Welcome to the Experiments section of the BARBIE Project. Here, you will find comprehensive protocols and procedures detailing the experiments conducted throughout this year’s journey. Each section is crafted to guide you through the methodologies and techniques we employed, featuring an interactive protocol* for deeper exploration and learning of protein production.

Materials

  • Equipment: Petri Dishes, Erlenmeyer Flask, Autoclave, Sterile Pipettes
  • LB Broth (To prepare 1 L ⇒ 10 g Tryptone; 5 g Yeast Extract; 10 g NaCl; 20 g Bacto Agar and complete with water)
  • Antibiotic (if needed)

Steps

  1. Prepare LB Broth: Combine 10 g tryptone, 5 g yeast extract, 10 g NaCl, and 20 g agar in 800 mL sterile water. Adjust volume to 1 L and pH to 7.0-7.5.
  2. Sterilisation: Autoclave the broth at 121°C for 25 minutes.
  3. Pour Plates: Add the antibiotics if needed. Pour ~20 mL of agar mixture into sterile Petri dishes and allow to solidify.
  4. Storage: Invert plates to prevent condensation, wrap or bag them, label, and store at 4°C.

Materials

  • Bacterial Strain: E. coli (e.g., DH5α)
  • Growth Medium: Luria Broth (LB)
  • Glycerol: 10% sterile solution
  • Equipment: Erlenmeyer Flasks, Centrifuge, Ice or Cold Room (4°C), Sterile Microcentrifuge Tubes, Electroporation Cuvettes

Steps

  1. Inoculate Bacteria: Streak a single colony of E. coli on an LB agar plate and incubate overnight at 37°C.
  2. Inoculate a Single Colony: Transfer into 3-5 mL of LB broth and shake overnight at 37°C.
  3. Dilute and Grow: Dilute the overnight culture 1:100 to 1:500 in fresh LB broth (0.5 to 1 L) and grow until OD600 reaches ~0.4-0.6 (mid-log phase), approximately 3-6 hours.
  4. Harvest Cells: Cool the culture on ice for 10-15 minutes.
  5. Transfer and Centrifuge: Transfer to pre-chilled centrifuge bottles and centrifuge at 5000 rpm for 5-10 minutes at 4°C to pellet cells.
  6. Wash Cells: Resuspend pellet in equal volume of ice-cold sterile deionized water and centrifuge again.
  7. Repeat Wash: Repeat the wash step two more times, gradually reducing the volume of water to about half the original culture volume.
  8. Glycerol Wash: Resuspend the final pellet in ~20 mL ice-cold sterile 10% glycerol solution.
  9. Centrifuge Again: Centrifuge at 10,000 rpm for 10-15 minutes at 4°C.
  10. Final Resuspension: Resuspend the pellet in a small volume of ice-cold sterile 10% glycerol (3-4 mL per liter of original culture).
  11. Aliquot: Aliquot suspension into pre-chilled microcentrifuge tubes (e.g., 40 µL per tube).
  12. Storage: Freeze aliquots in liquid nitrogen or dry ice/ethanol bath for long-term storage. Store at -80°C until needed.
  13. Using Competent Cells: Thaw an aliquot on ice, add salt-free DNA (1-5 µL), mix gently, and transfer to an electroporation cuvette. Electroporate as per manufacturer's instructions, then immediately add recovery medium (e.g., SOC) and incubate at 37°C for ~1 hour before plating on selective media.

Materials

  • Bacterial Strain: E. coli (e.g., DH5α)
  • Growth Medium: Luria Broth (LB)
  • Glycerol: 15% sterile solution
  • Calcium Chloride (CaCl₂): 100 mM solution, ice-cold
  • Equipment: Erlenmeyer Flasks, Centrifuge, Ice or Cold Room (4°C), Sterile Microcentrifuge Tubes, Spectrophotometer for Measuring Optical Density

Steps

  1. Inoculate Bacteria: Start with a single colony of E. coli grown on an LB agar plate. Inoculate into 10 mL of sterile LB broth and incubate overnight at 37°C with shaking (200 rpm).
  2. Dilute and Grow: Dilute the overnight culture by transferring 1 mL into 99 mL fresh sterile LB broth. Incubate at 37°C with shaking until OD600 reaches ~0.3-0.5 (mid-log phase), approximately 3-6 hours.
  3. Harvest Cells: Cool the culture on ice for ~10-15 minutes.
  4. Transfer and Centrifuge: Transfer to sterile centrifuge tubes and centrifuge at ~7000 rpm for 10 minutes at 4°C to pellet cells. Discard supernatant carefully.
  5. Wash Cells: Resuspend pellet in 20 mL ice-cold sterile 100 mM CaCl₂ solution. Gently mix and chill on ice for another 15 minutes.
  6. Centrifuge Again: Centrifuge at the same speed and temperature, then discard supernatant.
  7. Resuspend Pellet: Resuspend pellet in 5 mL ice-cold sterile 100 mM CaCl₂ supplemented with 15% glycerol and gently mix.
  8. Aliquot Cells: Divide resuspended cells into small aliquots (e.g., 50 µL) in sterile, ice-cold microcentrifuge tubes.
  9. Storage: Store aliquots at -80°C for long-term storage. Cells remain competent for at least one year.
  10. Transformation Efficiency Testing (Optional): Transform aliquots with known amount of plasmid DNA containing a positive selection marker and count transformant colonies after plating on selective media.

Materials

  • DNA Fragments:
    • Plasmid Backbone (Destination Vector)
    • Insert Fragments (PCR Products or Pre-cloned)
  • Enzymes:
    • Type IIS Restriction Enzyme (e.g., BsaI or BsmBI)
    • T4 DNA Ligase
  • Buffers:
    • 10X T4 DNA Ligase Buffer
    • Water: Nuclease-Free Water
  • Equipment:
    • Thermocycler
    • Ice or Cold Room
    • Microcentrifuge Tubes

Steps

  1. Design the Assembly: Ensure each insert and plasmid backbone have compatible overhangs generated by the chosen Type IIS enzyme. Recognition sites should flank the desired insert region.
  2. Prepare the Reaction Mix: In a total volume of 10 µL, combine:
    • Plasmid Backbone (75 ng/µL) ⇒ 1 µL
    • Insert Fragments (75 ng each) ⇒ Variable
    • 10X T4 DNA Ligase Buffer ⇒ 1 µL
    • T4 DNA Ligase (400 U/µL) ⇒ 1.25 µL
    • Type IIS Restriction Enzyme ⇒ 0.5 µL
    • Nuclease-Free Water ⇒ To 10 µL
  3. Set Up the Assembly Reaction: Combine all components in a microcentrifuge tube. Mix and briefly centrifuge to collect the liquid at the bottom.
  4. Perform the Assembly Reaction: Use thermal cycling conditions based on the number of inserts:
    • For 1 Insert ⇒ 37°C for 5 min (cloning) OR 37°C for 1 h AND 60°C for 5 min
    • For 2-4 Inserts ⇒ 37°C for 1 hr AND 60°C for 5 min
    • For 5-10 Inserts ⇒ (37°C for 1 min AND 16°C for 1 min) x30 AND 60°C for 5 min
    • For >10 Inserts ⇒ (37°C for 5 min AND 16°C for 5 min) x30 AND 60°C for 5 min
  5. Transform Competent Cells: After assembly, transform the mixture into competent E. coli cells using heat shock or electroporation. Plate-transformed cells on selective media to identify successful clones.

Notes:

  • Ensure total volume of unpurified PCR fragments in assembly reaction does not exceed 20% of total volume.
  • Use equimolar concentrations of all fragments for optimal results.
  • Include a positive control to verify assembly efficiency.

Materials

  • DNA Fragments:
    • Linearized Vector (Plasmid Backbone)
    • Insert Fragments (PCR Products or Other Linear DNA)
  • Gibson Assembly Master Mix
  • Buffers and Water:
    • Nuclease-Free Water
  • Equipment:
    • Thermocycler
    • Ice or Cold Room
    • Microcentrifuge Tubes

Steps

  1. Design Primers for PCR Amplification: Ensure overlap sequences (20-40 bp) for assembly of adjacent fragments.
  2. Prepare the Gibson Assembly Reaction: In a 20 µL volume, combine:
    • Gibson Assembly Master Mix ⇒ 10 µL
    • DNA Fragments (0.02–0.5 pmols for 1-2 fragments, or 0.2–1.0 pmols for 4-6 fragments) ⇒ Variable
    • Nuclease-Free Water ⇒ To 20 µL
  3. Incubate the Reaction: Incubate at 50°C for:
    • 15 minutes if assembling 2-3 fragments.
    • 60 minutes if assembling 4-6 fragments.
  4. Transform Competent Cells: After incubation, place reaction on ice. Transform into competent cells using standard protocols. Plate-transformed cells on selective media to identify successful clones.

Notes:

  • Total volume of unpurified PCR fragments should not exceed 20% of the total reaction volume.
  • Use equimolar concentrations of all fragments for optimal results.
  • Include a positive control to verify assembly efficiency.

Materials

  • Electrocompetent Cells: E. coli
  • DNA Sample: Plasmid or Other DNA for Transformation
  • Electroporation Cuvettes: 1 mm Gap
  • Recovery Medium
  • Buffers and Water:
    • Sterile Water for Dilutions
  • Equipment:
    • Electroporator
    • Ice or Cold Room
    • Microcentrifuge Tubes
    • 17 mm x 100 mm Round-Bottom Culture Tubes

Steps

  1. Preparation:
    • Pre-warm selective agar plates at 37°C for at least 1 hour.
    • Place recovery medium in a 37°C water bath.
    • Chill electroporation cuvettes and microcentrifuge tubes on ice.
  2. Thaw Competent Cells: Thaw electrocompetent E. coli cells on ice for ~10 minutes. Gently mix by flicking the tube.
  3. Prepare DNA Solution: Dilute a control plasmid (e.g., pUC19) to a final concentration of 10 pg/µL using sterile water.
  4. Mix Cells and DNA: Transfer 25 µL competent cells to a chilled microcentrifuge tube. Add 1 µL DNA solution and gently mix without introducing bubbles.
  5. Transfer to Cuvette: Carefully transfer cell/DNA mixture into chilled electroporation cuvette, ensuring cells settle without bubbles.
  6. Electroporate: Use the following conditions:
    • Voltage: 2.0 kV
    • Resistance: 200 Ω
    • Capacitance: 25 µF
  7. Recovery: Immediately add 975 µL pre-warmed recovery medium to cuvette and gently mix by pipetting up and down twice.
  8. Transfer and Incubate: Transfer mixture to pre-warmed round-bottom culture tube. Incubate at 37°C with shaking (250 rpm) for ~1 hour.
  9. Plating: Dilute cells as necessary and spread 100-200 µL onto pre-warmed selective agar plates. Incubate overnight at 37°C.

Materials

  • Competent Cells: E. coli (e.g., DH5α or Other Competent Strains)
  • DNA Sample: Plasmid DNA (1 pg to 100 ng)
  • Recovery Medium: LB Broth or SOC
  • Selection Plates: LB Agar Plates with Appropriate Antibiotic
  • Equipment:
    • Ice or Cold Room
    • Water Bath or Heat Block set at 42°C
    • Incubator set at 37°C
    • Microcentrifuge Tubes
    • Pipettes and Sterile Tips

Steps

  1. Preparation:
    • Thaw competent E. coli cells on ice for ~10 minutes.
    • Pre-warm LB broth or SOC medium in a water bath to 37°C.
  2. Mix Cells and DNA: Add 1-5 µL plasmid DNA to thawed competent cells (typically 50 µL) in a microcentrifuge tube. Gently flick to mix; do not vortex.
  3. Incubate on Ice: Incubate mixture on ice for 30 minutes to allow DNA uptake.
  4. Heat Shock: Transfer tube to a water bath or heat block set at 42°C for exactly 30-90 seconds (30 seconds typical).
  5. Cool on Ice: Immediately return tube to ice and cool for an additional 2-5 minutes.
  6. Add Recovery Medium: Add 500 µL pre-warmed LB broth or SOC medium to cells.
  7. Incubate: Incubate at 37°C with shaking (180 rpm) for 1 hour to allow recovery and expression of antibiotic resistance.
  8. Plating: After incubation, gently mix cells and perform serial dilutions if necessary. Spread 50-100 µL of cell suspension onto pre-warmed selective agar plates. Incubate overnight at 37°C.

Notes:

  • Ensure all materials are sterile to prevent contamination.
  • Use freshly prepared competent cells and avoid prolonged heat exposure during heat shock to maintain transformation efficiency.
  • Adjust DNA and competent cell volumes based on experimental needs; larger volumes may require longer incubation times.

Materials

  • Agarose Powder (1% or 0.8% depending on fragment size)
  • Buffer Solution: TAE or TBE
  • DNA Samples (PCR Products, etc.)
  • Loading Buffer
  • Ethidium Bromide or GelRed (optional for visualization)
  • Gel Casting Tray and Comb
  • Electrophoresis Apparatus
  • Power Supply
  • UV Transilluminator (for visualization)

Steps

  1. Prepare the Agarose Gel: Weigh agarose (1 g for 100 mL at 1%). Mix with TAE/TBE buffer and dissolve by microwaving. Cool to ~55°C, pour into casting tray, and let solidify.
  2. Prepare DNA Samples: Mix DNA with loading buffer. Include a molecular weight ladder as a control.
  3. Load the Gel: Place solidified gel in electrophoresis tank and cover with running buffer. Load DNA samples and ladder into wells.
  4. Run the Gel: Connect to power supply and run at 80-150 V until loading dye migrates 75-80% down the gel.
  5. Visualize DNA: Stain with ethidium bromide if necessary. Use UV transilluminator to visualize and document DNA bands.

Materials

  • Template DNA: Plasmid or PCR Product
  • Primers: Specific to Target Region
  • BigDye Terminator v3.1 Cycle Sequencing Kit
  • Nuclease-Free Water
  • Capillary Electrophoresis System
  • Microcentrifuge Tubes

Steps

  1. Prepare DNA Template: Purify and measure DNA concentration (aim for 10-100 ng/µL).
  2. Set Up Sequencing Reaction: Combine in microcentrifuge tube: Template DNA (10-100 ng), primers, BigDye Terminator mix, and nuclease-free water as per kit instructions.
  3. Perform Sequencing Reaction: Use thermal cycling conditions specified in the BigDye Terminator kit, typically including denaturation, annealing, and extension phases.
  4. Clean Up Reaction: Purify to remove unincorporated dNTPs and enzymes.
  5. Prepare for Capillary Electrophoresis: Resuspend purified product in loading buffer, heat at 95°C for 5 min, then place on ice.
  6. Run Capillary Electrophoresis: Load samples into the capillary system and run according to the manufacturer's instructions.
  7. Analyze Results: Use software to analyze chromatograms and determine the nucleotide sequence.

Materials

  • DNA Sample: Plasmid or PCR Product
  • Restriction Enzyme: Specific to Recognition Sites in DNA
  • Buffer: Appropriate Buffer for Restriction Enzyme (usually supplied with enzyme)
  • BSA (Bovine Serum Albumin): If required by enzyme
  • Nuclease-Free Water
  • Microcentrifuge Tubes
  • Pipettes and Tips

Steps

  1. Prepare Reaction Mix: In a microcentrifuge tube, combine:
    • Nuclease-Free Water ⇒ To reach final volume (e.g., 16-16.5 µL for a 20 µL reaction)
    • 10X Restriction Buffer ⇒ 2 µL
    • DNA Sample ⇒ 1 µL (~1 µg)
    • Restriction Enzyme ⇒ 0.5-1 µL (5-10 U)
  2. Mix Components: Gently mix by pipetting up and down. Avoid bubbles. Briefly centrifuge the tube to collect all liquid at the bottom.
  3. Incubate Reaction: Incubate at the optimal temperature for the specific restriction enzyme (usually 37°C) for 1 to 16 hours, depending on enzyme activity and DNA complexity.
  4. Stop Reaction (if necessary): Some enzymes can be heat-inactivated. If required, incubate at 65°C for 20 minutes or according to the enzyme's specifications.
  5. Analyze Digested DNA: Run an aliquot of the digested sample on an agarose gel to confirm successful digestion.
  6. Purify Digested DNA (if needed): Use a purification kit or perform ethanol precipitation to purify the digested DNA for subsequent applications.

Materials

  • Filter Paper
  • Electroblotting Equipment
  • Cassettes
  • PBS
  • Samples (Protein SDS-PAGE Gel)
  • Blocking Solution: 5% Skim Nonfat Dry Milk Powder in PBS-T
  • Primary Antibody Solution: 1:1000 dilution of primary antibody (mouse anti-hexahis antibody) in blocking solution
  • Secondary Antibody Solution: 1:5,000 dilution of secondary antibody (goat anti-mouse HRP conjugated) in blocking solution

Steps

  1. Make a Sandwich: Layer filter papers, place the membrane on top of the filter paper, transfer the SDS-PAGE gel onto the membrane, and cover with additional filter papers.
  2. Transfer the Sandwich to the Blotting Machine: Place the assembled sandwich into the "Trans-Blot Turbo" machine following the manufacturer's instructions. Select the appropriate settings based on the membrane type (mini or maxi).
  3. Transfer Proteins: Run the blotting process as per the machine's guidelines. Once completed, carefully remove the blotted membrane.
  4. Block the Membrane: Incubate the membrane in the blocking solution (5% skim nonfat dry milk powder in PBS-T) for 1 hour at room temperature or overnight at 4°C with shaking to prevent non-specific binding.
  5. Primary Antibody Incubation: Incubate the membrane with the primary antibody solution (1:1000 dilution of mouse anti-hexahis antibody in blocking solution) for 1 hour at room temperature or overnight at 4°C with shaking.
  6. Wash the Membrane: Wash the membrane three times for 5 minutes each with PBS-T to remove unbound primary antibodies.
  7. Secondary Antibody Incubation: Incubate the membrane with the secondary antibody solution (1:5,000 dilution of goat anti-mouse HRP conjugated secondary antibody in blocking solution) for 1 hour with shaking.
  8. Wash the Membrane Again: Wash the membrane three times for 10 minutes each with PBS-T to remove unbound secondary antibodies.
  9. Add Color-Developing Solution: Apply the color-developing solution to the blot to visualize the protein bands.
  10. Incubate in the Dark: Incubate the blot with the color-developing solution in the dark for no more than 5 minutes to prevent overdevelopment.
  11. Visualize the Blot: Place the blot on the gel imaging system to visualize and document the protein bands.

Materials

  • BCA Protein Assay Kit and Samples
  • 96-well Microplate
  • Protein Standards

Steps

  1. Prepare Protein Standards: Create a series of protein standards with different BSA concentrations as per the kit instructions.
  2. Add Standards and Samples: Add 10 µL of each standard and your GFP elution sample “E” into designated wells of the microplate in duplicate.
  3. Prepare Working Solution: Mix Reagent A and Reagent B in a 50:1 ratio to create the working solution.
  4. Add Working Solution: Add 200 µL of the working solution to each well containing standards and samples. Mix gently to ensure thorough mixing.
  5. Incubate: Place the microplate in a 37ºC incubator for 30 minutes to allow color development.
  6. Measure Absorbance: After incubation, measure the absorbance of each well at 562 nm using a plate reader.
  7. Calculate Protein Concentration: Generate a standard curve using the protein standards and apply the equation to determine the protein concentration of your samples.

Materials

  • 1X Running Buffer (0.5 mM Tris base, 192 mM glycine, 0.1% SDS, pH 8.3)
  • Sample Buffer (3X) - Blue
  • Ladder (usually 1 Kb)
  • Resolution (Bottom Gel):
    • Water
    • 1.5 M Tris-HCl pH 8.8
    • 30% Acrylamide
    • 10% SDS
    • 10% Ammonium Persulfate (APS)
    • TEMED
  • Stacking (Top Gel):
    • Water
    • 0.5 M Tris-HCl pH 6.8
    • 30% Acrylamide
    • 10% SDS
    • 10% Ammonium Persulfate (APS)
    • TEMED
  • Cassettes (Glass and Plastic)
  • Alcohol 98%

Steps

  1. Prepare the Gel: Calculate the required amount of polyacrylamide (8-15%) based on your needs. Fill the cassettes with the resolution (bottom) gel mix and add a few drops of 98% alcohol to keep the gel straight. Allow the gel to polymerize for 30 to 60 minutes.
  2. Prepare the Stacking Gel: Remove the alcohol and pipette the stacking (top) gel mix into the well area. Allow the stacking gel to polymerize for at least 45 minutes.
  3. Seal and Store the Gel: Seal the gel with wet paper (Running Buffer or water) and store it in the refrigerator if not used immediately.
  4. Prepare Samples: Mix 10 µL of Sample Buffer (3X) with 20 µL of each protein sample.
  5. Boil Samples: Boil the samples at 95ºC for 5 minutes to denature proteins.
  6. Load Samples: Apply the boiled samples into the wells of the gel.
  7. Run the Gel: Place the gel into the electrophoresis apparatus, fill with **1X Running Buffer (0.5 mM Tris base, 192 mM glycine, 0.1% SDS, pH 8.3)**, and run at 120 V for approximately 60 minutes. Monitor for bubble formation.
  8. Dye the Gel: Once electrophoresis is complete, stain the gel with Coomassie Brilliant Blue or another appropriate stain until protein bands are visible.
  9. Unstain the Gel: Remove excess dye by soaking the gel in destaining solution until clear bands are visible.
  10. Photograph and Analyze: Photograph the gel using a gel documentation system and analyze the protein bands.

Materials

  • LB
  • Competent Cells Expressing BARBIE or CBM Mad10 (BL21(DE3))
  • Flasks
  • Antibiotic (Ampicillin)
  • Shaker and Incubator
  • Spectrophotometer and Cuvette
  • Centrifuge
  • pH Meter
  • Freezer

Steps

  1. Pre-inoculum: Take the appropriate amount of LB media, add Ampicillin (100 ug/uL final concentration), and add 200 µL pre-inoculum cells per 10 mL LB.
  2. Grow Cells: Incubate the culture in a shaking incubator at 37°C, 220 rpm overnight.
  3. Inoculum Preparation: Add 10 mL of pre-inoculum to 500 mL LB supplemented with the proper antibiotic. Grow at 37°C, 220 rpm until OD600 reaches 0.8.
  4. Induce Protein Expression: Add 1 mM IPTG to the culture and incubate at 37°C for 3 hours.
  5. Harvest Cells: Centrifuge the culture at 5000 g for 15 minutes at 4°C the next day.
  6. Store Pellet: Store the cell pellet at -80°C until further processing.

Materials

  • LB
  • Competent Cells Expressing BARBIE or CBM Cys (BL21(DE3))
  • Flasks
  • Antibiotic (Kanamycin, 30 µg/mL final concentration)
  • Shaker and Incubator
  • Spectrophotometer and Cuvette
  • Centrifuge
  • pH Meter

Steps

  1. Pre-inoculum: Take the appropriate amount of LB media, add Kanamycin (30 µg/mL final concentration), and add 5 µL competent cells per 5 mL LB.
  2. Grow Cells: Incubate the culture in a shaking incubator at 30°C, 220 rpm overnight.
  3. Inoculum Preparation: Add 5 mL of pre-inoculum to 500 mL LB supplemented with the proper antibiotic. Grow at 37°C, 220 rpm until OD600 reaches 0.8.
  4. Induce Protein Expression: Add 0.3 mM IPTG to the culture and incubate overnight.
  5. Harvest Cells: Centrifuge the culture at 5000 g for 15 minutes at 4°C the next day.
  6. Store Pellet: Store the cell pellet at -80°C until further processing.

Materials

  • LB
  • Competent Cells Expressing Spidroin (BL21(DE3)pRARE2)
  • Flasks
  • Antibiotics (Kanamycin and Chloramphenicol, 30 µg/mL each final concentration)
  • Shaker and Incubator
  • Spectrophotometer and Cuvette
  • Centrifuge
  • pH Meter

Steps

  1. Pre-inoculum: Take the appropriate amount of LB media, add Kanamycin and Chloramphenicol (each 30 µg/mL final concentration), and add 20 µL competent cells per 1 mL LB.
  2. Grow Cells: Incubate the culture in a shaking incubator at 30°C, 220 rpm overnight.
  3. Inoculum Preparation: Add 5 mL of pre-inoculum to 500 mL LB supplemented with the proper antibiotics. Grow at 37°C, 220 rpm until OD600 reaches 0.8.
  4. Induce Protein Expression: Add 1 mM IPTG to the culture and incubate at 20°C overnight.
  5. Harvest Cells: Centrifuge the culture at 5000 g for 15 minutes at 4°C the next day.
  6. Store Pellet: Store the cell pellet at -80°C until further processing.

Materials

  • LB
  • Competent Cells Expressing Nt-CBM/Nt-Barbie1 (BL21(DE3)pRARE2)
  • Flasks
  • Antibiotics (Kanamycin and Chloramphenicol, 30 µg/mL each final concentration)
  • Shaker and Incubator
  • Spectrophotometer and Cuvette
  • Centrifuge
  • pH Meter

Steps

  1. Pre-inoculum: Take the appropriate amount of LB media, add Kanamycin and Chloramphenicol (each 30 µg/mL final concentration), and add 1 µL competent cells per 1 mL LB.
  2. Grow Cells: Incubate the culture in a shaking incubator at 30°C, 220 rpm overnight.
  3. Inoculum Preparation: Add 5 mL of pre-inoculum to 500 mL LB supplemented with the proper antibiotics. Grow at 37°C, 220 rpm until OD600 reaches 0.8.
  4. Induce Protein Expression: Add 1 mM IPTG to the culture and incubate for 4 hours.
  5. Harvest Cells: Centrifuge the culture at 5000 g for 15 minutes at 4°C the next day.
  6. Store Pellet: Store the cell pellet at -80°C until further processing.

Materials

  • AKTA Purification System
  • Nickel Column (HisTrap HP 5 mL)
  • Superloop (to inject sample)
  • Sonicator
  • Centrifuge
  • Buffer A and Buffer B (composition depends on your protein and objective)

Steps

  1. Prepare Cell Lysate:
    • Centrifuge cell pellet in 40 mL Buffer A.
    • Add the following to the resuspension for enhanced lysis and protease inhibition:
      • 1 mM PMSF (serine protease inhibitor)
      • 50 µg/mL Lysozyme (to break down the cell wall)
      • 20 U DNase Turbo (to degrade DNA and reduce viscosity)
      • 100 µM DTT (to prevent oxidation of cysteines and maintain reducing conditions)
      • 40 µL Leupeptin (1,000x stock concentration, a protease inhibitor)
    • Gently mix the suspension and incubate for 30 minutes at 4°C with constant agitation to facilitate lysis.
    • Sonicate the resuspended cells for 10 minutes using the following parameters:
      • 4 seconds ON, 4 seconds OFF pulse cycle to prevent overheating.
      • Amplitude set to 40% to ensure efficient cell disruption.
      • Carry out sonication on ice or in a cold environment to avoid heat-induced protein denaturation.
    • Centrifuge the lysate at 17,000 x g for 1 hour at 4°C to separate the soluble fraction from cellular debris.
    • Transfer the supernatant (soluble protein fraction) to a new tube without disturbing the pellet.
    • Filter the supernatant through a 0.45 µm syringe filter to remove any remaining particulates and ensure a clear solution for column loading.
  2. Set Up AKTA Purification:
    • Equilibrate the HisTrap column with Buffer A.
    • Load the filtered supernatant containing the protein into the Superloop for loading onto the column.
    • Allow the protein to bind to the column in Buffer A.
    • Perform several washes with Buffer A to remove unbound proteins and impurities.
    • Elute the His-tagged protein using Buffer B containing 300-500 mM imidazole to displace the protein from the nickel resin.
    • Collect the eluted fractions and analyze the protein content by UV absorbance at 280 nm or SDS-PAGE.
    • Pool the fractions containing the desired Barbie-Cys protein.
    • Concentrate the eluted protein if necessary and store at -80°C for future experiments.

Materials

  • AKTA Purification System
  • Superdex Column (200)
  • Centrifuge
  • GF Buffer (composition depends on your protein and objective; no Imidazole)

Steps

  1. Prepare Lysate: Centrifuge the affinity-purified supernatant to remove any remaining debris.
  2. Load the Column: Inject the clarified lysate into the Superdex column.
  3. Wash the Column: Wash the column with more than one column volume of GF Buffer to ensure the removal of unbound proteins.
  4. Collect Fractions: Collect the desired protein peaks based on the elution profiles.
  5. Final Steps: Combine and concentrate the fractions containing your target protein as needed.