Team:UNSW Australia/Experiments


Experiments

Cloning

Linearisation pETDuet1 and pRSFDuet1 plasmid backbones using PCR

  • Forward and reverse primers created for the plasmids
  • 2 uL of plasmid + 198 uL of water
Component 50 µl Reaction Final Concentration
Q5 High-Fidelity 2X Master Mix

 25 µl

 1X
10 µM Forward Primer

2.5 µl

0.5 µM

10 µM Reverse Primer

2.5 µl

0.5 µM

Template DNA

2 uL DILUTED

< 1,000 ng

Nuclease-Free Water

18 uL

  
STEP TEMP TIME

Initial Denaturation

98°C

30 seconds

25–35 Cycles

98°C

5–10 seconds

62

10–30 seconds

72°C

2 min

Final Extension

72°C

2 minutes

Hold

4–10°C

  

Plasmid digest (Dpn1 digest)

  • Set-up the reaction mixture:

    Restriction Enzyme

    1µl

    DNA

    1 µg

    10X Cutsmart

    5 µl (1X)

    Total Reaction Volume

    50 µl
  • Incubate for 1 hr at 37C
  • Heat inactivate at 80°C for 20 minutes

Agarose gel electrophoresis

  1. Combine 100x agarose powder with 1x TAE buffer in a microwavable flask (eg. 1 g of agarose for 100 mL of TAE). The volume of agarose gel will depend on the size of gel you are making.
  2. Microwave for 1-2 min until the agarose is completely dissolved (do not overboil the solution). Stop and swirl the flask every 20 seconds and until the solution is as clear as water.
  3. Let agarose solution cool down to about 50°C (when you can comfortably hold the flask with your hand), then add 1 uL of RedSafe to the agarose solution.
  4. Seal the ends of a gel tray using masking tape. Pour the agarose into the gel tray with a well comb in place. Let the gel sit at room temperature for 20-30 mins until the gel solidifies.
  5. Place the gel into the gel box, fill the gel box with 1 x TAE buffer until the gel is covered then remove the well comb.
  6. Mix 2 uL of the digest sample with 3 uL of H2O and 1 uL of 6x loading dye.
  7. Load c into the first lane of the gel. Load the remaining digest samples into the gel. Remember to include a negative control (non-digested plasmid).
  8. Connect the gel box to a power pack and run the gel at 100V for 1 hr. You should be able to see small bubbles rising in the buffer solution immediately after you turn the power pack on.
  9. Carefully take the gel tray to the spectrophotometer and analyse the DNA fragments with UV light. We expect to see a single clear band in digested samples, and a smear for the undigested plasmid at a higher position. Smear and a clear band indicates incomplete digestion.

Gibson Assembly

Materials:

  • 5X Isothermal Reaction Mix (6 mL total, Store at -2:0/lideg;C):
    • 3 mL 1 M Tris-Hcl (pH 7.5)
    • 300 μL 1 M MgCl2
    • 60 μL 100 mM dGTP
    • 60 μL 100 mM dATP
    • 60 μL 100 mM dTTP
    • 60 μL 100 mM dCTP
    • 300 μL 1 M DTT
    • 1.5 g PEG-8000
    • 300 μL 100 mM NAD
    • 360 µL water
  • Assembly Master Mix (1.2 mL total, store in 15 µL aliquots at -20°C.):
    • 320 μL 5X Isothermal Master Mix
    • 0.64 μL 10 U/μL T5 exonuclease
    • 20 μL 2 U/μL Phusion DNA Pol
    • 0.16 μL 40 U/μL T4 DNA Ligase
    • 860 μL water

Method:

  1. PCR or digest your fragment of choice and gel purify
  2. If PCR from a methylated DNA template (e.g. propagated plasmid), a DpnI digest can be used to remove the unwanted plasmid. Clean up afterwards.
  3. Thaw a 15 μl assembly mixture aliquot and keep on ice until ready to be used.
  4. Add 5 μl of DNA to be assembled to the master mixture.
  5. The DNA fragments should be in equimolar amounts.
  6. Small fragments (<1 kb) must be added in a five times excess
  7. You can calculate the quantity of each fragment using their molecular weights.
  8. Alternatively, you can use the length of each fragment as a proxy for the molecular weight (assuming similar GC content in all fragments).
  9. Use 10-100 ng of each ~6 kb DNA fragment. For larger DNA segments, increasingly proportionate amounts of DNA should be added (e.g. 250 ng of each 150 kb DNA segment).
  10. Incubate at 50°C for 15 to 60 min (60 min is optimal).

Heat shock transformation

  1. Incubate 50ng of plasmid construct with 25 µL of chemically competent E. coli T7
  2. Express cells or E. coli DH5α on ice for 30 minutes.
  3. Heat shock the cells for 45 seconds at 42C and placed back onto ice for 2 minutes.
  4. Allow cells to grow for 45 minutes in 200 µL of SOC outgrowth media (NEB) at 37°C and 200 rpm.
  5. Spread plate onto Luria broth (LB) agar plates containing 100 µg/mL of ampicillin and grown at 37°C overnight.

Colony PCR

  1. Pick up 5 individual bacterial colonies from each plate that was grown overnight using a pipette tip and dilute each colony into 50 uL of water. Label A, B, C, D, E etc.
  2. Create the colony PCR master mix. Determine volumes for the cPCR master mix by multiplying (the number of reactions + 2) by each volume below:
    • 18 uL Nuclease free water
    • 5 uL 5x Taq master mix
    • 0.5 uL 10 uM T7 promoter primer
    • 0.5 uL 10 uM T7 terminator primer

    Example, for 20 colonies, you would add 22 x 18 uL of water, 22 x 5 uL of 5x Taq master mix, 22 x 0.5 uL of forward primer and 22 x 0.5 uL of reverse primer.

  3. Add 1 uL of each bacterial dilution and 24 uL of cPCR master mix to a PCR tube.
  4. Run PCR with the following instructions (lid at 105°C and volume = 20 uL):
    • 95°C 5:00 mins
    • 95°C 0:30 mins
    • 55°C 0:30 mins
    • 68°C 2:00 mins
    • 68°C 5:00 mins
    • 4°C 0:00 mins (hold)
  5. Run PCR products on a 1% agarose gel, TAE running buffer, 100V, 1 hr. 10 uL of PCR product + 2 uL of 6x loading dye, and have 100bp ladder in the first lane.

Sequencing

  1. Transfer 10 uL of purified plasmid sample (50-100ng/uL) to an Eppendorf tube. Add 5 uL of one primer.
  2. Request sequencing.
  3. Label Eppendorf tubes with the order number, and 1, 2, 3, etc.
  4. Take Eppendorf tubes to the new Biosciences building Lvl 2 (at UNSW, Sydney, Australia), and store the samples in the fridge provided.
  5. Sanger sequencing is carried out following the provided protocol.

Restriction cloning

  1. Set-up the reaction:

    Restriction Enzyme

    1µl of each enzyme

    DNA

    1 µg

    10X Cutsmart

    5 µl (1X)

    Total Reaction Volume

    50 µl
  2. Incubate for 1 hr at 37°C
  3. Heat inactivate at 80°C for 20 minutes

Ligation

  1. Set up the following reaction in a microcentrifuge tube on ice:
    Component 20 μl Reaction

    T4 DNA Ligase Buffer (10X)*

    2 μl

    Vector DNA

    50 ng

    Insert DNA

    A molar ratio of 1:3 vector to insert should be used

    Nuclease-free water

    to 20 μl

    T4 DNA Ligase

    1 μl
  2. Gently mix the reaction by pipetting up and down and microfuge briefly.
  3. For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.
  4. Heat inactivate at 65°C for 10 minutes.
  5. Chill on ice and transform 1-5 μl of the reaction into 25 μl competent cells.

Miniprep

Protocols were followed from the Qiagen QIAprep Spin Miniprep Kit. No changes were made.

Protein Expression and Purification

Starter culture

One colony was selected from the plate grown overnight and grown in 2 mL of LB containing 2 uL of the appropriate antibiotic at 37°C and 200 rpm and left overnight.

Large-scale grow-up

  • Baffled shake flasks containing 500 mL of LB with 50uL of the appropriate antibiotic at 37°C are inoculated with the starter culture.
  • The cells are grown at 37°C and 200 rpm and OD600 is periodically measured.
  • Once OD600 reaches above 0.6, add IPTG of 1 mM concentration to induce the expression the proteins.
  • After induction, grow the cells overnight at 24°C, 200 rpm.

Collection of cells by centrifugation

  • Centrifuge the bacterial culture at 4600 x g for 20 minutes.
  • Collect cell pellet and resuspended in binding buffer (20mm NaH2PO4, 500mM NaCl, 10mM Imidazole).

Cell lysis by sonication

  • Lyse the cell pellet by sonication (Branson) for 10 minutes at 50% amplitude at alternating 2 second intervals, kept on ice.
  • Centrifuge the cell lysate at 15000 rpm for 45 minutes.
  • Collect the supernatant (soluble fraction).

IMAC

Immobilised metal ion affinity chromatography (IMAC) was performed to purify the expressed proteins.

  • His-tagged protein is bound to a 1 mL Ni-NTA Superflow Cartridge (Qiagen) by loading the soluble fraction of the cell lysate onto the column.
  • Wash with 10 mL of binding buffer (20mm NaH2PO4, 500mM NaCl, 10mM Imidazole).
  • Elute with 2 mL of elution buffer (same as binding, but with 500 mM imidazole).
  • Collect fractions with SDS-PAGE.

Buffer exchange

Column

  • Elutions were analysed with SDS-PAGE and buffer exchanged into PBS pH 8 using Pierce Protein Concentrators PES, 10K MWCO, 2-6 mL (Thermo Scientific).
  • Add protein to the column
  • Top up column with PBS buffer
  • Centrifuge column at 4600 x g for 20 minutes.
  • Repeatedly centrifuge, discard flow through, and top up with PBS buffer (pH 8) until dilution factor of 0.1 is reached. That is, there is 1% of the old buffer left in the solution.

Dialysis

  • Add 1mL of protein and 1mL of PBS buffer (PH 8) to a 15mL Falcon tube.
  • Add 2mL of the solution to a SnakeSkin™ Dialysis Tubing, 10K MWCO, 22 mm.
  • Use dialysis tubing clamps (one-piece polypropylene clamp) to further secure the solution inside the snakeskin dialysis tubing.
  • Add 500mL of PBS buffer, pH 8, (this is the buffer we want to exchange into) into a 500ml glass beaker.
  • Place the dialysis tubing with the solution into the beaker.
  • Place the beaker on top of a magnetic stirrer, 75 rpm, and leave overnight.

Western Blot

Materials

  • NuPAGE Bis-Tris gel
  • NuPAGE MES running buffer
  • Mini iBlotTM stack
  • TBS-T:
  • 1x TBS with 0.1% Tween20
  • Blocking Solution:
  • 5% skim milk in TBS-T
  • Antibody Solution:
  • 1:2000 dilution of HRP conjugated anti-His-tag antibody in TBS-T + 1% BSA
  • Chemiluminescent HRP substrate

Sample Preparation

  1. Add reducing buffer to the bacterial lysates
  2. Heat at 95 degrees for 5 minutes

SDA-Page Gel

  1. Remove the NuPAGE gel from its packaging and peel off the plastic strip from its base
  2. Place the gel inside the tank, and fill with NuPAGE MES running buffer
  3. Load 5uL of the protein standards ladder into the first well
  4. Load up to 20uL of each lysate sample into the wells
  5. Connect the gel tank to a power pack, and run at 160V for 40min

Protein Transfer

  1. Remove and rinse the gel in water
  2. Inside an iBlotTM Transfer Device, assemble the mini stack with the gel inside
  3. Run at 20V for 7min

Blocking

  1. Incubate the membrane for 1-2hrs in blocking solution at room temperature, shaking

Antibody Staining

  1. Incubate the membrane in antibody solution either at 4 degrees overnight, or at room temperature for 2 hours
  2. Wash the membrane in TBS-T three times for 10mins per wash at room temperature, shaking

Detection

  1. Remove membrane from the last wash and place in chemiluminescent image analyser
  2. Prepare HRP substrate according to manufacturer's instructions and add to the membrane
  3. Image

Assembly

Catcher and Tag Assembly

  1. IaaH fused with SpyTag and proteins fused to SpyCatcher
  2. aPFD-SpyCatcher, gPFD-SpyCatcher and SpyCatcher-gPFD-SpyCatcher were mixed at a concentration of 3 µM and 15 µM respectively in a total volume of 250 µL in PBS pH 8, and incubated at room temperature.
  3. After 0, 10, 20 and 30 minutes of incubation, a 10 µL sample was taken and boiled with 5 µL 4x Bolt LDS sample buffer for 10 minutes at 95oC to cease SpyCatcher reactivity while preserving any covalent interactions.
  4. The samples were then examined on SDS-PAGE.

Size Exclusion Chromotography (SEC)

We would like the thank Hélène Lebhar of the UNSW Recombinant Proteins facility for conducting SEC experiments for us.

TEM

We would like to thank Daniel Lorenz Winter for conducting the TEM experiments for us.

FRET

FRET Protocol for Negative Controls

Part 1

  • Dilute mCerulean3 (Cerulean) and mVenus (Venus) protein samples to 1 mg/mL (≈ 34 uM) with PBS pH 7.4
  • Create a serial dilution of both Cerulean and Venus from 1mg/mL down to 1ug/mL in an (ideally) black sided 96 well plate (200 uL of undiluted protein, transfer 20 uL to 180 uL PBS, and so on).
  • 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.
  • Blank appropriately with 180 uL PBS.

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)2

  • Dilute samples equally if concentration is too high for the fluorescence reader of the machine.

Part 2

  • Add 90 uL of 1 mg/mL protein to 90 uL PBS for each protein in triplicate.
  • Add 90 uL of Cerulean to 90uL Venus in triplicate.
  • Blank with 180 uL PBS.
  • Excite all wells at the ideal excitation value determined for Cerulean and scan wavelengths longer than this.
  • Please see supplementary data for specific parameters used.

Enzyme Assays

BCA analysis

Protocol followed as per Pierce BCA Protein Assay Kit 23225 micro-plate procedure:

  • Sample to working reagent ratio = 1:8
  • 96-well plate reader: SPECTROstar Nano BMG LABTECH
  • Software used: SPECTROstar Nano -> exported to excel 2017 for analysis.

Salkowski Assay

The Salkowski assay is a measurement-based protocol used to assess the quantity of indole-3-acetic acid within a sample. This particular method has been developed from processes from 2011 iGEM Imperial College London and Tang and Bonner’s 1947 paper3.

Cellular Protocol

  1. Pick a single colony of untransformed T7 and transformed T7 and inoculate a 2-5ml overnight culture in LB.
  2. Add antibiotics to the transformed culture.
  3. The next day, add 50ml of LB + 2.5mg/mL tryptophan to a 250ml shake flask labelled “Control”.
  4. Add 50ml of LB + 2.5mg/mL tryptophan + antibiotic to another baffled shake flask labelled “IAA”.
  5. Add 1ml of the respective overnight cultures to each flask. Incubate with shaking (200 rpm) at 37oC. Sample at 1h, 4h, 7h and 20-24h.
  6. For each sample: a. Measure the OD at 600 nm.
  7. Spin down 1ml. Retain pellet and freeze. Retain supernatant and store at 4 C. Label these 4.
  8. The next day: Add Salkowski reagent to supernatants at the ratio of 1ml per 0.5ml of supernatant. Incubate in the dark for 30 minutes
  9. Add 1ml of Salkowski reagent to each cell pellet.
  10. Vortex to mix.
  11. Incubate in the dark for 30 minutes.
  12. Centrifuge at full speed for 5 minutes.
  13. Measure the absorbance at 530 nm Measure appropriate controls as well
  14. Normalise readings at each time point against OD600.

The assay is performed by adding the Salkowski reagent to a prepared sample:

  1. To prepare the Salkowski assay reagent, slowly add 95% H2SO4 to MilliQ water in a ratio of 3:2, allowing time for heat dissipation between additions,
  2. Dissolve 4.5g/L of anhydrous FeCl3 into the solution. Store in the dark, wrapped in foil.
  3. Prepare the standard by using a 3mM stock, then diluted 1 in 10 for usage – with 300uM to 0uM concentrations.
  4. Prepare a standard curve.
  5. Samples are run by adding the sample (or standard) to the Salkowski reagent in a ratio of 1:2 in a time sensitive manner within a 96-well microtitre plate.
  6. Run the plate on a plate reader for absorbance at 530nm, after being left for 30 minutes in the dark.

High Performance Liquid Chromotography (HPLC)

  1. Acidify supernatant with 5M HCl to pH 2.5
  2. Extract supernatant with an equal volume of ethyl acetate
  3. Evaporate off the solvent by either drying it through a vacuum or dissolving in methanol & acetic acid at pH 4.5:
  4. 1mL of a solution of 25% methanol, 1% acetic acid, rest H2O
  5. 250uL methanol, 10uL acetic acid, 740uL H2O
  6. Run system with solution 72% of 1% acetic acid & 28% of 100% methanol for 0.8mL/minute with injection of 30

Plants

Seed Surface Sterilisation

  1. Wild type Arabidopsis thaliana seeds were surface sterilised by mixing in a solution of 2.5% sodium hypochlorite with a drop of Tween for 10 minutes.
  2. Seeds were subsequently washed eight times in sterile water.

Indole-3-aecetic Acid Filter Sterilisation

  1. 10 mM stock indole-3-acetic acid (IAA) was prepared by dissolving indole-3-acetic acid sodium salt purchased from Sigma Aldrich in ethanol.
  2. In a laminar flow hood, the solution was filter sterilised through a 0.22μm Millex® syringe driven filter into and stored at 4°C in a foil covered container.

MS Agar Plate Preparation

  1. Murashige and Skoog (MS) media was prepared using M5519 MS basal powdered medium purchased from Sigma Aldrich.
  2. In four separate Schott bottles, 250 mL of MS media was prepared using 1.1 g of MS basal salts (per manufacturer instructions) and Milli-Q purified water.
  3. Each mixture was supplemented with sucrose (1% w/v) and agar powder (0.8% w/v) and adjusted to pH 5.7 using KOH.
  4. Mixtures were autoclaved and allowed to cool to approximately 50°C (or until able to be held comfortably).
  5. Cooled bottles were transferred to a dark laminar flow hood where indole-3-aecetic acid solution was added to prepare four separate MS agar mixtures at 0 μM, 1 μM, 10 μM and 100 μM concentrations IAA respectively.
  6. In a dark laminar flow hood, 50mLs of media was pipetted into a square plate using a serological pipette. Four 50mL plates were prepared for each concentration of IAA.
  7. Plates were labelled, sealed with parafilm and stored wrapped in foil at 4°C until further use.

Liquid Culture Media Preparation

  1. MS media was prepared using M5519 MS basal powdered medium purchased from Sigma Aldrich.
  2. In an autoclavable Schott bottle, 500 mL 1/2 MS media was prepared using 1.1g of MS basal salts and 500 ml of Milli-Q purified water (4.4 g salts equivalent to 1L MS media per manufacturer’s instructions).
  3. Media was supplemented with sucrose (1% w/v) and adjusted to pH 5.7 using KOH.
  4. Media was autoclaved and stored at room temperature until further use.

Arabidopsis thaliana Growth on Agar Plates

  1. Surface sterilised seeds were added to a flask containing an autoclaved solution of Milli-Q water with 1% w/v sucrose in a laminar flow hood.
  2. This flask was covered with foil and seeds were stratified at 4°C for two days.
  3. Stratified seeds were then allowed to germinate under light at room temperature for _ days.
  4. In a dark laminar flow hood, seedlings were transplanted to MS media plates with varying concentrations of IAA. Approximately five seedlings were transplanted per plate using sterile tweezers and loops. Seedlings were arranged in a line, 2 cm from the top of each plate. Three plates with seedlings were prepared for each concentration.
  5. Plates were sealed with parafilm and covered with foil leaving only the top 2 cm exposed in order to limit IAA photodegradation and simulate the natural growth conditions of higher plants whereby only shoots are exposed to illumination.1 Plates were stored upright at an approximately 90° angle. The plated seedlings were grown under light at room temperature for 20 days.
  6. Primary root length and lateral root numbers were measured at 4, 6, 11 and 20 days following the initial seedling transplant.

Arabidopsis thaliana Liquid Culture

  1. In a laminar flow hood, 25 mL of 1/2 MS media with 1% sucrose was aliquoted into twelve 50 mL foil wrapped falcon tubes.
  2. Filter sterilised IAA was added to prepare duplicate tubes at 0 nM, 1nM, 10 nM, 100 nM, 1 μM and 10 μM concentrations of IAA.
  3. One hundred μL of surface sterilised A. thaliana seeds were added to each tube.
  4. Liquid cultures were stratified at 4°C for two days, then grown under light at room temperature for 9 days.
  5. Three seedlings from each tube were selected at random and measured for primary root length and shoot length.