Sisi Fan, Jemma Pilcher, Iain Bower, Matthew Chin ...2013.igem.org/files/presentation/Imperial_College_Championship.pdf · Sisi Fan, Jemma Pilcher, Iain Bower, Matthew Chin, ... The

Sisi Fan, Jemma Pilcher, Iain Bower, Matthew Chin, Margarita Kopniczky, Wenqiang Chi & James Strutt

Could Synthetic Biology transform all this rubbish (trash)…

…into these?

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

Resources are lost in waste

700 million tons of trash is generated per year

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

Module overview

PHB

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




Chemical products



PHB production


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


We created our models for the project from differential equations using MatLab


Glycolysis TCA Cycle

PHB synthesis

Glucose

Metabolic model of PHB production from glucose

Dixton et al., 2011

Dixton et al., 2011

Metabolism

Glucose 1

2

3 4



Metabolism

Glucose 1

2

3 4



PHB production is sensitive to PhaB


Sensitivity of [PHB]

Time (minutes)

Our model for the project

Metabolism

Glucose 1

2

3 4

PHB production is sensitive to PhaB


P3HB Concentration

(g/L)

Increasing PhaB level: Effect on PHB production


Constitutive Promoter, BBa_K1149052

Hybrid Promoter, BBa_K1149051

Model Prediction J23104 increase PHB production rate


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%


12x more PHB

The hybrid operon produces more PHB than the constitutive


Nile red stains PHB

Key Result PHB production from waste


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






PHB production


P(3HB) degradation

Chemical products

Module 2: Plastic Fantastic

The first Synthetic Biology PHB recycling platform

Requirement for PHB recycling

1. Express PHB-degrading enzymes

2. Be resistant to 3HB toxicity



PHB degradation

PHB degradation


Phaz1 - BBa_K1149010


Modelling PHB depolymerisation

Wet Lab data guided further model optimisation

Purified PHB depolymerase is active

Blank phaZ1

PHB depolymerase (phaZ1)

Empty vector

Substrate alone


para-Nitrophenyl butyrate 4-Nitrophenol

PHB depolymerase

Key Result PHB depolymerase (phaZ1) clears PHB emulsions


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.


Requirement for PHB recycling



1. Internalise 3HB monomer

2. Metabolise 3HB

3. Make PHB

Permease


Permease

PHB from 3HB

J23104 0034

Metabolic model showing the production of PHB from glucose

Glycolysis TCA Cycle


PHB synthesis

Metabolic model predicts that E.coli will produce PHB from 3HB


Key Result: Permease internalises 3HB


Permease

Empty vector Control

Increased growth on 3HB with permease

Decrease of 3HB

Decrease of 3HB outside of the cell


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

Industrialisation

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

The next step

Our bioreactor at Imperial College

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

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



57 M1: M1: Design Specifications Model Test M1:

Modelling PHB production from glucose

Purification of PHB from cells using SRF as a carbon source media


Hybrid produces more P3HB than J23104

EV

EV

Constitutive

Constitutive

Hybrid

Hybrid


Western Blot analysis suggests that the osmY fusion protein is secreted


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




64 M1: M1: Design Specifications Model Test M1:

Modelling PHB production from glucose

Purification of PHB from cells using SRF as a carbon source media


Hybrid produces more P3HB than J23104

EV

EV

Constitutive

Constitutive

Hybrid

Hybrid


Western Blot analysis suggests that the osmY fusion protein is secreted


PUR esterase in the supernatant

Bdh2 dehydrogenase: BBa_K1149050


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.

http://parts.igem.org/Part:BBa_K1149050

PhaZ1 depolymerase is functional

p=0.0248


***

Monomer detection

Yellow colour change when 3HB is present

Control. Remains colourless


Sisi Fan, Jemma Pilcher, Iain Bower, Matthew Chin ...2013.igem.org/files/presentation/Imperial_College_Championship.pdf · Sisi Fan, Jemma Pilcher, Iain Bower, Matthew Chin, ... The

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