Project Description

Why Recycling Matters in Space

As space exploration ventures beyond Earth orbit toward the Moon and Mars, mission planners face a critical constraint: resupply cost increases exponentially with distance. For the International Space Station (ISS), which orbits just 400 km above Earth, resupply costs roughly $10,000–$20,000 per kilogram. For future deep-space missions to Mars, requiring 6–9 months of transit, resupply becomes logistically impractical and economically prohibitive. A crewed Mars mission lasting 2–3 years will generate an estimated 7.5–8 tons of solid waste for a four-person crew, all of which currently cannot be recycled and must either be stored (consuming valuable habitat volume) or discarded.

The ISS itself provides a concrete example. Four astronauts generate approximately 2,500 kg of trash per year, or roughly two trash cans per week. The ISS accumulates up to 2 tonnes of rubbish on board for a total of 12 tonnes per year, and this garbage is typically collected and loaded into cargo spacecraft bound for atmospheric re-entry, where it burns up on descent. While this solves the on-station storage problem, it wastes a valuable resource and creates atmospheric pollution.

Environmental Cost of Current Disposal

When spacecraft and orbital waste re-enter Earth's atmosphere at thousands of degrees Celsius, they release not only carbon dioxide and water vapor (which are harmless) but also metal oxides and combustion products from polymers like PET. After end of life, satellites and rocket stages reenter Earth's atmosphere and inject a substantial amount of their matter into the mesosphere and lower thermosphere. Recent research has documented significant and even dominant injection of several metal elements regularly used in spacecraft compared to natural injection by meteoroids, posing substantial risks of long-term adverse effects on the atmosphere such as ozone depletion, radiative effects and changes in cloud formation.

For plastic waste specifically, combustion at re-entry temperatures (>1000°C) releases particulates and volatile organic compounds. A future Mars program with multiple resupply missions could contribute measurably to this pollution, a burden that should be minimized through recycling rather than disposal.

Current Industrial Recycling: Why It Fails in Space

On Earth, PET recycling relies on two primary pathways, each with significant limitations for space:

Mechanical Recycling:

Mechanical recycling requires heating plastic to 285°C and high pressure for extrusion, followed by pelletization and polycondensation at elevated temperatures. A major drawback of mechanical recycling is the degradation of PET properties with each cycle, leading to a decrease in elasticity and viscosity, resulting in recycled polymer losing value and eventually being sent to landfills after several cycles. This process consumes significant energy and requires heavy industrial equipment, incompatible with the mass and power constraints of spacecraft.

Chemical Recycling (Industrial Standard):

The most mature industrial method is glycolysis. Glycolysis is widely adopted by major companies like DuPont, Dow Chemicals, and Shell Polyester for large-scale PET recycling, and it offers the mildest operating conditions among chemical recycling methods, operating at lower cost and lower volatility of solvents. However, glycolysis typically requires temperatures between 180–250°C and the use of catalysts or high pressures. Alternative chemical methods like methanolysis demand even harsher conditions: conventional methanolysis processes typically require harsh conditions exceeding 200°C and 2–4 MPa pressure.

Why These Fail in Space:

• High temperatures require insulation and thermal management, adding mass and power consumption
• Catalysts (metal oxides, acids, bases) create corrosion risks and contamination hazards aboard spacecraft
• Equipment footprint is too large for missions with severe volume constraints (a Mars habitat has ~100 m³ total pressurized volume)
• Safety concerns with high-pressure reactors in microgravity environments (fluid dynamics change dramatically without gravity)
• Power consumption for heating would strain spacecraft electrical systems

There is a critical temperature gap: industrial recycling operates at 180–285°C, while safe spacecraft operations typically stay below 50°C.

We propose SynPETic, a system that closes this gap by harnessing biological catalysts (enzymes) to depolymerize PET at mild temperatures and pressures, making it safe and efficient for space-based recycling.

The Core Innovation: The PETosome

Our solution combines three engineered proteins into a multi-enzyme complex inspired by nature's cellulosomes, catalytic machines found in cellulase-producing bacteria that degrade cellulose through coordinated enzyme action.

Three Engineered Proteins:

1. ICCG-DoT (PETase with dockerin linker)
   • Source: Leaf-Branch Compost Cutinase (ICCG), engineered for PET specificity
   • Function: Breaks PET polymer chains at ester bonds → releases bis(2-hydroxyethyl) terephthalate (BHET) and smaller oligomers
   • Specific activity: ~100 mU/mL (milli-units per mL)
2. TfCa-DoG (MHETase with dockerin linker)
   • Source: Thermobifida thermostable cutinase (TfCa), modified for MHET hydrolysis
   • Function: Converts MHET → final monomers (terephthalic acid [TPA] and ethylene glycol [EG])
   • Specific activity: ~100 mU/mL
   • Note: Addition of 2 mM CaCl₂ in growth medium stabilizes the catalytic domain
3. ScafGVT (Trimeric Cohesin Scaffold)
   • Source: Inspired by Clostridium thermocellum cellulosome architecture
   • Function: Provides three cohesin domains (G, V, T) that bind dockerin-tagged enzymes via high-affinity, specific interactions
   • Role: Co-localizes both enzymes on a shared scaffold, enabling substrate channeling and sequential reaction steps

Why Dockerin-Cohesin Scaffolding Matters

When ICCG-DoT and TfCa-DoG act independently in solution, their reaction products mix inefficiently:

• ICCG-DoT rapidly produces BHET and MHET (intermediate)
• MHET then accumulates because free TfCa-DoG has slower access to the intermediate
• Result: Reaction bottleneck at the MHET→TPA step (67% MHET, 32% TPA, 1% BHET after 96 hours of free enzyme)

By fusing both enzymes to complementary dockerin/cohesin domains and loading them onto the ScafGVT scaffold, we achieve spatial co-localization: the intermediate product (MHET) released by ICCG-DoT is immediately adjacent to TfCa-DoG, enabling substrate channeling and more efficient sequential conversion:

PET → [ICCG-DoT] → MHET → [immediately adjacent TfCa-DoG] → TPA + EG
(much faster cascade)

Prior iGEM projects have explored PETase expression and basic enzyme assays. Several commercial efforts (e.g., Carbios in France, Quantumscape) focus on thermophilic variants or traditional chemical catalysis.

Our contribution is unique in three ways:

1. Enzyme pair is new to the iGEM registry: ICCG-DoT and TfCa-DoG have not previously been fused to dockerin/cohesin scaffolding in this combination. We're the first to pair a leaf-compost cutinase (ICCG) with a Thermobifida MHETase (TfCa) on a cellulosome-inspired scaffold.
2. Scaffolding approach is new: While BioBricks exist for individual PETase variants, coordinated multi-enzyme complexes for plastic degradation are rare in the registry. This composite part (ICCG-DoT + TfCa-DoG + ScafGVT) opens a new design pattern for other teams pursuing multi-enzyme plastic degradation.
3. Space-optimization is distinct: Our design emphasizes:
   • Low temperature (37–50°C operational range): compatible with spacecraft thermal budgets
   • Mild conditions: no strong acids, bases, or high pressures needed
   • Minimal equipment: enzymes can be produced via fermentation on or before missions
   • Product reusability: TPA and EG monomers can be rebuilt into PET using mild repolymerization routes (enzymatic or CDI-catalyzed)

Original Project Goals

1. Clone and express three recombinant proteins (ICCG-DoT, TfCa-DoG, ScafGVT) in E. coli
2. Characterize each protein for identity and purity
3. Measure PETase activity on real PET film
4. Demonstrate two-enzyme synergy on both defined substrates (BHET) and real PET
5. Validate composite part (PETosome assembly and functionality)
6. Complete repolymerization pathway (PET reconstruction from TPA + EG monomers)
7. Test in simulated space conditions (microgravity via clinostat; radiation exposure)

What We Achieved

Wet Lab:

• Successfully engineered and purified all three proteins from E. coli expression systems
• Confirmed protein identity via SDS-PAGE, Western blot (anti-His antibody)
• Measured enzymatic activity across multiple assay formats (qualitative plate assays, quantitative HPLC)
• Key result: ICCG-DoT + TfCa-DoG combination showed superior activity on BHET substrate compared to either enzyme alone (p < 0.000001 for both TPA and MHET production)
• Demonstrated statistically significant synergy in the two-enzyme system

Composite Part:

• Constructed and validated a novel dockerin-cohesin scaffold complex
• Confirmed binding specificity (scaffold with no dockerin shows no activity improvement; negative control passes)

Measurement:

• Targeting Best Measurement award: comprehensive characterization using SDS-PAGE, Western blot, quantitative BSA assay (protein concentration), HPLC (product quantification with standard curves)
• Replicate data (n=2–3 per condition) with statistical testing (nonlinear regression + extra sum-of-squares F-test)

What We Did Not Complete

1. Full PETosome assembly in microgravity: We designed a clinostat but did not complete spaceflight testing. This is forward work for future missions.
2. Repolymerization validation: External collaborators (Maroof, Trade Wind Bio) explored CDI esterification and DES + lipase routes; results are promising (FTIR shows ester formation) but incomplete (full GPC/NMR characterization pending).
3. End-to-end recycling loop in space: Our work validates the depolymerization half; a future team will need to integrate repolymerization and test the full cycle aboard ISS or a high-altitude platform.

For Space Missions

• Mars missions: 7.5–8 tons of waste from a 2–3 year mission can be recycled in-situ instead of requiring resupply
• ISS operations: ~12 tonnes/year of plastic waste could be processed into useful feedstock (e.g., filament for 3D printing, replacement components)
• Reduced launch mass: Every kilogram of waste recycled saves ~$10k–$20k in resupply costs and propellant

For Earth

• Atmospheric protection: Reducing orbital waste re-entry helps mitigate long-term ozone depletion and atmospheric chemistry changes
• Proof of concept: If enzymatic recycling works in the harsh space environment (vacuum, radiation, thermal extremes), it's a robust solution for terrestrial recycling in less ideal conditions
• Sustainable loop: TPA and EG monomers can feed back into PET production or high-value polymers, creating a true circular economy for plastics

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