Recycling Poly-3-hydroxybutyrate

Overview

Poly-3-hydroxybutyrate(P3HB) is a bioplastic, more specifically it is a polyester which is naturally produced inside bacteria such as Alcaligenes eutrophus. It is used as an energy store by these bacteria(1). It appears as globules inside the cell. PHB as a plastic has benefits over those derived from oils; it is produced from renewable resources which minimise the amount of fossil fuels required in plastic production(2,3) and can also biodegrade to non-toxic compounds which can be used as an energy source by organisms commonly found in the environment(4). Physically P3HB has properties which allow it to be used as a replacement for oil based plastics for certain applications e.g packaging(5).

We have made P3HB in E.coli and developed a system by which it can be recycled when products made from it come to the end of their life. In order to make P3HB we transferred three genes, naturally found in Ralstonia eutropha into E.coli MG1655. These encode the three enzymes necessary for P3HB production; polyhydroxyalkanoate synthase(phaC), 3-ketothiolase(phaA) and acetoacetyl coenzyme A reductase(phaB). These exist as a P3HB producing operon. We have tried to genetically maximise the production of P3HB as high yields are required for P3HB to be economically viable.

phaC, which encodes the polyhydroxyalkanoate (PHA) synthase, phaA, which encodes a 3-ketothiolase, and phaB, which encodes an acetoacetyl coenzyme A (acetoacetyl-CoA) reductase.

Specification

Degradation of P(3HB)

Our bacteria should be able to resist any potential toxicities that are associated with P(3HB) or 3HB

Our bacteria should be able to degrade (P3HB)

Synthesis of P(3HB)

Our bacteria should be able to take up and internalise 3HB from the surrounding media

Our bacteria could be able to utilise P(3HB) as a sole carbon source

Our bacteria should be able to synthesise intracellular P(3HB)

Modelling

Introduction to building a deterministic model

In engineering, when one wants to make a product to solve a particular problem it is common to adopt a strategic cycle to assist in the realisation of such product. The stages involved in the cycle are: (i) User-requirements/specifications, (ii) Design (of modules), (iii) Modelling, (iv) Implementation (wet lab), (v) Testing and verification and (vi) End-product.

A major aspect of how engineering is involved in synthetic biology is modelling. It plays an important role in the verification of modules designed to ensure that they are built correctly and meet the specification. Modelling also adds in the predictability throughout the design process by means of simulations with software. With the ability to predict, complexity of molecular biology can be masked and the pathways in a cell can be considered as systems or subsystems.

The fact that the modelling is program-based particularly suits the open-source theme as the codes can be shared on the wiki such that synthetic biologists working in the relevant areas can download them as templates for their models.

ODEs in Matlab Simbiology model: P(3HB) synthesis

Genetic regulations and expressions:

BDH2 – 3-hydroxybutyrate dehydrogenase

atoAD – Acetyl-CoA:acetoacetyl-CoA transferase (α and β subunits)

phaB – Acetoacetyl-CoA reductase

phaC – P(3HB) synthase

Enzyme kinetics:

BDH2 – 3-hydroxybutyrate dehydrogenase

atoAD – Acetyl-CoA:acetoacetyl-CoA transferase (α and β subunits)

phaB – Acetoacetyl-CoA reductase

phaC – P(3HB) synthase

Introduction to building a metabolic model

Design

Results

E.coli MG1655 containing the PhaCAB construct accumulates the bioplastic PHB inside its cells as illustrated in the figure below. A control strain with a plasmid containing the BBa_J23104 promoter and BBa_B0034 ribosome binding site and the strain containing PhaCAB were grown on plates with Nile Red stain in the media. Nile Red binds to the membranes surrounding the PHB and fluoresces.

Figure x. More intense fluorescence is seen for J and S which contain phaCAB and phaCB respectively. These operons allow them to produce the bioplastic PHB. PHB is stained by Nile Red which fluoresces when the plate is imaged at 473nm.

Protocols

LB-Agar Plates containing Nile Red stain

Nile Red final concentration in media should be 0.5µg/ml

1. After autoclaving 300ml of LB-Agar, add 600µl Nile Red solution(0.25mg/ml DMSO)
2. Add 150µl chloramphenicol
3. Pour plates in a clean fume cupboard

Nile Red Culture staining

Nile Red final concentration should be 20µg/ml media

Add 320µl to 4 ml overnight culture and incubate for 30mins at room temp.

Fluorescent Microscopy

Agar pads are first made

Extracting PHB

• Centrifuge settings: 4000RPM, 10mins.
• Scale as appropriate.
• After each centrifuge step the supernatant should be poured off.
• This should provide PHB with 99% purity and a high molecular weight.

1. Centrifuge 50ml culture and resuspend in PBS.
2. Resuspend pellet in 5ml Triton X-100(1% v/v in PBS) for 30mins at room temp.
3. Centrifuge, resuspend in 5ml PBS.
4. Centrifuge, add 5ml sodium hyperchlorite solution and incubate at 30˚C for 1 hour.
5. Centrifuge, wash with 5ml distilled water and 70% EtOH several times.
6. Allow powder to dry.

Relationship between OD and dry biomass

1. Grow 3 large cultures in 3% glucose 1L/300ml LB - inoculate with 1ml OD 0.1 and grow for 24h.
2. Calculate OD of original culture and dilute until there is a range of 0.2 - 1.0 OD(try to have large volumes for low OD).
3. Weigh centrifuge containers.
4. Transfer solutions to these containers, recording what volume of each OD is transferred. .
5. Centrifuge these solutions down and dry the pellet out overnight in a 50°C oven.
6. Dry cell mass = (container mass + dry cell mass)/container mass
7. Plot OD against dry cell mass.

Measure OD of culture. If OD>0.4 then dilute by a dilution factor d. Divide new OD by d to give the corrected OD.

Either create a calibration curve or assume dry biomass is proportional.

1. Weigh each container,then culture PHB producing microbes.
2. Calculate the culture’s optical density(OD).
3. Centrifuge culture and pour off supernatant.
4. Weigh container and pellet.
5. Heat in oven until weight is constant.
6. Re-weigh container and pellet, subtracting container weight to calculate biomass dry weight.

Measure OD of cultures before extracting PHB to estimate dry biomass.

Safety

Our project used several potentially harmful chemicals. Ensure you know the risks involved with chemicals you use by checking the full material safety data sheet(MSDS)

Dimethyl Sulfoxide(DMSO)

It has known carcinogenic effects and can permeate your skin, carrying other chemicals with it. Handle in a fume cupboard, wearing nitrile gloves and goggles.

Papers Referenced

• (1) ANDERSON A, DAWES E. Occurrence, Metabolism, Metabolic Role, and Industrial Uses of Bacterial Polyhydroxyalkanoates. Microbiol Rev 1990 DEC;54(4):450-472.
• (2) Harding KG, Dennis JS, von Blottnitz H, Harrison STL. Environmental analysis of plastic production processes: Comparing petroleum-based polypropylene and polyethylene with biologically-based poly-beta-hydroxybutyric acid using life cycle analysis. J Biotechnol 2007 MAY 31;130(1):57-66.
• (3) Kim S, Dale BE. Energy and Greenhouse Gas Profiles of Polyhydroxybutyrates Derived from Corn Grain: A Life Cycle Perspective. Environ Sci Technol 2008 OCT 15;42(20):7690-7695.
• (4) Jendrossek D, Handrick R. Microbial degradation of polyhydroxyalkanoates. Annu Rev Microbiol 2002;56:403-432.
• (5) Philip S, Keshavarz T, Roy I. Polyhydroxyalkanoates: biodegradable polymers with a range of applications. Journal of Chemical Technology and Biotechnology 2007 MAR;82(3):233-247.