Polystyrene: biologically producing self-folding plastics

Introduction

Polystyrene is one of the most widely used plastics today. As a synthetic polymer of styrene monomers, polystyrene exhibits self-folding properties when heated. We endeavored to biologically synthesize styrene by genetically engineering E. coli to produce the enzymes required in the pathway from L-phenylalanine to trans-cinnamic acid to styrene. Using this pathway we aim to produce the styrene monomer both in vivo and in vitro from renewable sources. After producing styrene biologically, we characterized a method for polymerizing styrene into the polymer, polystyrene, which is then ready for folding.

Styrene Synthesis: Engineering E. coli to produce the styrene monomer

From genes to proteins

The first step in making biologically produced polystyrene is to create the monomer styrene. After combing through the literature we found a two-step enzymatic pathway from the amino acid phenylalanine to styrene. The pathway had successfully been achieved in our host organism, E. coli, by the McKenna group from Arizona State University in 2011. As we read more about styrene synthesis we found that the two-step pathway was actually more complicated than it looked. The second enzyme in the pathway, Ferulic Acid Decarboxylase (FDC), was regulated by an unknown co-factor, and this co-factor was produced by a third enzyme UbiX. We later read in a paper published this June 2015, that the unknown co-factor was a prenylated riboflavin formed from dimethylallyl monophosphate (DMAP) and flavin mononucleotide (FMN). So we had found a complete pathway to produce styrene in E. coli. Now we just needed to get the three proteins PAL, FDC and UbiX. In order to obtain our enzymes we followed three steps: Step one, get the genes. Step two, put the genes into plasmids. And step three, turn genes into proteins.

Step one: Get the genes

In order to get the genes that encoded our three enzymes we either extracted the gene directly from a host organism, synthesized the gene using Integrated DNA Technologies (IDT), or ordered the preexisting part from the iGEM registry.

Our first enzyme PAL was previously characterized by the University of British Columbia 2013 iGEM team. We ordered their biobrick (BBa_K1129003) from the registry. However, this PAL gene was extracted from Streptomyces maritimus. In Rebbecca McKenna’s 2011 paper she found that the PAL gene from Streptomyces maritimus was nearly completely ineffective. So in addition to ordering UCB’s PAL biobrick we also ordered from IDT a codon optimized (for E. coli) version of the PAL gene from Anabaena variabilis. We chose the PAL gene from Anabaena variabilis because it was well characterized in literature and had been shown to be an effective phenyalanine ammonia lyase.

For our second enzyme FDC we both extracted the gene directly from Saccharomyces cerevisiae and ordered this gene codon optimized (for E. coli) from IDT.

For our last enzyme UbiX we both extracted the gene directly from E. coli and ordered this gene with a FLAG tag from IDT. As we will explain later on, we used a FLAG tag sequence at the end of all of our synthesized genes in order to extract and purify the protein for in-vitro assays.

Step two: Put the genes into plasmids

After obtaining our genes, we needed to insert them into the standard pSB1C3 backbone so we could submit them as biobricks. To do this we digested our linear gene and standard iGEM RFP plasmid (BBa_J04450) with a combination of EcoRI and SpeI or PstI restriction enzymes. We then ligated with T4 ligase and transformed into NEB 5-alpha competent E. coli cells. See below for our digestion, ligation, transformation protocol. We confirmed our gene insert through DNA sequencing with VF2 and VR primers.

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References

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