Linearisation pETDuet1 and pRSFDuet1 plasmid backbones using PCR

1. Forward and reverse primers created for the plasmids
2. 2 µl of plasmid + 198 µl of water
3. PCR amplification, according to the table below:

Plasmid digest (Dpn1 digest)

1. Set-up the reaction mixture:

   Restriction Enzyme	1µl
   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

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 42°C and place 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 appropriate antibiotics and grow at 37°C overnight.

Colony PCR

1. Pick up individual bacterial colonies from an agar plate that was grown overnight using a pipette tip, and dilute each colony into 50 μL of water.
2. Prepare the PCR master mix by multiplying the volume for each component shown below (for 1 reaction) by the number of colonies to sample +1 (i.e. number of reactions plus 1 excess).
   • 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
3. Add 1 uL of each diluted colony and 24 uL of PCR master mix to a PCR tube.
4. Run PCR with the following steps (lid at 105°C and volume = 20 uL):
   • Step 1: 95°C for 5 mins
   • Step 2: 95°C for 30 secs
   • Step 3: 55°C for 30 secs
   • Step 4: 68°C for 2 mins
   • 25-35 cycles of steps 2-4
   • Step 5: 68°C 5 mins
   • Then hold at 4°C
5. Run PCR products on a 1% agarose gel and image for analysis.

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.

Miniprep

Protocols were followed from the Qiagen QIAprep Spin Miniprep Kit Cat No./ID: 27104. No changes were made.

Starter culture

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

Large-scale grow-up

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

Collection of cells by centrifugation

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

Cell lysis by sonication

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

IMAC

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

1. 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.
2. Wash with 10 mL of binding buffer (20mm NaH2PO4, 500mM NaCl, 10mM Imidazole).
3. Elute with 2 mL of elution buffer (same as binding buffer, but with 500 mM Imidazole).
4. Collect fractions with SDS-PAGE.

Buffer exchange

Column

1. 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).
2. Add protein to the column
3. Top up column with PBS buffer
4. Centrifuge column at 4600 x g for 20 minutes.
5. Repeatedly centrifuge, discard flow through, and top up with PBS buffer (pH 8) until dilution factor of 0.1 is reached. That is, until there is 1% of the old buffer left in the solution.

Dialysis

1. Add 1mL of protein and 1mL of PBS buffer (pH 8) to a 15mL Falcon tube.
2. Add 2mL of the solution to a SnakeSkin™ Dialysis Tubing, 10K MWCO, 22 mm.
3. Use dialysis tubing clamps (one-piece polypropylene clamp) to further secure the solution inside the snakeskin dialysis tubing.
4. Add 500mL of PBS buffer, pH 8, (this is the buffer we want to exchange into) into a 500ml glass beaker.
5. Place the dialysis tubing with the solution into the beaker.
6. 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 iBlot 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

Catcher and Tag Assembly

1. Purified IaaH fused to SpyTag (IaaH-SpyTag) and prefoldin proteins fused to SpyCatcher (aPFD-SpyCatcher, gPFD-SpyCatcher and SpyCatcher-gPFD-SpyCatcher) were obtained
2. IaaH-SpyTag was mixed with either aPFD-SpyCatcher, gPFD-SpyCatcher and SpyCatcher-gPFD-SpyCatcher 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 obtained.
4. 5 µL of 4x Bolt LDS sample buffer was added to each sample, and then boiled for 10 minutes at 95°C to cease SpyCatcher reactivity whilst preserving any covalent interactions.
5. The samples were then examined via 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.

BCA Protein Assay

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

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

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 protocol has been adapted from the methods used by 2011 iGEM Imperial College London and Tang and Bonner’s 1947 paper^3.

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 37°C. 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