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Contents 1 Recycling Poly-3-hydroxybutyrate 1.1 Overview 1.2 Specification 1.2.1 Degradation of P(3HB) 1.2.2 Synthesis of P(3HB) 1.3 Design 1.3.1 Degradation of P(3HB) 1.3.2 Synthesis of P(3HB) from 3HB 1.4 Results 1.5 Protocols 1.6 Safety 1.6.1 Papers Referenced

Recycling Poly-3-hydroxybutyrate

Overview

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 in order to do this, we have engineered E.coli to break down P3HB extracellularly to release the monomers of 3-hydroxybutyrate(3-HB). E.coli in a different bioreactor will then use the 3-HB as their carbon source for the reproduction of P3HB. For this, we designed a pathway where a permease will take up 3HB, the bdh2 dehydrogenase enzyme will convert it to acetoacetate, which can be then used for PHB synthesis.

This is a rough diagram, something better is coming. right?

Specification

Degradation of P(3HB)

Our bacteria should be resist any toxic effects that are associated with P(3HB) or 3HB

Our bacteria should degrade (P3HB)

Synthesis of P(3HB)

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

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

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

Design

Degradation of P(3HB)

Extracellular expression of phaZ1, PHB depolymerase enzyme ([http://parts.igem.org/Part:BBa_K1149010 BBa_K1149010])

http://www.igem.org/wiki/images/8/86/Reaction_phaz.jpg

Synthesis of P(3HB) from 3HB

We designed a biobrick for the expression of a Putative permease identified from the literature (GI:158937168) for 3HB inport. We optimised the sequence for E.coli and planned experiments with the construct. However, the gene synthesis was delayed and we did not get to the stage of characterizing and submitting the part.

Intracellular expression of bdh2, 3HB dehydrogenase enzyme ([http://parts.igem.org/Part:BBa_K1149050 BBa_K1149050]).

http://www.igem.org/wiki/images/f/f0/Reaction_bdh.jpg

Results

Protocols

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)

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.