Restriction Digest

Materials:

Procedure:

1. Label PCR tubes.
2. Add all reagents to the tube.
3. Incubate for 30 minutes at 37°C. If both enzymes are Time-Saver Qualified, only incubate for 5-15 minutes.
4. Incubate for 20 minutes at 65°C.

Ligation

Materials:

Procedure:

1. Label PCR tubes.
2. Add all reagents to the tube.
3. Incubate for 10 minutes at room temperature.
4. Incubate for 10 minutes at 65°C.

Gibson Assembly

Materials:

Procedure:

• Into a clean 200ul PCR reaction tube
• Add: 5 ul of each fragment (part)
• Add: 10ul of Master Mix
• Add 10ul of dH20
• If you used 2-3 fragments, place in Thermocycler for 15 min. If you used 4 or more fragments, place in Thermocycler for 60 min with the appropriate cycle.

Golden Gate Assembly

Materials:

Procedure:

1. Label PCR tubes.
2. Add all reagents to the tube.
3. Mix gently
4. Place tubes on a thermocycler
5. Choose appropriate assembly protocol:
• For 1-4 Inserts: 37°C, 1 hr → 55°C, 5 min
• For 5-10 Inserts: (37°C, 1 min → 16°C, 1 min) x 30 → 55°C, 5 min
• For 11-20 Inserts: (37°C, 5 min → 16°C, 5 min) x 30 → 55°C, 5 min

Transformation

Materials:

• E. Coli Strains: BL21 (DE3)
• DNA Plasmids of Interest
• SOC Broth

Procedure:

1. Thaw a tube of BL21(DE3) Competent E. coli cells on ice until the last ice crystals disappear.
2. Mix gently by carefully tapping and carefully pipette 30 µl of cells into five transformation tubes on ice.
3. Label all tubes.
4. Add 5 µl containing 1 pg–100 ng of the respective plasmid DNA to each cell mixture. Do not add any plasmid to the Negative Control tube.
5. Carefully flick each tube 4–5 times to mix cells and DNA. Do not vortex.
6. Place the mixtures on ice for 30 minutes. Do not mix.
7. Heat shock at exactly 42°C for exactly 10 seconds. Do not mix.
8. Place on ice for 5 minutes. Do not mix.
9. Pipette 950 µl of room temperature SOC into each mixture.
10. Place at 37°C for 60 minutes. Shake vigorously or rotate (~5:12).
11. Transfer 100 µl of each dilution into separate labeled tubes with appropriate antibiotic and incubate overnight at 37°C.

Additional Notes:

• Thawing:
• Incubation of DNA with Cells on Ice:
• Outgrowth:
• Plating:

3% Glucose Media Recipe (Calgary 2017)

Materials:

• SOC
• Glucose
• Appropriate antibiotic if required (chloramphenicol, ampicillin)
• dH2O
• 1500-mL Erlenmeyer flask
• Stir bar
• Aluminum foil
• Hot water bath
• Overnight culture of PHB-producing bacteria

Procedure:

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.

Protein Miniprep using Nickel-Based Spin Columns

Materials:

• Lysis/Binding Buffer
• Wash Buffer
• Elution Buffer
• NEBExpress® Ni Spin Column
• Bacterial culture (sample)

Procedure:

1. Buffer Preparation

Buffer	2X IMAC Buffer	2M Imidazole	H2O	Total
Lysis/Binding Buffer	7.5 mL	0.025 mL	7.5 mL	15.0 mL
Wash Buffer	5.0 mL	1.25 mL	5.0 mL	10.0 mL
Elution Buffer	2.5 mL	5.0 mL	1.25 mL	5.0 mL

2. Sample Preparation
• Harvest cells by centrifugation at 4,000 x g for 15 minutes, store the pellet at -20°C or process immediately. Store supernatant as the extracellular component.
• Resuspend cell pellet in Lysis Buffer and lyse using the method of choice (use approximately 5 ml of lysis buffer per gram of cell paste).
• Note: Cells can be lysed by standard methods, including sonication, repeated freeze-thaw cycles, French press, etc. Other commercially available lysis reagents can also be used, following manufacturer’s instructions. It is recommended that imidazole be omitted from any lysis buffer.
• Centrifuge the sample at 12,000 x g for 15 minutes to pellet cellular debris. Remove the clarified protein lysate supernatant and transfer to a new microcentrifuge tube on ice. Insoluble cell components will be pelleted at the bottom of the tube.
• Note: A standard isolation typically employs 0.5 ml of clarified lysate.
• Collect supernatant containing soluble 6xHis-tagged proteins and transfer into a fresh tube.
3. Column Preparation
• Remove the bottom tab of the column by twisting, loosen the top cap, and place the column in the collection tube provided.
• Centrifuge the column at 800 x g for 1 minute to remove the storage buffer; discard the buffer.
• Add 250 μl of Lysis/Binding buffer to the column.
• Centrifuge the column at 800 x g for 1 minute; discard the Lysis/Binding buffer.
• Place the column in a new 2 ml microcentrifuge tube.
4. Lysate Binding
• Add up to 500 μl of the protein sample lysate to the column.
• Note: If the sample volume is greater than 500 μl, multiple applications can be performed; collect the flow through in separate microcentrifuge tubes.
• Tap the column to mix the lysate with the resin and allow binding for 3 minutes.
• Note: Binding of some His-tagged proteins can be increased with longer mixing times. Cap the column and seal the bottom using the plug provided. Mix end-over-end at 4°C for the desired time (typically 5-15 minutes). Prolonged mixing may result in more non-specific binding.
• Centrifuge the column at 800 x g for 1 minute; reserve the flow through.
• Place the column in a new 2 ml microcentrifuge tube.
5. Column Wash
• Add 250 μl of Wash Buffer to the column and centrifuge at 800 x g for 1 minute.
• Repeat the wash step twice more, collect each wash in the same 2 ml microcentrifuge tube.
6. Protein Elution
• Place the column in the same 2 ml microcentrifuge tube.
• Add 200 μl of Elution Buffer to the column. Mix the resin with the elution buffer thoroughly.
• Note: Elution volume can be reduced to 100 μl if a more concentrated protein sample is desired; the total target protein yield may be lower.
• Centrifuge at 800 x g for 1 minute; save the eluted sample.
• Place the column in a new 2 ml microcentrifuge tube and repeat the elution step. Typically, >90% of the bound protein is eluted following the second elution.
• Analyze the clarified cell lysate (load), flow through, washes, and eluates by SDS-PAGE.

Cell Lysis

Materials:

• Buffer NPI-10
• Lysozyme
• Benzonase®

Procedure:

1. Store the pellets at –20°C or at –70°C for at least 1 h.
2. Place frozen bacterial cells at room temperature and allow them to thaw for 15 min.
3. Add 100 µL buffer NPI-10 and 10 µL lysozyme solution (10 mg/ml) to the thawed cells. In addition, add 3 units of Benzonase for every ml of the original cell culture volume (for example, for a 100 ml cell culture, add 300 units of Benzonase).
4. Resuspend the pellet by pipetting up and down.
5. Incubate for 30 min at either 4°C or room temperature (15–25°C).

SDS Page Analysis

Materials:

• Electrophoresis Kit
• 1x SDS Glycine Buffer
• Page gel
• Aluminum foil
• Loading Dye

Procedure:

1. Set up the electrophoresis chamber by adding 400ml of 1x SDS - Glycine Buffer.
2. Unwrap a pre-made 15 well PAGE gel and place it in the buffer solution.
3. Carefully remove the comb and clear wells.
4. Using a hot plate or microwave, heat a beaker of water until it boils.
5. Cover with aluminum foil.
6. Tightly cap sample tubes. Push tubes through foil to suspend in the boiling water.
7. Incubate samples for 5 minutes.
8. Immediately proceed to loading the gel while the samples are still warm.
9. Connect the electrical leads to the power supply.
10. Set the voltage of the power supply and perform electrophoresis. Allow the proteins to separate on the gel for the recommended length of time, or until the tracking dye reaches the bottom of the gel.

Bradford Assay

Materials:

• 96 well plate reader
• 1x BSA Standard
• PBS
• Coomassie Blue G-250 (Bradford Dye)
• Sample with an unknown concentration of phasin

Procedure:

1. Fill lane A1 with 200μL of 1x BSA standard.
2. Fill lanes A2-A12 with 100μL of PBS.
3. Transfer 100μL of A1 into A2. Pipette up and down 3 times to mix, and using the same pipette, transfer 100μL of A2 into A3. Repeat up until A12, and after mixing A11, do not transfer to A12.
4. Pipette 20μL of the unknown sample into each individual well of the 96-well plate.
5. Add 40μL of Bradford dye to each well containing standard and sample.
6. Add 80μL of PBS to all unknown sample wells, bringing the final volume to 140μL.
7. Read absorbance at 595 nm without any prior incubation.

GFP Fluorescence Assay

Materials:

• 96 well plate reader
• Control Cell Extract Containing GFP
• 1x TE Buffer
• Sample with an unknown concentration of protein

Procedure:

1. Fill lane A1 with 200μL of GFP Standard.
2. Fill lanes A2-A12 with 100μL of TE buffer.
3. Transfer 100μL of A1 into A2. Pipette up and down 3 times to mix, and using the same pipette, transfer 100μL of A2 into A3. Repeat up until A12, and after mixing A11, do not transfer to A12.
4. Pipette 50μL of the unknown sample into each individual well of the 96-well plate.
5. Add 50μL of TE to all unknown sample wells, bringing the final volume to 100μL.
6. Read fluorescence at Blue 475 nm excitation (500-550 nm emission) without any prior incubation.

Sudan Black B Solution

Materials:

• Sudan Black B powder
• Propylene glycol

Procedure:

1. Add 0.7g of Sudan Black B powder into 100 mL of propylene glycol.
2. Heat to 100~110ºC and stir until fully dissolved.

Sudan Black B Staining for Microscopy

Materials:

• Bacteria Sample (in our case, E. Coli treated to produce PHB)
• Sudan Black B solution (0.3mg of solid Sudan Black B mixed with 100ml of 70% ethanol)

Procedure:

1. Staining will occur directly on petri dishes.
2. Directly smear alcoholic solution of Sudan Black (0.3%) on Petri dishes.
3. Incubate at 37 degrees C for 30 minutes.
4. Transfer one swab of bacteria from the petri dish to a microscope slide.
5. Swipe the slide over a Bunsen burner about 3 times.
6. Wash the stain with 96% ethanol, letting 96% ethanol run over the slide.
7. Observe with the naked eye if blue-black stains appear.
8. Move blue-black stained bacteria to slides.
9. Examine blue-black stained bacteria (PHB producers) under a microscope.

Sudan Black B Absorbance Assay

Materials:

• 96 well plate reader
• Sudan Black B (SBB) solution (0.7% w/v) in propylene glycol
• Distilled water
• Sample with an unknown concentration of protein

Procedure:

1. Fill lane A1 with 200μL of SBB solution.
2. Fill lanes A2-A12 with 100μL of dH2O.
3. Transfer 100μL of A1 into A2. Pipette up and down 3 times to mix and using the same pipette, transfer 100μL of A2 into A3. Repeat up until A12, and after mixing A11, do not transfer to A12.
4. Pipette SBB solution into unknown samples at a 1:10 ratio by volume of SBB solution to sample.
5. Mix either by slow centrifugation or pipetting up and down, and let sit for 40 minutes to stain.
6. Pipette 50μL of the unknown sample into each individual well of the 96-well plate.
7. Add 50μL of dH2O to all unknown sample wells, bringing the final volume to 100μL.
8. Read absorbance at 595nm without any prior incubation.