Recyling 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 some application e.g packaging(5) such as .

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

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

Implementation

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

Safety