sisi fan, jemma pilcher, iain bower, matthew chin...
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Plastic of the Future: Poly-3-hydroxybutyrate
PHB
• A bacterial energy store
• An alternative for petrochemical
• Biocompatible
• Produced from plant biomass
• Expensive
However…
PHB granules
In August we visited POWERDAY the largest Materials Recovery Facility in SE England
• Human practice identified Solid Recovered Fuel (SRF) • SRF is a non-recyclable product of material recovery facilities
Solid Recovered Fuel is the target
It costs POWERDAY over $28 million a year to ship SRF for incineration.
Human Practices
1/5 Plastics 4/5 Fibres Wood Paper
What is SRF made of?
320,000 tons per year
Simon Little, Marketing Manager
Human Practices
The Right use for SRF 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)
Human Practices
OUR AIM
Waste to Bioplastic to Product
Module 1: Making bioplastic from waste
Module 2: Recycling bioplastics
Module 2 Module 1
Our project is Human
Practices driven
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
M1: Resource-full Waste
M2: Plastic Fantastic
Non-recyclable, mixed waste
Wood, paper & fibres
Polyurethane degradation
PHB production
Control phaZ1 lysate
P(3HB) degradation
Module 1: Resource-full Waste
Chemical products
NATIVE
Chassis choice- E.coli MG1655
Our chassis and parts: -GRAS: Generally regarded as safe
-Already used in industry
Growing our E.coli on PBS and waste
Incubated and plated
The bacteria are still alive after 3 days
And after 6 days!
Waste conditioned
media
LB
No toxic effect
Our chassis thrives on waste
M1: Resource-full Waste
M2: Plastic Fantastic
Non-recyclable, mixed waste
Chemical products
Wood, paper & fibres
Polyurethane degradation
PHB production
Control phaZ1 lysate
P(3HB) degradation
PHB production from waste
1. Produce PHB
2. Produce PHB from the mixed waste
Requirement for PHB production
Our bacteria should:
Design Specifications Model Test M1:
Native operon, BBa_K934001 (Tokyo2012)
Control phaCAB
Specifications Model Test M1: M1: Design Specifications Model Test M1:
Nile red stains PHB
Metabolism
Glucose 1
2
3 4
Model-based flux optimisation
Design Specifications Model Test M1:
We created our models for the project from differential equations using MatLab
Design Specifications Model Test M1:
Glycolysis TCA Cycle
PHB synthesis
Glucose
Metabolic model of PHB production from glucose
Dixton et al., 2011
Dixton et al., 2011
PHB production is sensitive to PhaB
Design Specifications Model Test M1:
Sensitivity of [PHB]
Time (minutes)
Our model for the project
Metabolism
Glucose 1
2
3 4
PHB production is sensitive to PhaB
Design Specifications Model Test M1:
P3HB Concentration
(g/L)
Increasing PhaB level: Effect on PHB production
Design Specifications Model Test M1:
Constitutive Promoter, BBa_K1149052
Hybrid Promoter, BBa_K1149051
Model Prediction J23104 increase PHB production rate
Design Specifications Model Test M1:
Modelling data
Native Hybrid
Key Result Hybrid operon produces much more PHB
M1: M1: Design Specifications Model Test M1:
Imperial iGEM’s
Hybrid Promoter
phaCAB
Native promoter
phaCAB
Dry Biomass (g) 3.48 1.8 P3HB mass (g) 2.05 0.09 LB media volume (L) 1.2 1.2 Imperial’s P(3HB) mass/dry mass
cells (%)* 58.9 5
Tokyo Tech 2012 Highest P(3HB)
mass/dry mass cells(%)* 9.9
Imperial’s P(3HB) concentration 1.66 0.075 Tokyo Tech 2012 Highest P(3HB) 0.204
Key Result: 60% of biomass is P3HB
Native Hybrid
Optimised Literature values are between 80%
M1: M1: Design Specifications Model Test M1:
12x more PHB
The hybrid operon produces more PHB than the constitutive
M1: M1: Design Specifications Model Test M1:
Nile red stains PHB
Key Result PHB production from waste
M1: M1: Design Specifications Model Test M1:
PHB extracted from our bacteria grown on waste
Adapted 3-HB medical assay kit that turns yellow when 3HB is present
Monomer detection
Yellow colour change when 3HB is present
Control. Remains colourless
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
M1: Resource-full Waste
M2: Plastic Fantastic
Non-recyclable, mixed waste
Wood, paper & fibres
Polyurethane degradation
PHB production
Control phaZ1 lysate
P(3HB) degradation
Chemical products
Module 2: Plastic Fantastic
Requirement for PHB recycling
1. Express PHB-degrading enzymes
2. Be resistant to 3HB toxicity
Our bacteria should:
Design Specifications Model Test M2:
PHB degradation
Design Specifications Model Test M2:
Modelling PHB depolymerisation
Wet Lab data guided further model optimisation
Purified PHB depolymerase is active
Blank phaZ1
PHB depolymerase (phaZ1)
Empty vector
Substrate alone
Design Specifications Model Test M2:
para-Nitrophenyl butyrate 4-Nitrophenol
PHB depolymerase
Key Result PHB depolymerase (phaZ1) clears PHB emulsions
Design Specifications Model Test M2:
Day 0 Day 1 Day 3
3HB monomer detection
phaCAB
Empty Vector Control
OD600 at 6h
Testing for 3HB toxicity
No toxic effect until 10 mM of 3HB. In a bioreactor we would filter off 3HB to prevent it reaching this concentration.
Design Specifications Model Test M2:
Requirement for PHB recycling
Design Specifications Model Test M2:
Our bacteria should:
1. Internalise 3HB monomer
2. Metabolise 3HB
3. Make PHB
Metabolic model showing the production of PHB from glucose
Glycolysis TCA Cycle
Design Specifications Model Test M2:
PHB synthesis
Key Result: Permease internalises 3HB
Design Specifications Model Test M2:
Permease
Empty vector Control
Increased growth on 3HB with permease
Decrease of 3HB
Decrease of 3HB outside of the cell
Design Specifications Model Test M2:
As you now know from M1, we can make PHB
Now we have all the working parts to make the first synthetic biology PHB recycling platform.
• phaZ1 PHB depolymerase
• Permease
• pha CAB operon
Our system + cellulose hydrolysis will allow industrial development
Meeting Stuart Dunbar- Principal Scientist
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
Human Practices
Local solutions and future vision
Collaboration with
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?
CAD design
Human Practices
Communicating our project
BBC Radio 4
Interview with Adam Rutherford for the major national radio station
2 million listeners in the UK
Celebration of Science
GetSynBio article
Helping iGEM High school team
Human Practices
Thanks to our sponsors
Thanks to our advisors
Guy-Bart Stan Richard Kelwick Richard Kitney Kirsten Jensen Paul Freemont Alex Webb
Thanks to for this awesome experience
Our Achievement • Increased PHB production • Produced PHB from waste • The first ever Synthetic Biology PHB recycling platform • 15 Biobricks submitted
Trash to Treasure
Purification of PHB from cells using SRF as a carbon source media
Design Specifications Model Test M1:
Hybrid produces more P3HB than J23104
EV
EV
Constitutive
Constitutive
Hybrid
Hybrid
Design Specifications Model Test M1:
Western Blot analysis suggests that the osmY fusion protein is secreted
Design Specifications Model Test M1:
PUR esterase in the supernatant
94% 84%
95%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
DuraMem150 PBI-DBX TriSep RO X20
% Rejectrion of 3HB by membranes
Investigation into appropriate membranes for our system
Design Specifications Model Test M2:
Purification of PHB from cells using SRF as a carbon source media
Design Specifications Model Test M1:
Hybrid produces more P3HB than J23104
EV
EV
Constitutive
Constitutive
Hybrid
Hybrid
Design Specifications Model Test M1:
Western Blot analysis suggests that the osmY fusion protein is secreted
Design Specifications Model Test M1:
PUR esterase in the supernatant
Bdh2 dehydrogenase: BBa_K1149050
Design Specifications Model Test M2:
Bdh2 with no pelB secretion tag growth. Bdh2 MG1655 cells were grown in M9S media to gauge growth, the growth does not differ from the control as p = 0.5543. Data points show final time point after 6h growth for each concentration. Growth was at 37°C with shaking over 6h. Error bars are SEM, n=4.