Team:Stanford-Brown/PS

SB iGEM 2015

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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.

Now that our genes were in the pSB1C3 backbone we wished to add a promoter and RBS in front of our genes so that they could be functionally expressed in bacteria. To do this we attempted a “double digest” of linear TetR promoter and RBS DNA and our plasmids as shown in the graphic below. After attempting multiple times, we were unable to get the insertion to work. So we changed tactics. Instead of attempting to insert a 30 bp fragment, we decided to insert our gene product (between 500 and 1500 bp) into a plasmid from the registry with a T7 promoter and RBS (BBa_K525998). It worked!

The FDC gene we extracted from yeast has an illegal SpeI restriction enzyme site within its sequence. This made two of our plasmids not biobrick compatible. To solve this we attempted site directed mutagenesis of the 999th nucleotide in the FDC gene from an adenine to a cytosine. Based on a paper about efficient primer design for site directed mutagenesis, we designed two primers… We then attempted site directed mutagenesis and...

Step three: Turn genes into proteins

Now that we had all our genes in plasmids with a promoter and RBS we had to transform the plasmid and express the genes. We transformed our three synthesized genes (PAL, FDC, UbiX) into T7 expressing NEB E. coli separately. We used these transformed colonies to grow up larger cultures. We then initiated T7 polymerase gene expression by adding IPTG to our cultures. Because all of our synthesized genes had a FLAG tag at the end of their sequence, we were able to purify our proteins from the cell lysate. To do this we used the Anti-FLAG Tag protein purification method. We then used a BCA protein assay to determine the concentrations of our purified proteins. Finally we ran all three of our purified enzymes on SDS PAGE with a Mark 12 protein ladder to verify that our proteins were the correct molecular weight, which they were. See below for all of the specific protocols used in this section of our project.

Testing functionality in-vitro of enzymes and modeling pathway

Step one: Testing PAL

Once we were reasonably confident that we had successfully purified our PAL enzyme from the T7 expressing E. coli, the next logical step was to test the in vivo functionality of our protein extract. In order to do this, we made use of the fact that PAL’s reactant and product, namely phenylalanine and trans-cinnamic acid (tCA), have different characteristic absorbance spectra in the ultraviolet region. Notably, tCA has a large and easily distinguishable peak at 268 nm, whereas phenylalanine displays a much less noticeable peak just below 260 nm. Using the Beer-Lambert relation, which states that, all other factors held constant, concentration is directly proportional to absorbance, we could spectrophotometrically track the increase in absorbance at 268 nm. After creating a standard curve that tells us how to map absorbance at 268 nm to concentrations of tCA, we had everything we needed to track PAL’s conversion of phenylalanine to tCA in real time.

In an initial assay, we took absorbance spectra of the following reactions using a Nanodrop 2000 machine:

Phenylalanine alone: expect small peak just below 260 nm
PAL alone: expect small peak characteristic of proteins
tCA alone: expect a large peak characteristic of tCA at 268 nm
Phenylalanine and PAL together: expect a large peak characteristic of tCA at 268 nm

As you can see in the accompanying figure, this initial experiment provided good evidence that our PAL was in fact functioning. Our next step was to perform a kinetic time course experiment in order to obtain new data for our mathematical models (described below). In order to do this, we created reaction mixtures of PAL along with 8 different concentrations of its substrate, phenylalanine. With three replicates of each reaction, we had a total of 24 reactions that we then tracked in real time over the course of about 4 hours using a Spectramax Pro spectrophotometer. Not only did this experiment further demonstrate that our PAL was working, it also provided us with the necessary kinetic data to estimate PAL’s biochemical parameters.

Step two: Testing FDC/UbiX

Given the success of the spectrophotometric approach in our PAL assays, we attempted to proceed in a similar manner with FDC and UbiX. Unfortunately, there were several complicating factors.

In the case of UbiX, the main problem lay in the impossibility of distinguishing UbiX’s reactant from its product. UbiX catalyzes the prenylation of flavin mononucleotide. Unfortunately, this chemical transformation does not result in a change in the overall absorbance of the reaction solution that we are able to detect. The reactant and the product are simply too chemically similar. We believe that this is one reason why no isozyme of UbiX has ever been kinetically characterized.

In the case of FDC, we initially set out to use tCA’s unique absorbance spectrum to our advantage: whereas we measured an increase in tCA to track the activity of PAL (which produces tCA), we endeavored to measure a decrease in tCA to probe the activity of FDC (which consumes tCA). Unfortunately, FDC cannot function in the absence of its prenylated FMN cofactor, which is supplied by UbiX. This presents a challenge, since FMN has a strong absorbance peak in precisely the same region as tCA’s peak. Consequently, we could not apply the same spectrophotometric method to quantify FDC activity.

As an alternative, we adapted a purely computational approach to obtain at least some of the results that we would have gained from the same genre of experimental analysis that we performed in our PAL assay. Although we could not perform a multiplexed time course experiment on FDC or UbiX, we carried out a sensitivity analysis to determine FDC’s role in the overall synthetic pathway, as described below.

Step three: Modeling pathway

We had hoped that our in vitro assays would help us not only verify that our enzymes were functioning, but also assist us in determining each enzyme’s kinetic significance to the overall reaction. This knowledge would allow us to optimize in vivo styrene production by preferentially expressing the enzymes with the most significance. To do this, we planned on creating an operon of our three genes of interest, PAL, UbiX, and FDC. The genes closest to the beginning of the operon are transcribed more than those to the end. Therefore, we would place our most influential enzymes toward the beginning and our less important enzymes toward the end. However, without complete in vitro data we could not determine which enzymes were most significant.

There are 3! = 6 possible orderings of these three genes so we could potentially make 6 plasmids, test their styrene productivity levels, and submit the one which yielded the most styrene. The described process would have cost us weeks of our time as well as lab resources and money. Instead of creating all 6 plasmids and conducting the wet lab experiments, we instead turned to a mathematical model of our pathway with literature values of our enzymatic constants. In order to model our enzymatic pathway, we created a system of ordinary differential equations based on the Michaelis Menten enzyme model shown below. Numerically simulating the model in MATLAB gave us curves that represented the species concentration as a function of time over a specified period.

FDC and PAL’s influence on the overall reaction flux was tested by varying each of their concentrations while keeping the other constant. It was found that changing the concentration of FDC changed the rate of the reaction much more than changing PAL. Specifically, PAL and FDC started out at 3 nM protein concentration and 1nM of PAL concentration was added to one while keeping FDC constant. This process was repeated for the varying of FDC while keeping PAL constant. We then recorded the model’s prediction of styrene concentration after an hour of the reaction running and compared the two enzyme’s influence. The results can be seen in the graph below. Clearly, we can see that FDC had a much bigger effect on the overall reaction velocity.

We decided not to include UbiX in the model because we already knew that UbiX would correspond to active FDC in a 1 to 1 ratio, so we would never need more UbiX than FDC, and also because UbiX would always be right after FDC since UbiX is needed to activate FDC, our enzyme of most influence. In conclusion, our model helped us decide on the order FDC-UbiX-PAL for our combo plasmid without having to test out every combination possible.

GRRR now i have to make a table :(

In-vivo production and in-situ removal of Styrene

Step one: Making combo plasmid

Once we determined the gene order for our FDC-UbiX-PAL operon, we needed to make it. To do this we used the New England Biolabs Gibson Assembly kit. We designed eight primers according to a primer design protocol (also from NEB) for FDC, UbiX, PAL and RFP plasmids. We then ran a PCR extension on all four of the resulting segments. We ran these four products on a gel and gel extracted the bands that appeared at the desired migration distances. We used this purified product for the Gibson assembly and transformed our resultant plasmid into NEB 5-alpha competent cells. We ran a colony PCR and found three colonies with the expected band length of over 4,000 base pairs. We grew up liquid cultures of these three colonies, miniprepped, and ran sequencing using five internal primers designed specifically for the combo plasmid. We found that two of the three colonies had the correct sequence!

[We then transformed our combo plasmid into T7 expressing cells to extract our protein as before. Again, we ran our proteins sample on an SDS PAGE and confirmed that not only were all of our proteins expressing correctly, but that we had the most FDC and least PAL, as expected.]

Step two: In-situ removal

A major issue with producing styrene in vivo is that styrene is actually toxic to the cell. According to the McKenna (2011) styrene is toxic to the cell at concentrations as low as 300 mg/L. So in order to make styrene production practical in vivo we would have to either make our cells more resistant to styrene or find a way to remove the styrene as it was being produced. Because there is little known about why styrene is toxic to the cell, we decided to focus on the latter. We found a paper that used an immiscible solvent to remove styrene in situ from cell cultures that produced styrene: the ideas is that styrene, a nonpolar molecule, preferentially dissolves in the hydrophobic solvent over the aqueous cell culture. The solvent did not impede cell growth, but was able to more than double the total styrene produced. We decided to test out this result and purchased one of their reported solvents: n-dodecane (a nonpolar hydrocarbon). We then set up a cell growth assay with 48 liquid cell cultures (5 ml each). Half of the cultures had the solvent n-dodecane, while the other half had no solvent. We than exogenously added pure styrene to the cultures at varying concentrations from 0 mg/L to 4,800 mg/L. We let all of our cultures grow for 16 hours then we measured their optical density at 600 nm. We found that at high concentrations of styrene, the cultures without solvent had significantly lower absorbance levels than the cultures with solvent. This result confirmed that in situ removal of styrene using n-dodecane would effectively increase the styrene toxicity threshold for our cells, making in vivo production more effective.

Step three: In-vivo production

[Using our culture of transformed combo plasmid for protein extraction we designed an experiment to verify, without HPLC or GC-MS, that styrene could be produced in-vivo from our combo plasmid. We created four cultures…]

Styrene Polymerization: Polymerizing biologically produced styrene into polystyrene

Up until now we have focused on styrene synthesis. However equally important is the polymerization of styrene into polystyrene, our final product for folding. We researched many methods for styrene polymerization used both in labs and industry. We found that a free radical mechanism using the radical initiator azobisisobutyronitrile (AIBN) would be the simplest and easiest to test. So we ordered the initiator AIBN and compiled protocols.

Step one: Testing viability

Styrene has a conjugated pi bond system between its alkene and phenyl group. Because of this, the molecule can stabilize an electron radical allowing it to polymerize during propagation. As seen in the propagation mechanism to the right, this allows styrene to polymerize into long chains of polystyrene. However, in order to start the domino-like process of styrene polymerization there needs to be a radical initiator. This is where azobisisobutyronitrile (AIBN) comes in. AIBN is an unstable molecule that when heated to 60°C will decompose into two radical species. These radicals species can activate the polymerization of styrene. To test this we incubated pure styrene with AIBN at 60°C for one hour. We then precipitated out the polystyrene by using a methanol wash. We were able to do so because the styrene monomer is soluble in methanol, but polystyrene is not. Below is our dried sheet of polystyrene!

Step two: Optimizing protocol

[Now that we confirmed free radical polymerization of styrene using AIBN initiator worked, we wanted to optimize our protocols. We focused on three variables…]

Conclusion

We successfully cloned all the genes for PAL, FDC, and UbiX into plasmids submitted as biobricks to the registry. We purified our enzymes and confirmed the molecular weight on a SDS PAGE gel. The in vitro testing of PAL yielded quantitative information about the reaction velocity in the form of concentration curves that were fitted using Michaelis Menten models to determine reaction parameters. The parameters were then plugged into the model to make informed predictions about our specific enzymes and their influence on the overall pathway flux. The in vitro testing of FDC was more difficult in that it was almost impossible to measure the progress of the reaction using standard spectrophotometric methods. After using our model to make an informed prediction about which ordering of the operon would yield the largest amount of styrene, we used Gibson Assembly to create our desired configuration. After sequencing the product of this procedure, we found that we were successful in the assembly of our operon. We also refined procedures for extracting styrene as it is being produced bacterially and polymerize this styrene into polystyrene.

Some future work that could be done is in vivo testing of styrene production after transforming our operon-containing plasmid into E. coli. Once styrene is identified, our extraction procedure would need to be carried out in order to isolate the styrene from the cell while maintaining non-toxic levels in the cell’s environment to allow the cells to continue producing styrene. Finally, our polymerization method would need to be applied to our isolated styrene and then our polystyrene would be ready for folding and property testing.

Protocols Protocols: Materials: Equipment: References NEED TO ADD CITATIONS!!!!! (1) operon distance- Lim, H. N., Y. Lee, and R. Hussein. "Fundamental Relationship between Operon Organization and Gene Expression." Proceedings of the National Academy of Sciences (2011): 10626-0631. Print. (2) ubix pad1- Lin, Fengming, Kyle L. Ferguson, David R. Boyer, Xiaoxia Nina Lin, and E. Neil G. Marsh. "Isofunctional Enzymes PAD1 and UbiX Catalyze Formation of a Novel Cofactor Required by Ferulic Acid Decarboxylase and 4-Hydroxy-3-polyprenylbenzoic Acid Decarboxylase." ACS Chem. Biol. ACS Chemical Biology (2015): 1137-144. Print. (3) site directed mutagenesis- Liu, Huanting, and James H Naismith. "An Efficient One-step Site-directed Deletion, Insertion, Single and Multiple-site Plasmid Mutagenesis Protocol." BMC Biotechnology BMC Biotechnol: 91. Print. (4) styrene biosynthesis- Mckenna, Rebekah, and David R. Nielsen. "Styrene Biosynthesis from Glucose by Engineered E. Coli." Metabolic Engineering: 544-54. Print. (5) In situ removal - Mckenna, Rebekah, Luis Moya, Matthew Mcdaniel, and David R. Nielsen. "Comparing in Situ Removal Strategies for Improving Styrene Bioproduction." Bioprocess Biosyst Eng Bioprocess and Biosystems Engineering (2014): 165-74. Print. (6) new cofactor ubix- Payne, Karl A. P., Mark D. White, Karl Fisher, Basile Khara, Samuel S. Bailey, David Parker, Nicholas J. W. Rattray, Drupad K. Trivedi, Royston Goodacre, Rebecca Beveridge, Perdita Barran, Stephen E. J. Rigby, Nigel S. Scrutton, Sam Hay, and David Leys. "New Cofactor Supports α,β-unsaturated Acid Decarboxylation via 1,3-dipolar Cycloaddition." Nature (2015): 497-501. Print. (7) Anabaena PAL - Wang, Lin, Alejandra Gamez, Holly Archer, Enrique E. Abola, Christineh N. Sarkissian, Paul Fitzpatrick, Dan Wendt, Yanhong Zhang, Michel Vellard, Joshua Bliesath, Sean M. Bell, Jeffrey F. Lemontt, Charles R. Scriver, and Raymond C. Stevens. "Structural and Biochemical Characterization of the Therapeutic Anabaena Variabilis Phenylalanine Ammonia Lyase." Journal of Molecular Biology: 623-35. Print. (8) ubix cofactor- White, Mark D., Karl A. P. Payne, Karl Fisher, Stephen A. Marshall, David Parker, Nicholas J. W. Rattray, Drupad K. Trivedi, Royston Goodacre, Stephen E. J. Rigby, Nigel S. Scrutton, Sam Hay, and David Leys. "UbiX Is a Flavin Prenyltransferase Required for Bacterial Ubiquinone Biosynthesis." Nature (2015): 502-06. Print.

Abstract to create Cellulose using G. hansenii

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Experiment Engineering E. coli to produce polystyrene

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Data and Results Optimizing the production of biological PHA

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Protocols

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References

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