Team:UNSW Australia/Protocols


Team: UNSW Australia



Protocols

Overview

Cloning

The goal of the cloning experiments was to design gene constructs containing useful components for ease of cloning and protein production. LXYL-SpyTag, DBAT-SnoopTag, PAM-SnoopTag, TycA-SpyTag and mCerulean3-SnoopTag, proteins were ligated into the pET-19b expression vector via Gibson Assembly and transformed into the expression host, T7 Express E. coli. Colonies were screened for recombinant plasmids via colony PCR and sequences verified by Sanger Sequencing.

Protein Expression & Purification

The goal of the protein expression and purification experiments was to express and purify the scaffolding proteins as well as the key enzymes involved in Paclitaxel production pathways. Expression was induced via IPTG induction in T7 Express E. coli and proteins were purified using Immobilised Metal Affinity Chromatography (IMAC).

Scaffold Assembly

The spontaneous formation of the hexameric structure between α-prefoldin-SpyCatcher (αPFD-SpyCatcher) and β-prefoldin-SnoopCatcher (βPFD-SnoopCatcher) subunits was examined by Native-PAGE (polyacrylamide gel electrophoresis).

Catcher-Tag Conjugation

To create the enzyme-scaffold complex, the alpha and beta prefoldin hexamer are to be covalently attached to the enzymes through spontaneous and specific isopeptide bond formation between the Snoop or SpyCatcher and its corresponding Snoop or SpyTag. The Catcher-Tag conjugation between βPFD-SnoopCatcher with mCerulean3 as well as αPFD-SpyCatcher with mVenus-SpyTag was examined by SDS-PAGE.

Enzyme Kinetics

To examine the functionality of our scaffolded system, the biosynthesis of Paclitaxel was tested. Enzyme concentration was determined by BCA assay. Ellman's assay, detects free thiol (-SH) groups in solution was used as an indirect measure of DBAT activity.

The product turnover rate of PAM would have been examined by measuring the integration of the signals corresponding to alpha- and beta-phenylalanine using 1H Nuclear Magnetic Resonance Spectroscopy (NMR)1.

Cloning

Linearisation of pET-19b Plasmid Backbones Using PCR (Phusion)

Prior to the PCR reaction, pET-19b plasmid DNA was obtained by a small scale grow up of pET-19b containing E. coli, extracted and purified by miniprep (QIAGEN). Plasmid concentration was determined by nanodrop.

Recommended to read the Thermo Scientific Phusion High-Fidelity DNA Polymerase protocol prior to PCR for more details and troubleshooting methods.

  1. Design and purchase forward and reverse primers for plasmid backbone amplification.
  2. Vortex mix and centrifuge all master mix components prior to addition.
  3. Prepare master mix on ice, add all components in the following order:

    Refer to "example" column for exact volumes used to amplify pET-19b of 24.7 ng/uL concentration.

    Component per 50 uL rxn Final conc. Example
    Nuclease-Free Water add to 50 uL 32.5
    5X Phusion HF Buffer 10 1X 10
    10 mM dNTPs 1 200 uM 1
    Fwd Primer (10 uM) 2.5 0.5 uM 2.5
    Rev Primer (10 uM) 2.5 0.5 uM 2.5
    Template DNA variable* <250 ng 1
    Phusion DNA polymerase** 0.5 0.02 U/uL 0.5

    *The volume of template DNA required varies depending on the concentration of the template DNA. Ensure the volume added contains <250 ng of DNA. For plasmid DNA, 1-10 ng per 50 uL is recommended.
    **note: Phusion DNA polymerase should be added last, and right before thermocycling as it is heat sensitive. Polymerase must be kept on ice, or in a -20°C minicooler at all times.

  4. Place PCR tube(s) in the thermocycler and run at the following conditions:
    Cycle Step Temperature Time Cycles
    Initial Denaturation 98°C 30 seconds
    Denaturation 98°C 10 seconds 30X
    Annealing 62°C* 30 seconds 30X
    Extension 72°C 4 minutes** 30X
    Final extension 72°C 5 minutes
    Hold 4°C ∞

    *Annealing temperature varies depending on the primer pair. If using a different primers, use the Thermofisher scientific Tm Calculator to determine the recommended annealing temperature. If primers do not anneal, a temperature gradient can be used to empirically find the appropriate annealing temperature.
    **Extension time varies depending on the length of the desired amplicon. 15-30 seconds per kb is recommended. Refer to secion 5.4 of the Thermo Scientific Phusion High-Fidelity DNA Polymerase protocol for more details.

  5. Store at -20°C until further use.

Agarose Gel Electrophoresis

Preparing 1% agarose gel

  1. Add 15 mL 1X TAE buffer to a conical flask.

    Adjust volume of agarose gel depending on the size of gel you are making.

  2. Add 0.15 g of agarose powder to the flask. Weigh both flask and contents.

    Generally, 0.01 g of agarose powder per 1 mL of 1X TAE buffer.

  3. Heat until the agarose is completely dissolved. Stop and swirl the flask every 20 seconds to prevent vigorous boiling. Add milliQ water to make up the volume lost by evaporation (measured weight in step 2).
  4. Allow agarose solution cool until comfortable to hold, then add 1.5 uL of 10,000X GelRed to a final concentration of 1X to the agarose solution, swirl to mix.
  5. Seal the ends of a gel mold using masking tape. Pour the agarose into the gel mold with a well comb in place. Let the gel solidify at room temperature for 20-30 minutes.
  6. Place the gel into the gel tank, with wells closer to the negative electrode. Fill the gel tank with 1X TAE buffer until the gel is covered then remove the well comb.

Sample Preparation and loading

  1. Add 2 uL of PCR product, 9uL of H2O and 1 uL of 6X loading dye (NEB #B7024S) to a new microfuge tube, mix by pipetting and centrifuge to collect solution at the bottom of the tube.
  2. Load 5 uL of 2-log DNA ladder (NEB #N3200S) into the first lane of the gel. Load 10 uL of PCR samples into new wells.
  3. Connect the gel tank to a power pack and run the gel at 100V for 1 hour. Check for bubbling from the electrodes to ensure the gel is running.
  4. Turn off the powerpack and unplug the wires before transferring the gel to a square petridish, wash with RO water.
  5. Gel is ready to be imaged under the transilluminator setting.

Plasmid Digest (DpnI Digest - NEB)

  1. Set up the reaction mixture:
    Restriction Enzyme (DpnI) 1 uL
    DNA 1 ug
    10X CutSmart Buffer 5 uL (1X)
    Total Reaction Volume 50 uL
  2. Incubate for 1 hour at 37°C.
  3. Perform PCR clean up immediately, or heat inactivate at 80°C for 20 minutes.
  4. Store at -20°C.

PCR Clean Up

Performed following QIAquick PCR & Gel Cleanup Kit Quick-Start protocol.

  1. Prepare PE and PB buffer according to manufacturer's protocol.
  2. Add 150 uL Buffer PB to 50 uL PCR sample, mix by inverting.
  3. Load sample to the column and centrifuge for 1 minute. Discard flow through and replace column onto collection tube.
  4. Add 750 uL Buffer PE to the column. Centrifuge for 1 minute, discarding flow through and replacing column.
  5. Centrifuge for 1 minute to remove residual buffer.
  6. Place column into a clean 1.5 mL microcentrifuge tube.
  7. Add 50 uL of heated nuclease-free water or EB Buffer to the centre of the membrane, let the column stand for 1 minute. Then centrifuge for 1 minute.
  8. Nanodrop to determine the DNA concentration.

Ligation by Gibson Assembly

Materials

  • Linearised pET-19b backbone - prepared above
  • Desired insert gene
  • 2X Gibson master mix (NEB)

Preparing Plasmid Backbone

Linearised pET-19b backbone prepared by PCR linearisation, DpnI digest and PCR clean up.

Preparing Desired Insert Gene

  1. Design and order new gene sequence from gene synthesis company.

    UNSW iGEM 2019 obtained 5 insert sequences: LXYL-P1-2, DBAT, PAM, TycA and mCerulean3.

  2. Centrifuge tubes to ensure DNA pellets are contained at the bottom of the tube. This prevents loss of sample as the pellet could be dislodged during delivery.
  3. Resuspend pellet in nuclease free water to a final concentration of 10 ng/uL.
  4. Volume for resuspension varies depending on the molecular weight of the sequence ordered. Follow manufacturer's protocols for more detailed resuspension and storage instructions.
  5. Mix well by pipetting, or vortexing and centrifuge to collect solution at the bottom of the tube. Store at -20C°.

Gibson Assembly

Assembly performed following manufacturer's protocol.

  1. Set up the following on ice:
    2-3 Fragment Assembly Volume (uL)
    PCR Fragment(s) + Linearised vector X 0.02 - 0.5 pmol
    Gibson Assembly Master Mix (2X) 10
    Deionised H2O 10 - X
    Total Volume 20 uL
  2. Nanodrop DNA fragments. Prepare 50-100 ng of DNA mix containing vector DNA and at least 3-fold excess insert in nuclease-free water to a total volume of 10 uL.

    Molar ratio is determined by the sequence length of each fragment.

  3. Add 10 uL 2X Gibson Assembly Master Mix for a total reaction volume of 20 uL.
  4. Incubate at 50°C for 15 to 60 minutes (60 minutes is optimal).

    *Troubleshooting tips: for larger insert genes, extend incubation time. For smaller insert genes, increase to 5-fold excess.
    **For assembly with more than 3 fragments, refer to manufacturer's protocol.

Heat Shock Transformation

Preparing Agar Selection Plates

  1. Heat Millers LB Agar until completely liquid. Pause and swirl at 30 second intervals to prevent vigorous boiling. Allow to cool in a 55°C water bath
  2. Prepare sterile conditions for the following steps: Sterilise bench, equipment and gloves with 70% ethanol before use. Perform the following steps under a bunsen flame
  3. Pour equivalent of 20 mL liquid agar per plate into an autoclaved flask or sterile falcon tube.

    e.g. 100 mL is sufficient to make 5-6 selection plates.

  4. Add appropriate antibiotic to working concentration, swirl to mix.
  5. Some antibiotics are heat sensitive, so ensure agar is cooled prior to addition to prevent lowering the effectiveness of the antibiotic.
  6. Pour enough agar to sterile circle petri dishes to completely cover the bottom of the plate and allow the agar to solidify at room temperature.

    Leave gap when covering dish to prevent condensation in the plate.

  7. Safety Considerations
    • Be careful while heating, as the agarose bubbles vigorously. Leave cap on (sterile), but unscrewed (to release pressure).
    • Do not leave the flame unattended. While allowing plates to set, if needed, completely cover plates before turning off the flame. Alternatively, condensation can be removed by placing solidified agar plates in a 37°C incubator upside down (agar side up).

Transformation into T7 Express Cells

Transformation protocols vary per cell type, refer to manufacturer's protocols for optimal transformation.
Transformation into High Efficiency T7 Expression Competent E. coli following manufacturer's protocols.
*DH5-alpha cells can be used if higher plasmid recovery is desired.

  1. Set waterbath to 42°C.
  2. Thaw X tubes of T7 Express Competent E. coli cells on ice for 10 minutes.
  3. Use 1 tube per transformation sample, it is recommended to perform positive and negative transformation controls.
    • pUC19 positive control DNA - provided with the cells.
    • Linear pET-19b negative control.
      •
  4. Add 2uL of plasmid DNA to the cell mixture. Flick the tube 4-5 times to mix cells and DNA. Do not vortex.
  5. Place the mixture on ice for 30 mins. Do not mix.
  6. Heat shock at exactly 42°C for exactly 10 seconds. Do not mix.
  7. Place on ice for 5 minutes. Do not mix.
  8. Under a flame, pipette 950 uL of room temperature SOC into the mixture.
  9. Place at 37°C for 60 minutes. Shake vigorously at 250 rpm or rotate.
  10. Warm selection plates to 37°C
  11. Mix the cell thoroughly by flicking the tube. Centrifuge at 13,000 rpm for 1 minute to pellet cells.
  12. Remove 900 uL of SOC media, and resuspend cells in the remaining 100 uL.
  13. Spread the 100 uL resuspension onto a selection plate and incubate overnight at 37°C.

    pET-19b vectors should be plated on ampicillin supplemented agar plates.

Colony PCR - Screening for Successful Transformations

Preparation

  1. Design and purchase forward and reverse primers for insert amplification.
    • Primers below are designed to bind to the pET-19b backbone and amplify the insert sequence.
    • Fwd Primer: 5'-ATGCGTCCGGCGTAGA
    • Rev Primer: 5'-CCGTTTAGAGGCCCCAAG
  2. Preheat thermocycler to 98°C
  3. Vortex mix and centrifuge all master mix components prior to addition.

PCR Master Mix

  1. Make a mastermix for X.5 samples, where 'x' is the desired number of PCR samples.

    Recommended to screen 5-15 colonies.
    Recommended controls:

    • Positive control: known successful transformant
    • Negative control: no template DNA, to observe if there is contaminating template DNA in the master mix
    • Negative control: circular pET-19b from which the linear template was obtained, for a visual reference of non-DpnI digested pET-19b transformants.
      •

  2. Assemble the components in the following order, excluding template DNA and Phusion DNA polymerase.
  3. Aliquot 19.8 uL of Master Mix to each prelabelled PCR tube.
    Component per 20 uL* rxn Final conc.
    Nuclease-Free Water 13.4
    5X Phusion HF Buffer 4 1X
    10 mM dNTPs 0.4 200 uM
    Fwd Primer (10 uM) 1 0.5 uM
    Rev Primer (10 uM) 1 0.5 uM
    Template DNA**
    Phusion DNA polymerase** 0.2 0.02 U/uL

    *20 uL samples are recommended for screening purposes.
    **Template DNA introduction is described in the "Patching" section below.
    ***note: Phusion DNA polymerase should be added last, and right before thermocycling as it is heat sensitive. Polymerase must be kept on ice, or in a -20°C minicooler at all times.

Patching

  1. Draw grids on an agar selection plate supplemented to ampicillin. Enough for all the colonies to be screened.
  2. Using a sterile pipette tip or inoculation loop, touch a single colony and inoculate the master mix by swirling the tip in solution. Do not discard tip.
  3. Using the same tip, touch the agar 3-5 times within a single grid. Discard tip. Grids should be labelled corresponding to the colony used.
  4. Repeat for all colonies to be screened. Incubate plate at 37°C overnight.

Thermocycling

  1. Add 0.2 uL of Phusion DNA polymerase to each PCR tube. Begin thermocycling step immediately.
  2. Place PCR tube(s) in the thermocycler and run at the following conditions:
    Cycle Step Temperature Time Cycles
    Initial Denaturation 98°C 3 minutes*
    Denaturation 98°C 10 seconds 30X
    Annealing 67.6°C** 30 seconds 30X
    Extension 72°C 15-30 seconds per kb*** 30X
    Final extension 72°C 5 minutes
    Hold 4°C ∞

    *Initial denaturation step is abnormally long for colony PCR to lyse the cells.
    **Annealing temperature varies depending on the primer pair. If using different primers, use the Thermofisher scientific Tm Calculator to determine the recommended annealing temperature. If primers do not anneal, a temperature gradient can be used to empirically find the appropriate annealing temperature.
    ***Extension time varies depending on the length of the desired amplicon. 15-30 seconds per kb is recommended. Refer to secion 5.4 of the Thermo Scientific Phusion High-Fidelity DNA Polymerase protocol for more details.

  3. Run PCR products on a 1% agarose gel and image on a GelDoc for analysis.

Sanger Sequencing

Small Scale grow up

  1. Prepare X number of 15 mL falcon tubes containing 5 mL Luria Broth supplemented with the appropriate antibiotic at working concentration.
  2. Use a sterile pipette tip or inoculation loop to inoculate the broth with a single colony of bacteria, swirl tip in broth.
  3. Incubate for 8-16 hours at 37C°, shaking at 250 rpm.

    12 hours is optimal, prevent exceeding 16 hours.

Miniprep - Plasmid DNA Extraction & Purification

Performed following manufacturer's protocols.
All centrifugation steps performed at 13,000 rpm (17,900xg) in a table-top microcentrifuge, unless otherwise stated.

  1. Prepare Buffer P1 and Buffer PE following manufacturer's protocol.
  2. Pellet 5 mL bacterial overnight culture by centrifugation at >8,000 rpm (6,800xg) for 3 minutes at room temperature (15-25°C). Discard broth into LB waste.
  3. Resuspend pelleted bacterial cells in 250 uL Buffer P1 and transfer to a microcentrifuge tube.
  4. Add 250 uL Buffer P2, mix thoroughly by inverting until the solution becomes clear. Do not allow lysis solution to proceed for more than 5 minutes.
  5. Add 350 uL Buffer N3 and mix immediately by inverting. Centrifuge for 10 minutes.
  6. Apply 800 uL supernatant to QIAprep spin column by pipetting. Centrifuge for 1 minute, discard flow through.
  7. Add 500 uL Buffer PB to spin column. Centrifuge for 1 minute, discard flow through.
  8. Add 750 uL Buffer PE to spin column. Centrifuge for 1 minute, discard flow through.
  9. Centrifuge for 1 minute to remove residual buffer.
  10. Place spin column in a clean 1.5 mL microcentrifuge tube. Add 50 uL of heated* nuclease-free water, or Buffer EB, to the centre of the spin column. Let stand for 1 minute, centrifuge for 1 minute.

    *Heating elution solution aids in releasing DNA from the membrane, increasing yield.

  11. Nanodrop elution to determine DNA concentration.

Submission to Ramaciotti Centre for Genomics

  1. Request sequencing at the UNSW Centre for Genomics via the online portal.
  2. Label clean 1.5 mL microcentrifuge tubes as per order instructions.
  3. Add 8 uL miniprepped sample DNA to its corresponding tube.
  4. Add 2 uL of 10 uM of one primer (either forward or reverse).
  5. Take microfuge tubes to the Ramaciotti Centre for Genomics Level 2 (UNSW, Sydney Australia). Store samples in the fridge provided.
  6. Sanger Sequencing is carried out by the Centre staff following the provided protocol.

Protein Expression & Purification

Starter Culture

  1. Using a pipette tip or innoculating loop, add a colony of bacteria to 2 mL of Luria Broth in a 15 ml falcon tube with an apropriate antibiotic to a working concentration. Perform this step aseptically next to a bunsen burner flame.
  2. Incubate for 2-4 hours in the shaking incubator at 37°C at 160-200 rpm overnight (10-15 hours).

Large-Scale Grow-Up

  1. Perform the following steps in a biosafety cabinet or next to a bunsen burner and ensure conical flasks have be autoclaved.
  2. Pour approximately 200 mL of Luria Broth into a 1 L conical flask with required anitbiotic to a working concentration.
  3. Innoculate broth with starter culture (add approximatley 1ml of starter culture per 100 ml of LB).
  4. Cover with a foil lid and incubate in a shaking incubator at 37°C at 140-160 rpm.
  5. Measure optical density (OD) of inoculated broth every hour. As the OD reaches 0.1, measure the OD more regularly (every 20-30 minutes).
  6. When OD of broth reaches 0.6 add IPTG to a concentration of 0.5 mM (or whichever concentration is required for the specific protein).

    7. Note: If performing bug buster to check soluble expression of proteins, before adding IPTG, transfer the equivalent of 1 ml of cells at 0.6 OD into a microfuge tube to act as the before IPTG induction sample. Centrifuge for 1 minute at 15,000 rpm, remove supernatant and freeze pellets until needed.

Harvesting of Cells

  1. Transfer culture to centrifuge bottles or tubes and centrifuge cells at 4,600 x g for 20 minutes to pellet cells. Cell pellet can be stored at -40°C until needed

    2. Note: If performing bug buster to check soluble expression of proteins, before pelleting cells, transfer the equivalent of 1ml of cells at 0.6 OD into a microfuge tube to act as the after IPTG induction sample. Centrifuge for 1 minute at 15,000rpm, remove supernatant and freeze pellets until needed.

Bug Buster

  1. Resuspend pellet of before and after IPTG samples in 100 uL of 100% Bug Buster.
  2. Incubate for 5 minutes at room temperature.
  3. Take out 20 uL of supernatant from each sample and place into microfuge tubes labelled as "total protein" and either "before" or "after IPTG induction".
  4. Centrifuge the remaining 80 ul of the "after IPTG induction" samples at 15,000 RPM for 20 minutes.
  5. Transfer 20 uL of each sample's supernatant to new tubes labelled "soluble protein after IPTG induction". Discard rest of supernatant.
  6. Add 600 uL of 10% bug buster to resuspend pellet.
  7. Centrifuge for 20 minutes at 15,000 RPM. Discard supernatant.
  8. Resuspend pellet in 100 uL of 10% bug buster.
  9. Remove 20 ul and place into new tube labelled "insoluble protein after IPTG induction".
  10. Follow steps of SDS-PAGE protocol to visualise bug buster samples on protein gel (samples include; total protein before IPTG, total protein after IPTG, soluble protein after IPTG and insoluble protein after IPTG).

SDS-PAGE

Prepare samples and buffer.

  1. Add 20 uL of 6x SDS to 20 ul of sample.
  2. incubate samples with SDS for 10 minutess at 95°C in a heating block.
  3. Prepare 1X MES running buffer (appropriate volume necessary for gel tank).

    4. Prepare gel cassette

  4. Cut open the 4-12% Bolt Bis-Tris 12-well gel cassette pouch and remove the cassette. Remove the tape covering the slot at the lower portion of the cassette.
  5. Lower the gel cassette into the tank, with the wells facing towards you. And half-fill tank with 1x MES running buffer.
  6. Remove the gel comb by sliding the comb up vertically (Be careful not to damage the wells when removing the comb, or samples may leak from one well to the other).
  7. Use a 1ml pipette and rinse the wells with 1X running buffer.
  8. Ensure cassette is resting at the base of the tank and clamp is holding cassette in place. Fill the tank with running buffer to the 'fill' line. (ensure back of tank is filled to at least half-way with buffer).

    9. Loading Samples

  9. Make sure that the wells are completely filled with 1X running buffer.
  10. Load 5 ul of Spectra BR ladder in appropriate wells.
  11. Load 20 ul of sample in each well.

    12. Electrophoresis

  12. Place the lid securely on the electrophoresis tank.
  13. With the power off, connect the electrode cords to power supply.
  14. Turn on the power. Run Bolt 4-12% Bis-Tris gels in 1X MES buffer at 180 V, 500 mA for 35 minutes.

    15. Note: different buffers and gels require different running conditions (voltage, amps, time).

    Gel Staining

  15. Crack the gel cassette open and release the gel into a square petri dish or similar container.
  16. Wash the gel 3 times in milliQ water (discard liquid into MES buffer waste).
  17. Fix gel in 25% isopropanol, 10% acetic acid for 15 minutes on platform rocker.
  18. Discard solution into isopropanol waste container.
  19. Rinse with milliQ water twice for 10 minutes each (on platform rocker).
  20. Discard water and stain the gel overnight with Coomassie blue stain.
  21. Destain gel by adding milliQ water to cover gel and a kim wipe folded at the top of the gel to absorb stain. Change kim wipe after the first 30 minutes of destaining and repeat after 1 hour if required.
  22. Once blue colour has been washed from gel background, discard water into coomassie stain waste.
  23. Gel is now reading for imaging under white light.

Cell Lysis by Sonication

  1. Re-suspend cell pellet in Binding Buffer.
  2. Sonicate cells for 8 minutes at 2 second intervals and 40% amplitude. Keep on ice while sonicating.
  3. Centrifuge cell lysate for 30 minutes at 10,000 x g to seperate soluble protein from cell debris/insoluble protein.

His-tag Protein Purification by AKTA Start Machine

  1. Prepare, filter and de-gas binding buffer, elution buffer: 0.5 M NaOH, 1xPBS and 20% Ethanol.
  2. Filter sample though 0.22 um filter.
  3. Follow machine instructions to collect fractions, clean and store column.
  4. Collect 20 ul of fractions which may contain desired protein as determined by AKTA. Start chromatogram and follow SDS-PAGE protocol to determine which fractions contain desired protein with minimal contaminants.

Buffer Exchange and Protein Concentration

  1. Add desired fractions to Amicon Ultra 15 ml, 10 KD concentrating column. Fill to 15 ml mark with desired buffer and centrifuge for 5 minutes at 4,600 x g. Repeat until liquid is below 1.5 ml.

    Note: column size is determined by size of fractions and desired final volume/concentration of protein needed and molecular weight cut-off is determined by size of protein being collected.

  2. Discard flow though and top up with fresh buffer up to 15 ml mark. Centrifuge for 5 minutes at 4,600 x g. Repeat until liquid is below 1.5 ml. Repeat this step 2 more times with exchange buffer.
  3. Continue centrifugation until desired volume of sample is reached. BCA assay can then be performed on the concentrated sample to determine protein concentration.

Measurement

BCA Assay

Protocol followed as per Pierce BCA Protein Assay Kit 23225 to determine protein concentration in solution:

  1. Sample to working reagent ratio = 1:8
  2. 96-well plate reader: SPECTROstar Nano BMG LABTECH
  3. Software used: SPECTROstar Nano

Native PAGE

  1. Estimate concentration of pure αPFD-SpyCatcher and βPFD-SnoopCatcher using Thermo Fisher Pierce BCA kit.
  2. Calculate the volume of αPFD-SpyCatcher concentrate required to make 10 uL of 2.5 uM solution. Calculate the amount of βPFD-SnoopCatcher concentrate such that the molar ratios of α:β are as follow: 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5
  3. Add enough αPFD-SpyCatcher solution to make 10 uL of 2.5 uM solution to 9 PCR or microfuge tubes.
  4. Add the calculated volumes of βPFD-SnoopCatcher and label the tubes with the corresponding molar ratio.
  5. Make a control of for αPFD-SpyCatcher and βPFD-SnoopCatcher by adding required volume for 10 ul of 2.5 uM.
  6. Fill all tubes up to 10 uL with 20 mM sodium phosphate, pH 8.
  7. Incubate overnight at 4°C.
  8. After incubation, add 2 uL of 6X Native PAGE loading dye to each sample and mix well.
  9. Load 5 uL of the NativeMark Unstained Protein Standard in the first well and 12 uL of sample into subsequent wells.
  10. Run the gel at 80 V for 30 minutes at room temperature.
  11. Move the gel and power pack to the cold room and run at 80 V for 2 h 45 minutes.

Catcher-Tag Assembly

  1. Mix purified protein fused to Snoop or Spy Tag with αPFD-SpyCatcher or βPFD-SnoopCatcher at a concentration of 5uM in a total volume of 10uL topped up using PBS at pH8.
  2. Incubate at room temperature overnight.
  3. Add 2uL of 6x SDS loading to the samples and incubate for 10 minutes at 95°C.
  4. Perform SDS-PAGE to visualise conjugation of proteins to prefoldins.

FRET

  1. Dilute αPFD-SpyCatcher and mVenus-SpyTag to 5uM at a total volume of 1ml with 1xPBS.
  2. Dilute βPFD-SnoopCatcher and mCerulean3-SnoopTag to 5uM at a total volume of 2ml with 1xPBS.
  3. Create the following master mixes using the stocks prepare in steps 1 and 2.
    Volume of stock to add (ul)
    Master Mix 1x PBS buffer αPFD-SpyCatcher βPFD-SnoopCatcher mVenus-SpyTag mCerulean3-SnoopTag
    A 800 400 800
    B 800 200
    C 600 400
    D 1200 600 1200
    E 800 200
    F 600 400
  4. Using the master mixes above create the final mixes as below.
    Volume of stock to add (ul)
    Final Mix 1x PBS buffer A B C D E F
    1 250 250
    2 250 250
    3 250 250
    4 250
    5 250 250
    6 250 250
    7a 200 50 250
    7b 150 100 250
    7c 100 150 250
    7d 50 200 250
    7e 250 250
  5. Add 200ul of each Final Mix to a black-96-well plate in duplicate.
  6. Scan the excitation and emission spectra of these wells to determine the ideal values of excitation and emission for each value, using the values from Markwardt et al. and Jonáš et al. as shown in the table as a starting point.
    Excitation Emission
    mCerulean3 433 (400-465) 475 (465-525)
    mVenus 515 (500-525) 528 (520-550)

    The values in these tables were taken from, Markwardt et al. (2011)1 and Jonáš et al. (2014)1.

Ellman's Assay

Performed following manufacturer's protocol.

Materials

  • Reaction Buffer: Tris-HCl (262 mM), EDTA (13 mM), pH 8.0
  • DTNB (3.5mM) in methanol
  • Isoamyl alcohol (70mM) in H2O
  • Acetyl CoA in H2O: 0.38mM final concentration
  • Purified enzyme: Concentration determined by BCA
  • Microtiter Plate

Ellman's Assay

  1. Add CoA-SH to separate wells of a microtitre plate at the following concentrations: 0, 0.2, 0.4, 0.8, 1.2, 1.6, 2mM. Supplement with water to a total volume of 350 uL.
  2. Measure the absorbance at 412nm and construct a standard curve to determine limitations of CoA-SH detection.
    • 240 uL Reaction Buffer
    • 10uL Isoamyl alcohol
    • 20uL Acetyl CoA
    • 30uL Purified enzyme (water was substituted in a blank)
  3. Add the following in triplicate:
  4. Add 50 uL of DTNB to each well.
  5. Absorbance was measured at 412nm at 30 second intervals for one hour.

NMR

  1. Make a 1% stock of DMSO by mixing 10 uL of 100% DMSO with 990 uL of water.
  2. Add 42.62 uL of 1% dimethyl sulfoxide (DMSO) (final concentration: 10 mM) and 60 uL D2O (final concentration: 10%) to a microcentrifuge tube.
  3. Transfer 497.38 uL of sample to the tube and invert several times to mix.
  4. Transfer 600 uL of the mix to an NMR tube and label accordingly.
  5. Analyze the sample using H1 NMR with water expression via excitation sculpting.

References

  1. Jonáš, A. et al. In vitro and in vivo biolasing of fluorescent proteins suspended in liquid microdroplet cavities. Lab Chip 14 3093-3100 (2014).