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 11 μL DNA containing 1μL 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

Phosphorylated ends of DNA and RNA were removed, preventing unwanted ligation of linearized DNA.

Materials

Digested DNA vector

10X Antarctic phosphatase buffer

Antarctic phosphatase

ddH2O

Protocol

  1. To vector tube from restriction digest, add:

    • 1μL 10X Antarctic phosphatase buffer

    • 1μL Antarctic phosphatase

    • 8μL ddH₂O

  2. Incubate tube at 37°C for 30 minutes.
  3. Deactivate Antarctic phosphatase via heat shock by incubating tube at 80°C for 20 minutes.

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 Brilliant Blue or sivler salts, fluorographed, autoradiographed or used for immunoblotting.

Secretion

PHB Synthesis

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)

  • campicillin (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

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 Imperial College iGEM Team, 2013.

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 add 5mL 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.
  8. Allow powder to dry overnight in an open tube.

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

Process