Team:CCA San Diego/Experiments

With the limited space and time available to us because of COVID-19, we had just over two months to perform our experiments. Still, we developed procedures from various sources to make Collagene through all steps of our Project Design. Here, you can find them categorized into protocols for expression (of the protein), implementation (gels and threads), and general procedures for the wet lab.

Implementation

Procedure

  1. Powdered ECM samples were solubilized with 1.0 mg/ml pepsin in 0.01 M HCl at a concentration of 10 mg ECM/ml
  2. Stirred at room temperature for 48 h to form a pre-gel solution
  3. To pH 7.4 with 0.1 N NaOH, isotonically balanced with 10x PBS(pH ~ 1.5–2.5)
  4. Diluted with 1x PBS to the final concentration of 8 mg/ml, which allows in vivo gelation
  5. Kept at 37 °C for ~120 min together with either genipin
  6. Keep genipin in 50% DMSO for genipin-crosslinked hydrogel (ECM/G) or with EDC (Sigma)

Materials

  • 100% Acetone (500mL)
  • NaCl (40g)
  • Deionized Water
  • Porcine Skin Sample
  • Citrate Buffer (800mL)
    • 8g Sodium Citrate
    • 800mL Deionized Water
  • Pepsin

Procedure

    Preparation of Porcine Skin

  1. Thoroughly wash a skin sample obtained from a deceseased pig.
  2. Use a razor or knife to slice off any muscle or fat layers beneath the dermis of the pig.
  3. Scrape both sides of the remaining skin layer to remove hair and subcutaneous fat.
  4. Cut the skin into tiny pieces (a blender can be used as well).
  5. Prepare a 100% acetone bath in a sealable container. Put the skin pieces into the acetone bath, seal it, and store in -20°C freezer for 30 minutes.
  6. Let the sealed acetone bath rest at room temperature for 1 hour.
  7. Remove the porcine skin from the container and rinse with deionized water.
  8. Prepare a 10% NaCl bath in a sealable container. Put the skin pieces into the NaCl bath, seal it, and store in a 4°C refrigerator for 24 hours.
  9. Remove the porcine skin from the container and rinse with deionized water.
  10. Make the sodium citrate buffer with 8g sodium citrate and 800mL distilled water. Make adjustments to reach pH 4.3.
  11. Separate porcine skin into separate 50mL tubes with 5g skin per tube. Pour 10mL citrate buffer into each tube. Vortex the solution lightly.
  12. Store the tubes in a 4°C refrigerator for 48 hours.

    13. Pepsin Digestion

  13. Each tube was filled to 30mL acetic acid and half received 600mg Pepsin too.
  14. Centrifuge the tubes at 3500rpm for 6 minutes.
  15. Prepare 20mL of 1.5M NaCl solution in deionized H2O.

Materials

  • Constant rate of Extension Tensile testing instrument with the following:
  • Load cell with a maximum of 1,000N capacity
  • 50mm/minute constant rate of extension
  • Distance between upper and lower sets of grips is 100 mm
  • Pneumatic Rubber faced grips (Rear 75 x 25mm and 25 x 25mm front)
  • For PC operated machines, appropriate software (to BS EN ISO 13934 Part 2;1999 )
  • Printer capability
  • Calibrated metal ruler

Procedure

  1. Cut 3 rectangular specimens parallel to the warp direction and 3 rectangular specimens parallel to the weft direction.
  2. Each Specimen should measure 150mm in length and 100mm in width
  3. Place a direction line parallel to the warp on each specimen prior to removing from the fabric
  4. Condition the specimens for a minimum of 4 hours in a conditioned atmosphere of 20 ± 2°C and 65 ± 2% RH before testing.
  5. Using an Automatic Constant Rate of Extension Tensile Tester
    1. The programme is automatic. The test speed is 50mm per minute.
    2. Use the 75mm x 25mm rubber faced grip on the rear and 25mm x 25mmm grip on the front.
    3. With the correct grips in place set the distance between upper and lower sets of grips to 100mm, this should be checked using a calibrated metal ruler.
    4. Select the correct programme and input the appropriate machine settings and fabric details etc.
    5. Fix the test specimen centrally in the upper grips, so that the 38mm line drawn on the specimen coincides with one edge of the grips.
    6. Close the upper grips.
    7. Avoiding any pretension, adjust the specimen along the guide line into the lower grips.
    8. Close the lower grips
    9. Set the cross head in motion.
    10. The maximum force at which the fabric ruptures (breaks) will be recorded.
    11. Repeat the above procedure for the remaining specimens.
  6. Any break which occurs within 5mm of the clamping line of the grips should be reported as a grip break
  7. If the grip break falls below the lowest normal break results then it should be disregarded and further samples tested
  8. If it falls above the lowest normal break result, then it can be included

Expression

Materials

  • Transformed Yeast Cells
  • Eppendorf tubes
  • Acetone
    • 80% acetone/li>
    • 20% TCA/Acetone
    • -20oC Acetone
    • (Supplement with 5 mM DTT)
  • SDS Extraction Solution
    • 1% SDS
    • 0.1 M Tris-HCl (pH 6.8)
    • 2 mM EDTA-Na2
    • 20 mM DTT
    • 2 mM PMSF (add before use)
  • All organic solvents pre-chilled at -20oC and contained 5 mM DTT added before use.

Procedure

  1. Prepare Crude Extract
    • (Note: 0.1-0.5 g sample in 1.0-1.5 mL buffer of choice). Homogenize in a SDS extraction buffer (in a mortar on ice)
    • Transfer the extract into eppendorf tubes
    • Centrifuge (15000g, 5 min, 4 C)
    • Pipette the supernatant to new tubes
  2. Supernatant
    • Add 20% TCA/acetone (1:1), fully vortex, and place on ice for 5 min
    • Centrifuge (15000g, 3 min, 4 C) and discard supernatant
  3. Protein Precipitate
    • Briefly wash with acetone and centrifuge (15000g, 2 min, 4 C). Discard supernatant.
    • Repeat Steps 8-9 at least one more time using 80% acetone
    • Air-dry proteins for 3 minutes (AVOID OVER-DRYING).
    • Dissolve proteins in buffer of choice.
  4. Protein Determination
    • Keep samples for SDS-PAGE or treat with proteolysis to remove globular domain.

Materials

  • Lysis Buffer
    • 50 mM Tris-HCl pH 8.0
    • 1% DMSO
    • 50-200 mM NaCl
    • 1 mM EDTA
    • 1 mM PMSF
    • 1 ug/ml leupuptin
    • 1 ug/ml pepstatin A
  • Equipment
    • Vortex or Bead Beater
    • Glass Beads (500 uM)
  • Protease inhibitor solution
    • (Always prepare solution fresh just before using. Place on ice to prechill).
    • Pepstatin A
    • Leupeptin
    • Benzamidine
    • Aprotinin
  • PMSF Stock solution (Appendix D.A; Add as necessary throughout the protocol.)
    • (Always prepare solution fresh just before using. Place on ice to prechill).
    • Dissolve 0.1742 g PMSF in 10 mL isopropanol. Wrap tube in foil and store at RT. PMSF primarily inhibits serine proteases. This is a 100x stock solution. PMSF is hazardous.

    Procedure

      Precipitation

    1. Prepare the glass beads by washing them in concentrated HCl, followed by extensive rinsing (check that then pH is neutral) and drying. The dried beads should be chilled before use.
    2. Resuspend cells in equal amount of chilled lysis buffer and place suspension in sturdy non-glass tube. Add PMSF (10 uL/ml of cell suspension) at this point
    3. Add 1-3 g of chilled glass beads/gram of cell wet weight.
    4. Vortex 3-5 times for 1 minute, keep cells on ice for 1 minute between vortexes. Use highest setting.
    5. Remove glass beads.
    6. Remove cell debris by ultracentrifugation at 4oC for 30 min at 45000 rpm using a 45 Ti rotor (Beckman).

      7. Pepsin Digestion

    7. 1 g of cell paste resuspended in 20 mL of 50 mM acetate buffer at pH 2, 3, and 4.
    8. Cells ruptured by sonication.
    9. Lysate clarified by centrifugation (15,000 x g for 30 min, 4 C).
    10. Held at 4 C for 16 h and any precipitate formed removed by centrifugation.
    11. Treat supernatant with proteolysis (.01 mg/ml pepsin was added and mixture incubated for 16 h at 4 C).

Materials

  • Dpn1 Enzyme (from -20 C: keep on ice)
  • PCR Product
  • 37 C Incubator

Procedure

  1. Add 2 uL of the enzyme to the completed PCR reaction. (1 uL if 20uL, 2 uL if 50 uL).
  2. Use P20 set set to -5uL, and pipette up and down 7 times. (P20 to 10uL if the reaction is 20uL, P200 to 25uL if the reaction is 50uL).
  3. Incubate at 37 degrees Celsius for 1-3 hours.

Materials

  • Wide Orifice Tips
  • "Hockey Stick" Inoculation Loops
  • SOC
  • High-Efficiency NEB5 Alpha Cells (stored at -80°C)

Procedure

  1. Put ice in an ice bucket.
  2. Transfer a tube of E. coli competent cells (high-efficiency NEB5 alpha cells) from -80°C to ice.
  3. Label a PCR strip and place it on ice for pre-cooling the PCR strip. Close the lid of the parts to be used.
  4. Place the DNA sample used for transformation on ice.
  5. Keep the tube of competent cells, the empty PCR strip, and the DNA sample on ice for 5 minutes.
  6. Using a wide-orifice tip (to be gentle to the cells) and a pipette set to 10 ul, pipette the cells up and down 5 times to make the suspension even.
  7. Add 2 ul of the E. coli cell suspension to a tube within the PCR strip.
  8. Using an inoculation needle, pin-transfer the DNA sample into the cell suspension, Tap the mixture inside the tube using the inoculation needle 10 times to mix.
  9. Keep the PCR strip on ice for 30 minutes.
  10. Return the tube of competent cells to -80°C.
  11. Use a thermocycler and set the temperature of the heat block to 42°C.

Materials

  • Dpn1 Enzyme (from -20 C: keep on ice)
  • PCR Product
  • 37 C Incubatorn

Procedure

  1. Add 2 uL of the enzyme to the completed PCR reaction. (1 uL if 20uL, 2 uL if 50 uL).
  2. Use P20 set set to -5uL, and pipette up and down 7 times. (P20 to 10uL if the reaction is 20uL, P200 to 25uL if the reaction is 50uL).
  3. Incubate at 37 degrees Celsius for 1-3 hours. prior to removing from the fabric

Materials

  • 1L Column (alternatively scale down to a syringe with small cotton bed)
  • Cell Lysate
  • 50% Ni-NTA Agarose Beads Slurry (50mL)
  • His-Wash Buffer (750mL)
    • 50 mM Tris-HCl pH 8.0
    • 10 mM Imidazole
    • 500 mM NaCl
    • 1 mM PMSF
    • Fill to deionized H2O
  • His-Elution Buffer (300 mL)
    • 500 mM Tris-Cl pH 8.0
    • 300 mM Imidazole
    • 50 mM NaCl
    • .1 mM EDTA
    • 1 mM PMSF
    • Fill to 300 mL deionized H2O

    Procedure

    1. Have an ice bucket ready on your bench.
    2. Prepare the wash and elution buffers with the necessary materials and molarities.
    3. Resin Preparation
      • Make sure your column is stably suspended in the air (preferably use a clamp stand). Place a waste beaker underneath the column.
      • Slowly pipette 50mL of 50% Ni-NTA Agarose Beads into the column. Wait for the liquid to drip out of the column. All that is left is 25mL of agarose beads (a bllue tint means they contain nickel).
      • Slowly pipette 200mL of Wash Buffer into the column. Pipette gently onto the side of the column. Wait for the liquid to drip out of the bottom.
    4. Purification
      • This step is optional: you can replace the waste beaker for a separate vial if you wish to sample the eluted E.coli particles for an SDS page.
      • Slowly pipette your cell lysate into the column (do not go over column capacity). If you are using a smaller column, you can do multiple lysate pours.
      • Slowly pipette 250mL of Wash Buffer into the column using the same gentle procedures. Wait for the liquid to drip out of the column. Do this same step a second time.
      • Remove the current collection vial from under the column. Prepare 20 labeled vials for protein collection (alternatively, you can use one vial).
      • Put the first of 20 vials under the column. Gently pipette 15mL of Elution Buffer into the column and wait for all liquid to drip out. Place the protein vial in the ice bucket.

Materials

  • LiOAc Mix
  • PEG Mix
  • YPD
    • li>
  • Salmon Sperm DNA
  • ddH2O
  • SD Dropout Plates
  • Yeast strain
  • Plasmid/DNA

Procedure

  1. Pick colony from plate into 5mL YPD for overnight growth in 30°C shaker.
  2. Back dilute over night culture to OD 0.175 in 15mL growth media.
  3. Grow up yeast to OD600 ~0.75 at 30°C. Takes 4-5 hours for WT in YPD.
  4. Pellet at 2000 rpm for 10 min in a large centrifuge with swinging buckets.
  5. Prepare lithium acetate transformation mix while harvesting cells.
    • 50% w/v PEG-3350
    • LiOAc 1.0 M
    • 10 mg/mL ssDNA
    • Plasmid DNA + H2O
  6. Pour off the medium, resuspend the cells in .5x volume of sterile water and repeat step 4.
  7. Pour off the water, resuspend the cells in 0.5x volume 100 mM LiAc and repeat step 4.
  8. Final suspension in 0.01x volume 100 mM LiOAc (100 uL per transformation - around 300 uL for 15 mL culture)
  9. Pellet at 8000 rpm for 1 min at 10 C on small tabletop centrifuge.
  10. Resuspend in the lithium acetate transformation mix. I suggest resuspending with the plasmid DNA and ddH2O (54 μL) first, then mixing with the LiOAc+PEG+ssDNA mixture (306 μL) afterwards, as it is too viscous to easily disrupt the pellet.Incubate 30°C for 30 min.
  11. Incubate for a further 17 min at 42°C in water bath/heat block.
  12. Centrifuge 8000 rpm for 2 min and remove supernatant.
  13. For a prototrpohic or auxotrophic marker, resuspend in 200 uL of H2O and incubate at RT for 10 min.
  14. Plate desired volume on plates. Typically do half. Incubate 30C for 2 days - small colonies should appear on the second day.

Materials

  • 1L Distilled Water
  • 20 g Powdered Agar
  • 28.4g CM Broth minus URA
  • 2 L Conical Flask
  • Petri Dishes

Procedure

  1. Pour 1L of distilled water into the concical flask. Measure 20g and 28.4g of Powdered Agar and CM Broth in weighboats.
  2. Pour the CM Broth powder into the conical flask. Dissolve the powder by shaking the flask.
  3. Shake the flask while pouring in the Agar powder. Mix the flask with a large magnet spinner for 3 minutes at 1000rpm, or until all agar clumps are dissolved.
  4. Autoclave on an autoclavable tray at 121°C, S:20. Leave in autoclave for the full cycle (52 minutes).
  5. Take the media out to cool down for 20-30 minutes after autoclave is done.
  6. While cooling the media, set the petri dishes under the fume hood and sterilize with UV light for 15 minutes. Pipette 25 mL of media into each dish, avoiding any bubbles.
  7. Leave the plate lids off until their moisture dissipates, and wait for agar to dry. Store at 4C.

Materials

  • Yeast Lysis Buffer
    • 20 mM HEPES, pH 7.6
    • 50 mM NaCl
  • Yeast Suspension Buffer
  • Zymolyase 20T
  • Beta-mercaptoethanol

Procedure

  1. Spheroplast Formation
    • Pellet yeast cells (bacterial culture, OD600 1.5-2.0) by centrifugation at 200-500 x g for 5-10 minutes. Suspend the cell pellet in an equal volume of Yeast Suspension Buffer. Add 1 µl of ß-mercaptoethanol per 100 µl yeast suspension.
    • Gently pipet up and down until the cell suspension is homogeneous. Incubate the suspension for 5 minutes at 4°C. Gently pipet again to suspend the cells.
    • Flick the vial containing Zymolyase® to mix the solution. Add 10 µl Zymolyase® for each 100 µl cell suspension in Yeast Lysis Buffer. Gently mix the content.
    • Incubate the suspension at 37°C for 30-60 minutes. Note: (OPTIONAL). Lysis can be monitored by taking 25 µl of suspension and mixing with 1 ml Yeast Lysis Buffer and reading the optical density at OD 800nm.
    • At the end of incubation, centrifuge the suspension at 10,000 x g for 5 minutes. Remove and discard the supernatant carefully, leaving the spheroplast pellet in the tube.
  2. Spheroplast Lysis.
    • For lysis, suspend the spheroplast pellet in an appropriate volume of the Yeast Lysis Buffer (2-3 times the volume of spheroplast pellet). Pipet the suspension up and down a few times. Vortex periodically and incubate on ice for 30 minutes. The lysis may be further facilitated by incubating the cells for 1-3 minutes at 37°C or a brief sonication step. Sonication is necessary for shearing genomic DNA. Please note, the higher Yeast Lysis Buffer to spheroplast pellet ratio the better the cell lysis.
    • Centrifuge the lysed cells at 20,000 x g, for 30 minutes at 4°C and collect the clear lysate. Note: Additional volume of Yeast Lysis Buffer can be purchased separately for downstream applications, e.g. chromatography and dialysis, etc.

Materials

  • 0.2 M Lithium acetate 1% SDS solution
  • Ethanol 96-100^ and 70%
  • Heat Block

Procedure

  1. Place one yeast colony from the plate or spin down 100-200 uL of liquid yeast culture (Od600 ~0.4). Suspend cells in 100 uL of 200 mM LiOAc, 1% SDS solution.
  2. Incubate for 5 minutes at 70 C.
  3. Spin down DNA and cell debris at 15000 g for 3 minutes
  4. Wash pellet with 70% ethanol.
  5. Dissolve pellet in 100 uL of H2O or TE and spin down cell debris for 15 seconds at 15000 g.
  6. Use 1 uL of supernatant for PCR.

General

Materials

  • 0.8 g Agarose (for 0.8% agarose)
  • TAE 0.5x Buffer
  • 6 uL DNA Ladder (mixed w/ Loading Dye)
  • 1 uL DNA samples
  • Gel Casting Tray
  • Gel Chamber
  • UV Transilluminator
  • Power Supply (15 W)

Procedure

  1. Mix 0.8g agarose with with 100 mL 0.5x TAE buffer.
  2. Heat by microwave for 1 min 45 sec at ~15-30 sec intervals.
  3. Pour the agarose solution into a gel tray with the well comb in place.
  4. Keep newly poured gel at room temperature for 20-30 min.
  5. Aliquot 1 uL of DNA samples and add 4 uL ddH2O and 1 uL 6x DNA Loading Buffer.
  6. Vortex and centrifuge.
  7. Remove comb once gel is solidified and pour rest of 1x TBE (75 mL into gel box). Make sure gel is fully emersed.
  8. Load samples into lanes, flanked by 6 uL DNA Ladder.
  9. Ensure power to the gel box is turned on. (The side where the DNA is placed is negatively charged - black- and the opposite side is positive - red).
  10. Run at 120 V for 40-60 min.

Materials

  • Nanodrop Machine
  • Elution Buffer
  • Wash Buffer
  • PCR Product (DNA fragment binding buffer to sample 5:1)

Procedure

  1. Nanodrop
    • Select "Nucleotide" on the machine.
    • Make sure the reading is 0 ng/ul (or a negative value) meaning that the machine is clean. If not, grab a kimwipe, wipe the pedestal (top and bottom) and add 2ul of water on the pedestal.
    • Click on “Measure.” Once you see the 0 ng/ul reading, proceed to wiping the water from the pedestal.
    • Add 2ul of Qiagen Elution Buffer to the pedestal, this will be your “Blank.” Once you see the 0ng/ul from your blank, wipe the liquid off the pedestal. Now you are ready to measure your samples
    • Load 2 ul of your sample on the pedestal, click “Measure” and write down the ng/ul value. Don’t forget to use a kimwipe to clean the pedestal after measuring each sample.
    • Finally, when you are done using the nanodrop machine, load 2 ul of water, click “Measure” and leave a kimwipe on the pedestal so that the next person using it will know it is clean.
    • DNA should have a 260/280 ratio between 1.8-2.0. RNA should be above 2.0. Contaminated DNA will have a low 260/280 of below 1.8.
  2. DNA Elution
    • In a 1.5mL microcentrifuge tube, add 2- 7 volumes of DNA binding buffer to each volume of DNA sample(Mix briefly by vortexing).
    • Transfer mixture to a Zymo-Spin column in a collection tube.
    • Centrifuge for 30 seconds. Discard the flow through.
    • Add 200uL DNA Wash Buffer to the column. Centrifuge for 30 seconds. Repeat the wash step.
    • Add >= 6uL DNA Elution Buffer directly to the column matrix and incubate at room temperature for one minute. Transfer the column for 30 seconds to elute the DNA.

Materials

  • Thin-walled 200 uL PCR tubes
    Table 2
    Reagent Volume
    Sterile Water 7.8 uL
    PrimeStar Max 10 uL
    Forward Primer (100 uM Stock) 0.6 uL
    Reverse Primer (100 uM Stock) 0.6 uL
    Template DNA 1 uL
    Final volume 20 uL
  • Thermocycler

Procedure

  1. In PCR tubes, add all the reagents listed in Table2.
  2. Briefly spin down the PCR tubes (2-3 s) using a tabletop microcentrifuge in order to ensure that all of the reagents are in the reaction mixture
  3. Place the PCR tubes into the selected thermocycler.
  4. Once the lid to the thermocycler is properly closed, start the required amplification program, as in table 3.
    5. Table 3
    Cycle Step Cycling Temperature Time Number of Cycles
    Denaturation 98 C 15 s 35
    Annealing 55 C 10 s 35
    Extension 72 C 35 s 35
    Final Extension 72 C 5 min 1
    Hold 12 C ∞ 1
  5. If the amplified DNA is NOT to be immediately used, label the tubes with your initials and date and temporarily store at 4°C or freeze at -20°C for long-term storage in assigned storage box

Materials

  • 1x Running Buffer
    • 950 mL water
    • 50 mL MOPS SDS Running Buffer
  • NuPAGE LDS Sample Buffer (4x)
  • NuPAGE Reducing Agent (opt.)
  • Deionized Water
  • Protein Sample
  • 8-15 BIS-TRIS gels from 4 C
  • Coomassie Blue Stain

Procedure

  1. Preparing the Samples
    • Prepare samples according to the table below.
    • Heat the samples at 70 C for 10 minutes.
  2. Preparing the Buffers
    • Add 50 mL of 20X NuPAGETM MES or MOPS SDS Running Buffer to 950 mL of deionized water to prepare 1X SDS Running Buffer.
    • For reduced samples, add 1 mL of NuPAGETM Antioxidant to 400 mL 1X SDS Running Buffer.
    • Preparing the Gel
      • Remove the comb, and rinse the gel wells three times using 1X Running Buffer.
      • Remove the white tape near the bottom of the gel cassettes.
      • Place the gels in the mini gel tank.
    • Loading the buffers
      • Fill the chambers with the appropriate 1X running buffer.
        • Mini Tank: Add 400 mL of buffer to each chamber.
        • XCell SureLockTM Mini-Cell: Add 600 mL of buffer to the lower chamber, and 200 mL to the upper chamber (for reduced samples, use running buffer with antioxidant in the upper chamber).
    • Loading the Samples and Ladders
      • Load the appropriate volume of the samples in the appropriate wells.
      • Load the protein ladder in the appropriate well.
    • Running the gel
      • Optimal run times vary depending on gel percentage and power supply used for electrophoresis.
        • If using MES Running Buffer, run for 35 minutes at 200 V constant.
        • If using MOPS Running Buffer, run for 50 minutes at 200 V constant.
        • Note: If not using a Thermo Fisher ScientificTM power supply, install NovexTM Power Supply Adapters.