Protocol for Assay

Protocol for Assay

Draft v 2

Experimental Protocol: CBM27_RGD_MaSp1_4x Fusion Expression and Tremella Composite Formation

Construct: CBM27_RGD_MaSp1_4x_Fusion
Vector: pET28a (NdeI/XhoI, C-terminal 6xHis tag)
Protein MW: ~37 kDa (340 aa)
Expression system: Cell-free protein synthesis (CFPS) — see Aim 2 context below

Aim 2 Context: Why This Assay Exists

This protocol is the experimental component of Aim 2a of the Exoskin project. Aim 1 established the computational and synthesis-feasibility foundation: the CBM27_RGD_MaSp1_4x construct has been designed in Benchling, codon-optimised for E. coli K12, validated structurally by AlphaFold3 (CBM27 pLDDT >90, RGD solvent-exposed, MaSp1 disordered as expected), and confirmed orderable by Twist Biosciences at 6,297 bp in pET28a ($145.45, Clonal Gene). HADDOCK docking of CBM27 against Tremella β-(1→3)-D-mannan is pending due to a documented gap in carbohydrate force field databases for fungal mannans; this makes wet lab confirmation of polysaccharide binding the critical validation step.

Primary execution route: Ginkgo Bioworks cloud lab automation (Aim 2a)

The protocol below is designed for submission to the Ginkgo Bioworks (via HTGAA Twist aim 1 order submission to be confrimed) automated CFPS platform. Steps are written to be compatible with liquid-handling automation and standard Ginkgo reagent sets. Where a step requires upfront specification (e.g. chaperone supplementation), this is flagged explicitly, because automated cloud lab runs cannot be modified mid-protocol after submission.

Alternative execution route: If Ginkgo partnership is not confirmed in time, this protocol can be run manually using NEB PURExpress E6800 in any standard molecular biology lab. All volumes and conditions are identical between routes.

Step 1: Cell-Free Protein Synthesis

Materials needed:

• PURExpress Solution A
• PURExpress Solution B
• Murine RNase Inhibitor
• Template DNA: pET28a_CBM27_RGD_MaSp1_4x plasmid (circular, 250 ng) or linear PCR product
• DnaK chaperone mix (supplementary, see note below)
• Nuclease-free water
• Ice

Chaperone specification note (required for Ginkgo submission): MaSp1 repeat regions are prone to aggregation and beta-sheet stacking due to their glycine/alanine-rich repetitive sequence. DnaK/DnaJ/GrpE chaperone supplementation should be specified upfront in the Ginkgo protocol submission. Typical addition: 2 µM DnaK, 0.4 µM DnaJ, 0.1 µM GrpE in the final reaction volume. If using manual PURExpress, add chaperones after assembling the base reaction and before incubation.

Protocol:

1. Thaw Solutions A and B on ice. Do not vortex.
2. Assemble the following reaction on ice in this exact order in a 1.5 ml microcentrifuge tube:
   • 10 µl Solution A
   • 7.5 µl Solution B
   • 0.5 µl RNase Inhibitor (20 units)
   • 2 µl template DNA (250 ng)
   • 0.5 µl DnaK chaperone mix (see note above)
   • 4.5 µl nuclease-free water
   • Total volume: 25 µl
3. Mix gently by pipetting up and down 5 times. Do not vortex.
4. Incubate at 37°C for 2 hours.
5. Place on ice immediately after incubation.

Run alongside: one negative control reaction with no template DNA, identical volumes and conditions.

Step 2: His-Tag Purification (Mandatory Before Step 3)

This step is required before proceeding to Tremella composite formation. The raw PURExpress reaction contains many E. coli ribosomal and accessory proteins. Mixing unpurified reaction with Tremella polysaccharide risks false-positive gel formation from non-specific protein-polysaccharide interactions. Ni-NTA purification isolates only the 6xHis-tagged fusion protein.

Materials needed:

• Ni-NTA agarose resin (e.g. Qiagen, cat. 30210) or magnetic Ni-NTA beads
• Binding buffer: 50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, pH 8.0
• Wash buffer: 50 mM NaH₂PO₄, 300 mM NaCl, 20 mM imidazole, pH 8.0
• Elution buffer: 50 mM NaH₂PO₄, 300 mM NaCl, 250 mM imidazole, pH 8.0
• Spin columns or magnetic rack
• PBS pH 7.4 for buffer exchange

Protocol:

1. Dilute the 25 µl CFPS reaction with 225 µl binding buffer (1:10 dilution).
2. Add 25 µl pre-equilibrated Ni-NTA resin slurry. Mix on rotary mixer for 30 minutes at 4°C.
3. Centrifuge 700 × g, 2 minutes. Remove supernatant (flow-through).
4. Wash resin twice with 200 µl wash buffer. Centrifuge 700 × g, 2 minutes each wash.
5. Elute with 50 µl elution buffer. Incubate 5 minutes at room temperature, then centrifuge 700 × g, 2 minutes.
6. Buffer exchange into PBS pH 7.4 using a 10 kDa MWCO spin concentrator to remove imidazole.
7. Recover purified protein in approximately 20–25 µl PBS.

Reserve 2.5 µl for SDS-PAGE (Step 3). The remaining ~18–22 µl proceeds to Step 4.

Step 3: SDS-PAGE Confirmation

Materials needed:

• 10-20% Tris-glycine precast gel
• SDS loading buffer (4x)
• Protein ladder (10-250 kDa range)
• Running buffer (Tris-glycine-SDS)
• Coomassie Blue stain (or silver stain for higher sensitivity)

Protocol:

1. Take 2.5 µl of the purified eluate and add 2.5 µl of 2x SDS loading buffer (or 0.83 µl of 4x buffer + 1.67 µl nuclease-free water to reach 1x final concentration).
2. Heat at 95°C for 5 minutes to denature proteins.
3. Load onto gel alongside protein ladder and a lane with the same volume of purified negative control eluate.
4. Run at 200V for 35 minutes.
5. Stain with Coomassie Blue for 1 hour. Destain with water overnight, or use methanol/acetic acid destain for faster results.
6. Look for a band at approximately 37 kDa.

Success criterion: A visible band at 37 kDa in the expression lane that is absent in the negative control lane. Proceed to Step 4 only if this criterion is met.

If no band is observed: Do not proceed to composite formation. Troubleshoot expression first: check template DNA integrity by nanodrop and gel, verify T7 promoter orientation in the construct, and consider a second run with increased template (up to 500 ng) or extended incubation (4 hours at 37°C). If aggregation is suspected (smear or high-MW band), confirm DnaK chaperone supplementation was included.

Step 4: Tremella Composite Formation (only if Step 3 successful and not confirmed if can be run after above if at Ginkgo Bio)

Materials needed:

• Tremella fuciformis polysaccharide: minimum 95% purity, fungal-derived, CAS 9083-80-1 (note: use a supplier that specifies β-(1→3)-linked mannan as the primary backbone structure; Sigma-Aldrich or Funakoshi are appropriate sources — confirm catalogue specification before ordering)
• PBS buffer pH 7.4
• Purified fusion protein from Step 2 (~18–22 µl)
• Microcentrifuge tubes
• Rotary mixer
• Positive control: PURExpress negative control eluate (same volume as fusion protein sample)

Protocol:

1. Prepare a 1% w/v Tremella polysaccharide solution by dissolving 10 mg dried Tremella polysaccharide in 1 ml PBS pH 7.4. Stir gently at room temperature for 2 hours until fully dissolved. The solution should be visibly viscous. If it does not dissolve fully, warm to 37°C for 30 minutes before cooling back to room temperature.
2. Take the purified fusion protein from Step 2 (~18–22 µl). Record exact volume.
3. Add an equal volume of the 1% Tremella polysaccharide solution to the protein. Mix gently by pipetting 10 times. Do not vortex.
4. Prepare a parallel control by mixing the same volume of the purified negative control eluate with the same volume of 1% Tremella solution.
5. Incubate both tubes at room temperature for 30 minutes on a rotary mixer at low speed, to allow CBM27 domain binding to the Tremella β-(1→3)-D-mannan backbone.
6. Assess viscosity using the tube inversion test: invert each tube 180° and observe flow behaviour over 10 seconds. A gel or high-viscosity hydrogel will not flow freely; a low-viscosity solution will run to the cap immediately.
7. As a semi-quantitative measure, attempt to aspirate 10 µl from each tube using a standard 20 µl pipette tip. Note resistance to aspiration on a simple 0/1/2 scale (0 = no resistance, 1 = moderate resistance, 2 = cannot aspirate). Record for both the fusion protein tube and the control tube.

Success criterion: Visible increase in viscosity or gel formation in the fusion protein tube that is absent or substantially lower in the negative control tube. A tube inversion score of 1 or 2 in the fusion protein tube versus 0 in the control tube constitutes a positive result.

If no gel formation is observed: CBM27 may not be folding correctly under cell-free conditions, or the Tremella preparation may not contain sufficient β-(1→3)-mannan backbone to support CBM27 binding. Troubleshooting options: (1) repeat with a different Tremella polysaccharide source or lot; (2) run a second CFPS reaction with extended incubation and additional DnaK; (3) attempt CD spectroscopy on the purified protein to assess CBM27 secondary structure if equipment is available.

What Each Result Means

Ginkgo Submission Checklist

If submitting to Ginkgo Bioworks cloud lab, confirm the following are specified in your protocol submission before sending:

• Plasmid sequence file (Benchling export, .gb or .fa) for CBM27_RGD_MaSp1_4x in pET28a
• DnaK chaperone supplementation requested (2 µM DnaK, 0.4 µM DnaJ, 0.1 µM GrpE)
• Expected band size stated as 37 kDa (for SDS-PAGE automated imaging parameters)
• Ni-NTA purification step requested prior to composite assembly
• Tremella polysaccharide source and CAS number specified (CAS 9083-80-1, minimum 95% purity, β-(1→3)-mannan backbone confirmed)
• Negative control (no-template CFPS) requested in parallel