors](https://2017.igem.org/Team%3ACalgary/Sponsors)
* Human
  Practices
  + [HP Summary](https://2017.igem.org/Team%3ACalgary/Human_Practices)
  + [HP Silver](https://2017.igem.org/Team%3ACalgary/HP/Silver)
  + [HP Gold/Integrated HP](https://2017.igem.org/Team%3ACalgary/HP/Gold_Integrated)
  + [Collaborations](https://2017.igem.org/Team%3ACalgary/Collaborations)
  + [Engagement](https://2017.igem.org/Team%3ACalgary/Engagement)
  + [Safety](https://2017.igem.org/Team%3ACalgary/Safety)
* Judging
  + [Medal Requirements](https://2017.igem.org/Team%3ACalgary/MedalRequirements)
  + [Interlab](https://2017.igem.org/Team%3ACalgary/InterLab)

# Results

### Synthesis Results

### Characterization of pET29B(+)-phaC1J4

[BBa\_K2260001](http://parts.igem.org/Part%3ABBa_K2260001)

The objective of our project was to genetically engineer *E. coli* to synthesize PHB. We designed a pET29b(+)-phaC1J4 construct to allow our engineered *E. coli*  to employ the beta-oxidation pathway to break down volatile fatty acids (VFAs) and use it to synthesize PHB. We have successfully ligated and transformed this part into *E.coli*. The figure below shows the digestion confirmation of the vector pET29B(+)-phaC1J4. We have also sequence verified this part.

![Gel Confirmation](https://static.igem.org/mediawiki/2017/a/a0/Calgary2017_PHACJCONFIRMGEL.png)

**Figure 1:** To confirm transformation of the pET29B(+) vector containing phaC1J4 into competent *E. coli* cells, a double digest confirmation was performed using the restriction enzymes NotI and HindIII. DNA samples were run on 1% agarose gel run at 100 V for 40 minutes.

To confirm the production of protein, we induced gene expression in  *E. coli* BL21(DE3) transformed with pET29B(+)-phaC1J4 with IPTG and performed SDS-PAGE. Our results are shown in the picture below. The SDS-PAGE results indicated that our protein PhaJ4 was being expressed. It was difficult to distinguish PhaC1 amongst the other protein bands. However, because PhaJ4 is downstream of PhaC1, it is likely that PhaC1 was being expressed as well, but further SDS-PAGE runs must be performed for confirmation. Furthermore, our PHB synthesis experiment with  *E. coli* containing pET29B(+)-phaC1J4 produced PHB as shown in Figure 3. The successful synthesis of PHB indicated the functionality of our exogenous proteins.

![SDS-PAGE](https://static.igem.org/mediawiki/2017/0/0e/Calgary2017_Oct24SDSPAGE.png)

**Figure 2:** SDS-PAGE of soluble (S) and insoluble (I) proteins obtained from *E. coli* containing pET29B(+)-phaC1J4 induced with 0.1 mM of IPTG at 37°C. Cells were lysed with lysozyme and sonication to harvest proteins. The gel was run at 30 mA for 50 minutes and stained with Coomassie Blue.

To confirm the ability of *E. coli* transformed with our pET29B(+)-phaC1J4 construct to produce PHB, we grew O/N cultures for 24 hours and induced them with 0.1 mM of IPTG while supplementing them with chemical media mimicking fermented synthetic poop supernatant (FSPS). Cells were then left to culture for 16 hours. To collect the resulting PHB, we performed sodium hypochlorite extraction on the cultures, which can be found on the [Experiments](https://2017.igem.org/Team%3ACalgary/Experiments) page.

![](https://static.igem.org/mediawiki/2017/3/33/Beta_ox_results.png)

**Figure 3:** PHB extracted from *phaC1J4*-expressing cells cultured for 24 hours and supplemented with FSPS for 16 hours. PHB was extracted using TritonX-100, sodium hypochlorite, and ethanol in a series of washes and incubation. The negative control tube contained *E. coli* transformed with the pET29B(+) vector (without insert).

To confirm that the identity of the white powder made by the transformed *E. coli* was actually our desired product of PHB, we ran our results alongside a sample of pure industrial-grade PHB provided to use by PolyFerm by dissolving PHB in sulphuric acid, which can be detected via HPLC as crotonic acid. The analysis confirmed the identity of our PHB and its similarity to industrial-grade material because the presence of crotonic acid was indicated by the peaks in the spectrum below.

![hplc](https://static.igem.org/mediawiki/2017/2/25/Abeta_hplc.png)

**Figure 4:** HPLC spectra of pure PHB powder from PolyFerm (left) and PHB powder produced from our phaC1J4-expressing bacteria (right) digested with crotonic acid.

**Detailed results of these experiments can be found on this part's [Registry](http://parts.igem.org/Part%3ABBa_K2260001) page.**

### Characterization of pET29B(+)-phaCBA

[BBa\_K2260000](http://parts.igem.org/Part%3ABBa_K2260000)

As part of our project, we also used a pathway that relied on acetyl-CoA, a metabolite from the breakdown of glucose and some VFAs. We designed a pET29B(+)-phaCBA construct so that the *E. coli*  could convert acetyl-CoA to PHB. We have successfully ligated and transformed this part into *E. coli*. The figure below shows the digestion confirmation of the vector pET29B(+)-phaCBA. We have also sequence verified this part.

![Gel Confirmation 2](https://static.igem.org/mediawiki/2017/2/26/Calgary2017_RESULTSphaCBAConfirmGel.png)

**Figure 5:** To confirm transformation of the pET29B(+) vector containing phaCBA into competent *E. coli*, a double digest confirmation was performed using the restriction enzymes NotI and KpnI. DNA samples were run on 1% agarose gel.

We then cultured these transformed cells following the same method described above to product and extract PHB.

![Gel Confirmation](https://static.igem.org/mediawiki/2017/7/71/Results_phacba_phb_production.png)

**Figure 6:** PHB extracted from *phaCBA*-expressing cells cultured for 24 hours and inoculated with FSPS for 16 hours. PHB was extracted using TritonX-100, sodium hypochlorite, and ethanol in a series of washes and incubation. The negative control tube contained *E. coli* transformed with the pET29b(+) vector (without insert).

Again, To confirm that the identity of the white powder made by the transformed *E. coli* was PHB, we ran our results alongside a sample of pure industrial-grade PHB provided to use by PolyFerm by dissolving it in sulfuric acid. This leads to the production of crotonic acid peaks after HPLC analysis. The analysis confirmed the identity of our PHB, as the presence of crotonic acid was indicated by the peaks.

![Gel Confirmation](https://static.igem.org/mediawiki/2017/7/74/Cba_hlpc_results.png)

**Figure 7:** HPLC analysis of pure PHB powders from PolyFerm (left) and PHB powders produced from *phaCBA*-expressing bacteria (right) digested with crotonic acid.

**Detailed results can be found on this part's [Registry](http://parts.igem.org/Part%3ABBa_K2260000) page.**

### Secretion Results

### Characterization of pSB1C3-Phasin-HlyA Tag

[BBa\_K2260002](http://parts.igem.org/Part%3ABBa_K2260002)

In order to secrete PHB, we engineered the fusion protein phasin-HlyA tag first into *E. coli* DH5α for plasmid propagation then into *E. coli* BL21(DE3) for IPTG-inducible protein expression. Figure 8 shows the successful digestion of the phasin-HlyA tag construct with EcoRI and SpeI ligated in the pSB1C3 backbone.

![Secretion Journal Phasin Gel](https://static.igem.org/mediawiki/2017/5/58/Calgary2017_SecretionJournalPhasinHlyAGel.png)

**Figure 8:** Screening results of *E. coli* transformed with pSB1C3-phasin-HlyA tag. The plasmid was double digested with EcoRI and SpeI (DD) then run on a 1% agarose gel at 100 V for 30 minutes. The molecular ladder (L) is visible on the far left and the expected band sizes, obtained from Benchling Virtual Digest, are visible in the diagram on the right. Undigested plasmid (U) was used as a control and the double digests (which had successfully received our part) are visible in lanes 3 and 4.

To confirm the functionality of the phasin-HlyA protein, a "super" plasmid that contains both PHB-producing and PHB-secreting genes was made. This plasmid was created by ligating the phasin-HlyA construct into pSB1C3-phaCAB. *PhaCAB* is a PHB-producing BioBrick from [Imperial College, 2013](http://parts.igem.org/wiki/index.php?title=Part:BBa_K1149052)). This plasmid was transformed into *E. coli* BL21(DE3). The successful ligation of the "super" plasmid was confirmed with NotI restriction digest, as shown in Figure 9.

![Secretion phaCAB-phasin gel](https://static.igem.org/mediawiki/2017/1/15/Calgary2017_SecretionCAB_PhasinGel.png)

**Figure 9:** Screening results of 4 colonies of *E. coli* transformed with pSB1C3-phaCAB-Phasin-HlyA. Plasmid from the colonies were digested with NotI (D) then run on a 1% agarose gel at 100 V for 30 minuntes. The molecular ladder (L) is visible on the far left and the expected band sizes, obtained from Benchling Virtual Digest, are visible in the diagram on the right. Undigested plasmid (U) was used as a control and the digests from colony 6 are visible in lanes 6-7.

For a secretion assay, cultures of *E. coli* BL21(DE3) containing the "super" plasmid were inoculated in LB media + chloramphenicol + 3 % glucose. pSB1C3-pPhaCAB without the phasin-HlyA construct in *E. coli* BL21(DE3) was used as a control, and the experiment was run in triplicates. The bacteria were induced with IPTG, then incubated at 37°C for either 24 hours or 48 hours. After that time, the samples were separated into intracellular and secreted PHB fractions. CaCl2 was added to promote PHB agglomeration in the secreted fraction so that it could be separated from cells via differential centrifugation. The amount of PHB collected in each fraction was measured and those results are presented below (Table 1).

The addition of CaCl2 to the secreted fractions results in a pellet being obtained that contains both PHB and CaCl2. To account for this, the mass of secreted PHB was corrected for the mass of CaCl2 added (0.05549 g). The corrected results are presented in Table 2.

The assay showed that cells with PHB-secreting genes secreted more PHB than control cells which did not contain these genes, but this difference only became apparent after 48 hours of incubation at 37°C. At 24 hours, there was no meaningful difference between the amounts of PHB in the media of both *E. coli* variants (0.12851 g for the control and 0.12417 for the positive PHB-secreting samples). At 48 h, control cells had only 0.08051 g of PHB in the media, but the PHB-secreting cells secreted 0.17251 g. This is a 114% increase in the amount of PHB secreted by cells with Phasin-HlyA Tag compared to the control. This indicates that our part, Phasin-HlyA Tag, improves secretion of PHB after an incubation time of at least 24 hours.

**Table 1:** Experimental results of secretion assay. (-) is used to denote negative control, BL21(DE3) with pSB1C3-PhaCAB and (pSB1C3-Phasin) is used to denote PHB-secreting strains, BL21(DE3) with pSB1C3-PhaCAB-Phasin-HlyA Tag.

![](https://static.igem.org/mediawiki/2017/8/86/Calgary2017_SecretionCalculated.jpg)

**Table 2:** Corrected experimental results of secretion assay. (-) is used to denote negative control, pSB1C3-PhaCAB and (pSB1C3-Phasin) is used to denote PHB-secreting strains, pSB1C3-PhaCAB-Phasin-HlyA Tag. The amount of PHB secreted was corrected to account for mass of the CaCl2 that was added to promote PHB agglomeration.

![](https://static.igem.org/mediawiki/2017/2/2b/Calgary2017_CorrectedSecretion.jpg)

![](https://static.igem.org/mediawiki/2017/d/d1/Secretionfinal.JPG)

**Figure 10:** Experimental results of secretion assay. (-) is used to denote negative control, pSB1C3-PhaCAB and (pSB1C3-Phasin) is used to denote PHB-secreting strains, pSB1C3-PhaCAB-Phasin-HlyA Tag. There is a 114 % increase in the amount of PHB secretion by (pSB1C3-Phasin) compared to (-) when time = 48 hours (0.17251 g and 0.08051 g of secreted PHB, respectively).

**These results can also be found on this part's [Registry](http://parts.igem.org/Part%3ABBa_K2260002) page.**

### Process Development Results

### Methods for VFA quantification and characterization

As mentioned in our [Notebook](https://2017.igem.org/Team%3ACalgary/Journal), determination of the total VFA concentration in the solution was an important step in the process – knowing how to quantify total VFAs in the solution helped to prove that the fermentation of human feces with naturally-occurring bacteria increases the VFA concentration, as well as it helped to prove VFA presence in both fermented and unfermented synthetic feces.

**Titration** is commonly employed by wastewater treatment plants to give a rapid estimate of the VFA concentration in sample solution. Toe measure VFA concentration, we performed Simple Titration procedures, which can be found on our [Experiments](https://2017.igem.org/Team%3ACalgary/Experiments) page. The results in Table 3 indicate that this method tends to give a slight overestimate of the total concentration; however, it can be used for quick estimations and show changes in overall VFA concentration.

**Table 3:** Titration experiments results

| Experimental Conditions | Trial 1 | Trial 2 | Trial 3 |
| --- | --- | --- | --- |
| Actual VFA Conentration (mg/L) | 60 | 60 | 60 |
| Sample volume (mL) | 40 | 40 | 40 |
| Acid normality | 0.1 | 0.1 | 0.1 |
| **Results** |  |  |  |
| Original pH | 6.61 | 6.6 | 6.61 |
| Volume of acid added to titrate to pH 5 (mL) | 0.53 | 0.53 | 0.536 |
| Volume of acid added to titrate to pH 4.3 (mL) | 0.745 | 0.75 | 0.785 |
| Volume of acid added to titrate to pH 4 (mL) | 0.825 | 0.830 | 0.858 |
| Calculated VFA concentration (mg/L) | 66.1 | 67.7 | 74.9 |

**HPLC** is another method commonly employed in laboratory setting for the VFA concentration determination. The advantage of the method is the fact that it provides the concentration of different volatile fatty acids in the solution.

Table 4 shows VFA concentrations in fermented synthetic feces supernatant as measured by the HPLC. Since the amount of VFAs in the supernatant was unknown, different dilution factors were tested to ensure the VFA concentrations are within the optimal HPLC detection range.

**Table 4:**  VFA concentrations in fermented synthetic feces supernatant as measured by the HPLC at different dilutions.

| Sample Dilution | Acetate (mM) | Propionate (mM) | Butyrate (mM) |
| --- | --- | --- | --- |
| Undiluted | 108.9 | 197.55 | 12.65 |
| 1:32 | 37.15 | 99.27 | 8.03 |
| 1:128 | 41.10 | 109.17 | 9 |
| 1:256 | 40.53 | 111.77 | 11.67 |

### VFA fermentation results

We fermented synthetic feces to mimic the fermentation by natural gut flora that produce VFAs in the supernatant. In our process, these VFAs can later be used as a feedstock for our PHB-producing *E. coli*. First, we determined VFA production by leaving synthetic feces to ferment at 22 and 37°C. This experiment was performed before the Synthesis team had successfully created our PHB-producing parts, so we borrowed those of other teams available in the iGEM Registry (from [Imperial College 2013](http://parts.igem.org/wiki/index.php?title=Part%3ABBa_K1149051) and [Tokyo 2012](http://partsregistry.org/wiki/index.php?title=Part%3ABBa_K934001 )). The first VFA fermentation experiment showed higher VFA production at 37°C than at 22°C (Figure 11). The control condition was *E. coli*  BL21(DE3) transformed with pET29B(+) vector without any inserts (non-PHB producing), "Imperial condition" was PHB-producing *E. coli* transformed with a PHB-producing part from [Imperial College 2013](http://parts.igem.org/wiki/index.php?title=Part%3ABBa_K1149051), and "Tokyo condition" was PHB-producing *E. coli* transformed with a PHB-producing part from [Tokyo 2012](http://partsregistry.org/wiki/index.php?title=Part%3ABBa_K934001 ). The reported results are an average of 3 titrations performed for each sample.

![](https://static.igem.org/mediawiki/2017/2/2c/Calgary2017_VFAFermentResults1.jpeg)

**Figure 11:** VFA fermentation results for the first experiment.

We performed a second VFA fermentation experiment confirmed higher VFA production at 37°C than at 22°C (Figure 12). In addition to a 3-day fermentation (denoted as D3), a 5-day fermentation was also introduced during this experiment (denoted as D5). Higher VFA concentrations were observed after 5 days of fermentation. Similarly to the first experiment, the reported results are an average of 3 titrations performed for each sample.

![](https://static.igem.org/mediawiki/2017/5/53/Calgary2017_VFAFermentResults2.jpeg)

**Figure 12:** VFA fermentation results for the second experiment.

In subsequent experiments, the supernatant from fermented synthetic feces was collected after the VFA fermentation experiments, sterilized, and cultured with PHB-producing bacteria for 2 – 3 days at 37°C. PHB-producing bacteria cultured in the supernatant from synthetic feces fermented at 37°C had little to no growth, resulting in little to no PHB produced.
Although the supernatant from synthetic feces fermented at 37°C had higher VFA concentration (Figure 11, Figure 12), we believe the low pH of these samples prohibited bacterial growth, resulting in no PHB production. Consequently, the synthetic feces supernatant fermented at 22°C was selected as preferred VFA fermentation temperature due to optimal pH for bacterial growth, although it had a lower VFA concentration.

### Liquid-solid separation results

The first experiments we performed for solid-liquid separation of synthetic feces from supernatant were Gravity-Driven Sedimentation and Gravity-Driven Filtration; both protocols can be found on the [Experiments](https://2017.igem.org/Team%3ACalgary/Experiments) page. Both methods rely on gravity to settle solids in a phase separate from that of the liquid. The results of these experiments are summarized in Table 5 below:

**Table 5:**  Gravity driven filtration and sedimentation results

| Gravity driven filtration | Weight of water present in sample (g) | Weight of liquid recovered after 24 hours (g) | Percent of liquid recovered (%) |
| --- | --- | --- | --- |
| Sample 1 | 15 | 0 | 0.00 |
| Sample 2 | 40 | 20.4 | 51.00 |
| Sample 3 | 65 | 52.5 | 80.77 |
|  |  |  |  |
| Gravity Driven sedimentation | Weight of water present in sample (g) | Weight of liquid pipetted out after 24 hours (g) | Percent of liquid recovered (%) |
| Sample 1 | 15 | 0 | 0.00 |
| Sample 2 | 40 | 21.4 | 53.50 |
| Sample 3 | 65 | 47.5 | 73.08 |

It is clear the gravity alone did not sufficiently separate solids from liquids; therefore, the Staged Filtration [Experiment](https://2017.igem.org/Team%3ACalgary/Experiments) was conducted using 25 g of synthetic feces (Recipe 2). This method relied on using a series of filters with sequentially smaller pores to separate solids from liquid in multiple steps. The original sample contained 15 g of water, yet only 10% of it was recovered, meaning that a more advanced and power intensive technology has to be considered for this stage of the process.

**Table 6:**  Staged filtration experiments results

| Filtration type | Weight of liquid recovered (g) | Liquid lost due to transfer (g) |
| --- | --- | --- |

 comments || Strainer | 18.6 | 1.1 | Yellow thick liquid went through. Yeast bodies we visible in the filtrate. |
| "Paper towel" filter | 13.9 | 1.8 | A thick creamy-yellow sludgy layer remained on the filter and could be scraped down. Yeast bodies could still be visible |
| Coffee filter | 8.6 | 1.5 | Another similar looking creamy-yellow layer was scraped down. The yeast bodies were not visible in the liquid any more |
| 11 micron filter | 5.8 | 1.2 | Had to press very hard on the top of the filter to push the liquid through. |
| 0.2 micron filter | 1.5 |  | The majority of the liquid was not recovered because the filter got clogged. The recovered liquid had a brown tint, but appeared clear and transparent. |

Finally, we decided to investigate the efficiency of centrifugal-based extraction methods using the Centrifugation for Solid-Liquid Separation [Experiment](https://2017.igem.org/Team%3ACalgary/Experiments). When a 50 g undiluted sample of synthetic feces (Recipe 2) was tested, the mass of water recovered was 19.6 g, while the mass of initial water present in the sample was 30 g, meaning that there was 65% recovery of water. This result indicated that centrifugal based solid-liquid separation technology would be the best fit for our application.

### PHB Extraction Results

#### Chemical Coagulation Results

We demonstrated in the lab that adding calcium chloride and then centrifuging increased the amount of PHB removed from a suspension in water, as CaCl2 assists in PHB agglomeration. We carried out centrifugation at various speeds and then collected absorbance data for the samples to measure relative quantity of PHB in solution. The lower the absorbance reading, the higher the amount of PHB that was removed.

![](https://static.igem.org/mediawiki/2017/8/80/Calgary2017_ChemicalCoagulationTubes.JPG)

**Figure 13:** From left to right, PHB suspension after centrifugation at 1000 RPM, PHB suspension after centrifugation at 3750 RPM, PHB suspension after addition of 10 mM final concentration of CaCl2 and centrifugation at 3750 RPM.

Details concerning the methods of this experiment can be found [here](https://2017.igem.org/Team%3ACalgary/Experiments). The absorbance readings and the standard deviations of the various conditions tested are shown in the figure below:

![]( https://static.igem.org/mediawiki/2017/a/af/Calgary2017_ChemicalCoagulation.jpg)

**Figure 14:** Absorbance readings and their standard deviations for the various extraction methods tested.

As expected, centrifugation at higher RPM resulted greater removal of PHB from solution. The addition of CaCl2 increased PHB agglomeration, resulting in additional PHB extraction.

#### Electrocoagulation

We then looked at the possibility of using electrocoagulation to remove PHB from solution. We tested electrocoagulation on microscale PHB particles in suspension in water, nanoscale PHB particles in suspension in water, synthetic feces supernatant, and a 1:1 mixture of microscale PHB suspension and synthetic feces supernatant.

![](https://static.igem.org/mediawiki/2017/c/ce/Calgary2017_Electrocoagulation.JPG)

**Figure 15:** From left to right, electrocoagulation set-up using nanoscale PHB suspension in water, electrocoagulation set up using 1:1 mixture of PHB suspension, and synthetic feces supernatant.

From our preliminary experiments with just PHB particles in a water suspension, we were able to demonstrate that PHB particles do settle out via electrocoagulation. After we stopped running electricity through our apparatuses, we were able to observe a layer of PHB at the bottom of the container. However, after a few hours a layer of brown powder settled on top of the white layer of PHB. We hypothesized that this was probably iron(III) hydroxide, formed from the excess iron ions released by the anode that did not bind to the PHB.

![](https://static.igem.org/mediawiki/2017/1/18/Calgary2017_ElectrocoagulationMicroPHB.JPG)

**Figure 16:** From left to right, PHB settling via electrocoagulation. Layers of PHB and brown powder settled after electrocoagulation.

From our experiments with synthetic feces supernatant, we found that a layer of brown sludge settled at the bottom each time. Even with the 1:1 mixture there was no discernable layer of PHB within the sludge. We tried washing the sludge with dilute acid to remove the metal salts that might have been present in the sludge. However, we were still unable to separate PHB from the sludge.

The electrocoagulation experiments led us to conclude that while it is possible to settle out PHB using electrocoagulation, this method was not suitable for our media which contained a number of salts that interfered with the coagulation process and caused the formation of sludge.

### High Performance Liquid Chromatography (HPLC) Analysis of PHB

Crotonic acid is the product of PHB digestion with sulphuric acid, and itcan be detected using HPLC. During the first HPLC run with our PHB samples extracted from cell culture, we didn’t observe crotonic acid peaks as expected. This was likely due to digesting our PHB in sulphuric acid for 3 hours, which was likely too long, and resulted in degradation of crotonic acid in solution.

For our second run of HPLC trials, we tested two different sulphuric acid digestion times (20 minutes and 30 minutes) and observed peaks for crotonic acid for samples obtained from our [phaCBA part](http://parts.igem.org/Part%3ABBa_K2260000) and **confirmed that PHB was present in the our samples**. We also ran HPLC trials on PHB produced using [Imperial College's 2013 BioBrick](http://parts.igem.org/wiki/index.php?title=Part%3ABBa_K1149051) and observed crotonic acid peaks as well.

Although we were successful at detecting PHB using HPLC, quantification of PHB using HPLC would require further development. In particular, an optimal digestion time needs to be selected where all of the PHB in the sample is converted to crotonic acid **and** crotonic acid does not start to degrade. Nonetheless, we attempted to quantify our PHB samples by assuming an 80% conversion of PHB to crotonic acid, a value reported by Karr *et al.* (1983). However, conversation factors in our HPLC runs ranged from 40% to 60% for the standard PHB samples, resulting in inaccurate dilution factor predictions, and we were not able to quantify our samples. Accurate dilutions are important to ensure the sample concentration is within the HPLC detection range.

For our third HPLC run, we digested PHB samples from our beta-oxidation [BioBrick](http://parts.igem.org/Part%3ABBa_K2260001) for 15 and 30 minutes and observed clear peaks for crotonic acid. Again, we based our dilution factors on 60% conversion of PHB to crotonic acid. More details about the HPLC results can be found on the [Beta-Oxidation](http://parts.igem.org/Part%3ABBa_K2260001) and [Glycolysis](http://parts.igem.org/Part%3ABBa_K2260000) part pages.

### Tools from PHB

We used commerical PHB, the same type of bioplastic that was produced in the lab by genetically engineered bacteria, to make a 2.5 cm (length) by 1.1 cm (width) wrench (Figure 17). The wrench was made by melting PHB into a wrench-shaped mold and letting the plastic to cool.

![Wrench](https://static.igem.org/mediawiki/2017/d/dc/Calgary2017_Wrench.jpg)

**Figure 17:** A wrench molded from commercial PHB, the same type of plastic that was produced by engineered bacteria.

## Works Cited

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.

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