Sisi Fan, Jemma Pilcher, Iain Bower, Matthew Chin, Margarita Kopniczky, Wenqiang Chi & James Strutt
Could Synthetic Biology transform all this rubbish (t rash)…
…into these?
Plastic of the Future: Poly-3-hydroxybutyrate PHB • A bacterial energy store • An alternative for petrochemical • Biocompatible PHB granules However… • Expensive • Produced from plant biomass
Resources are lost in waste 700 million tons of trash is generated per year
Human Solid Recovered Fuel is the target Practices • Human practice identified Solid Recovered Fuel (SRF) • SRF is a non-recyclable product of material recovery facilities It costs POWERDAY over $28 million a year to ship SRF for incineration. In August we visited POWERDAY the largest Materials Recovery Facility in SE England
Human What is SRF made of? Practices 1/5 Plastics 4/5 Fibres Wood Simon Little, Paper Marketing Manager 320,000 tons per year
Human The Right use for SRF Practices Consulted with the local government Greater London Authority at City Hall “Imperial’s research project… …sits well with achieving the Mayor’s vision” - Doug Simpson (Principal Policy and Programme officer, Waste and Energy Team)
Our project is OUR AIM Human Practices driven Module 1: Making bioplastic from waste Module 2: Recycling bioplastics Module 1 Module 2 Waste to Bioplastic to Product
How can we turn SRF into a recyclable resource? By engineering E. coli to: • Breakdown SRF to produce products and feedstock • Convert feedstock into bioplastic • Continually reprocess bioplastic Waste to Bioplastic to Product
Module 1: Resource-full Waste Non-recyclable, mixed waste M1: Resource-full Waste Polyurethane Wood, paper Chemical degradation & fibres products M2: Plastic Fantastic Control phaZ1 lysate NATIVE PHB production P(3HB) degradation
Module overview PHB
Chassis choice- E.coli MG1655 Our chassis and parts: -GRAS: Generally regarded as safe - Already used in industry
Our chassis thrives on waste The bacteria are still alive after 3 days No toxic effect LB Waste Incubated conditioned and plated media Growing our E.coli on PBS and waste And after 6 days!
PHB production from waste Non-recyclable, mixed waste M1: Resource-full Waste Chemical Polyurethane Wood, paper products degradation & fibres M2: Plastic Fantastic Control phaZ1 lysate P(3HB) degradation PHB production
Requirement for PHB production Our bacteria should: 1. Produce PHB 2. Produce PHB from the mixed waste M1 : Design Specifications Test Model
Native operon, BBa_K934001 (Tokyo2012) Control phaCAB Nile red stains PHB Specifications Model Test M1: M1: M1 : Design Specifications Test Model
Model-based flux optimisation 3 4 2 Metabolism 1 Glucose We created our models for the project from differential equations using MatLab M1 : Design Specifications Test Model
Metabolic model of PHB production from glucose Glucose TCA Cycle Glycolysis Dixton et al., 2011 PHB synthesis Dixton et al., 2011 M1 : Design Specifications Test Model
Model-based flux optimisation 3 4 2 Metabolism 1 Glucose M1 : Design Specifications Test Model
Model-based flux optimisation 3 4 2 Metabolism 1 Glucose M1 : Design Specifications Test Model
PHB production is sensitive to PhaB Sensitivity of [PHB] Time (minutes) M1 : Design Specifications Test Model
PHB production is sensitive to PhaB 3 4 2 Metabolism 1 Glucose Our model for the project M1 : Design Specifications Test Model
Increasing PhaB level: Effect on PHB production P3HB Concentration (g/L) M1 : Design Specifications Test Model
Model Prediction J23104 increase PHB production rate Modelling data Constitutive Promoter, BBa_K1149052 Hybrid Promoter, BBa_K1149051 M1 : Design Specifications Test Model
Key Result Hybrid operon produces much more PHB Native Hybrid M1: M1: M1 : Design Specifications Test Model
Key Result: 60% of biomass is P3HB Imperial iGEM’s Native promoter Hybrid Promoter phaCAB phaCAB Dry Biomass (g) Native Hybrid 3.48 1.8 P3HB mass (g) 2.05 0.09 LB media volume (L) 1.2 1.2 Optimised Literature Imperial’s P(3HB) mass/dry mass 58.9 5 values are between cells (%) * 80% Tokyo Tech 2012 Highest P(3HB) 9.9 mass/dry mass cells(%) * 12x more PHB Imperial’s P(3HB) concentration 1.66 0.075 Tokyo Tech 2012 Highest P(3HB) 0.204 M1: M1: M1 : Design Specifications Test Model
The hybrid operon produces more PHB than the constitutive Nile red stains PHB M1: M1: M1 : Design Specifications Test Model
Key Result PHB production from waste PHB extracted from our bacteria grown on waste Monomer detection Adapted 3-HB medical assay kit that turns yellow when 3HB is present Yellow colour change Control. Remains when 3HB is present colourless M1: M1: M1 : Design Specifications Test Model
Successes in Module 1 1. PHB production 2. Massively improved PHB production 3. PHB from waste To the best of our and our advisors knowledge, this is the first time anyone has made PHB from SRF using Synthetic Biology
Module 2: Plastic Fantastic Non-recyclable, mixed waste M1: Resource-full Waste Polyurethane Wood, paper Chemical degradation & fibres products M2: Plastic Fantastic Control phaZ1 lysate PHB production P(3HB) degradation
The first Synthetic Biology PHB recycling platform
Requirement for PHB recycling PHB degradation Our bacteria should: 1. Express PHB-degrading enzymes 2. Be resistant to 3HB toxicity M2 : Design Specifications Test Model
PHB degradation Phaz1 - BBa_K1149010 M1: M1: M2 : Design Specifications Test Model
Modelling PHB depolymerisation Wet Lab data guided further model optimisation M2 : Design Specifications Test Model
Purified PHB depolymerase is active PHB depolymerase 4-Nitrophenol para-Nitrophenyl butyrate PHB depolymerase (phaZ1) Empty vector Substrate alone Blank phaZ1 M2 : Design Specifications Test Model
Key Result PHB depolymerase (phaZ1) clears PHB emulsions 3HB monomer detection Day 0 Day 1 Day 3 M2 : Design Specifications Test Model
Testing for 3HB toxicity OD600 Empty Vector at 6h Control phaCAB No toxic effect until 10 mM of 3HB. In a bioreactor we would filter off 3HB to prevent it reaching this concentration. M2 : Design Specifications Test Model
Requirement for PHB recycling Our bacteria should: 1. Internalise 3HB monomer 2. Metabolise 3HB 3. Make PHB M2 : Design Specifications Test Model
PHB from 3HB Permease J23104 Permease 0034 M1: M1: M2 : Design Specifications Test Model
Metabolic model showing the production of PHB from glucose TCA Cycle Glycolysis PHB synthesis M2 : Design Specifications Test Model
Metabolic model predicts that E.coli will produce PHB from 3HB M2 : Design Specifications Test Model
Key Result: Permease internalises 3HB Permease Increased growth on Empty vector 3HB with permease Control Decrease of 3HB Decrease of 3HB outside of the cell M2 : Design Specifications Test Model
As you now know from M1, we can make PHB • phaZ1 PHB depolymerase • Permease • pha CAB operon Now we have all the working parts to make the first synthetic biology PHB recycling platform. M2 : Design Specifications Test Model
Industrialisation
Human Our system + cellulose hydrolysis will Practices allow industrial development Commercial Viability Requires: 150 - 300,000 tons of sugar 50,000 tons of PHB 390,625 - 781,000 tons SRF 2.11% SRF to PHB Meeting Stuart Dunbar- Principal Scientist
Human The next step Practices Our bioreactor at Imperial College
Human Local solutions and future vision Practices CAD design Find out more about M.A.P.L.E. in our booklets Appliances that transform domestic waste into new 3D printed bioplastic objects. What would you 3D print? Collaboration with
Human Communicating our project Practices GetSynBio article BBC Radio 4 Interview with Adam Rutherford for the major national radio station Helping iGEM High school team Celebration of Science 2 million listeners in the UK
Thanks to our advisors Richard Kelwick Paul Freemont Richard Kitney Alex Webb Kirsten Jensen Guy-Bart Stan Thanks to our sponsors Thanks to for this awesome experience
Sisi Fan
Our Achievement • Increased PHB production • Produced PHB from waste • The first ever Synthetic Biology PHB recycling platform • 15 Biobricks submitted Trash to Treasure
M1: M1: M1 : Design Specifications Test Model
M1: M1: M1 : Design Specifications Test Model
Modelling PHB production from glucose M1: M1: M1 : Design Specifications Test Model 57
Purification of PHB from cells using SRF as a carbon source media M1 : Design Specifications Test Model
Hybrid produces more P3HB than J23104 EV Constitutive Hybrid Constitutive Hybrid EV M1 : Design Specifications Test Model
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