Part:BBa_K1692004

codon optimized PAL with T7 promoter and Flag Tag

Overview

Phenylalanine ammonia lyase (PAL) catalyzes the conversion of L-phenylalanine to trans-cinnamic acid, the first step in our styrene synthesis pathway. Our PAL construct is codon-optimized for expression in E. coli. The original sequence is derived from Anabaena variabilis. We chose the A. variabilis variant of PAL because the literature has characterized it as functioning well, in contrast to University of British Columbia’s 2013 PAL biobrick part (BBa_K1129003) from Streptomyces maritimus, which has much lower activity [1]. This construct includes a T7 inducible promoter, a ribosome binding site, and a FLAG-tag peptide sequence for easy and efficient protein purification. We have sequenced our construct and verified that all these components are indeed present. We were able to successfully clone our construct into E. coli and induce expression with isopropyl β-D-1-thiogalactopyranoside (IPTG). A sodium dodecyl sulfate polyacrylamide gel electrophoresis confirmed that our FLAG-tag extraction selectively purified the PAL enzyme. With the solution resulting from our extraction, we were able to perform kinetic time course assays on PAL at various concentrations of its substrate phenylalanine. These results constitute functional evidence that our PAL works as expected.

Styrene synthesis pathway The enzymes of interest are phenylalanine ammonia lyase (PAL), ferulic acid decarboxylase (FDC), and a flavin prenyltransferase involved in ubiquinone biosynthesis called UbiX. PAL catalyzes the conversion of phenylalanine to trans-cinnamic acid, while FDC catalyzes the conversion of trans-cinnamic acid to styrene [1]. Recently, it has been discovered that a cofactor is required to activate FDC. This cofactor is a product of the reaction between dimethylallyl monophosphate (DMAP) and flavin mononucleotide (FMN), which is catalyzed by the enzyme UbiX [2].

Experiments and Results

After obtaining our synthesized gene, we needed to insert it into the standard pSB1C3 backbone so we could transform it and submit as a biobrick. 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. Now that we had our gene in a plasmid with a promoter and RBS we transformed it into T7 expressing NEB E. coli. We grew up large cultures, which we 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.

This is a SDS PAGE gel with purified PAL, FDC and UbiX protein. We ran a Mark 12 protein ladder to verify that our proteins were the correct molecular weight.

Once we were confident that we had successfully purified our enzymes from the T7 expressing E. coli, the next logical step was to test the in vivo functionality of our proteins. In order to test PAL’s functionality, 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 [1]. Notably, tCA has a large and easily distinguishable peak at 268 nm, whereas phenylalanine displays a much less noticeable peak just below 260 nm. In an initial assay, shown below, we took absorbance spectra from a reaction mixture of PAL with phenylalanine and noticed a large peak at 268 nm, suggesting the presence of tCA.

In an initial assay, we took absorbance spectra of the following reactions using a Nanodrop 2000 machine. [Left] Phenylalanine and PAL alone: a negative control to show the absorbance of pure phenylalanine and pure PAL in tris buffer (ph 8.0). [Center] Phenylalanine and PAL together: we observed a very large absorbance peak at 268 nm, suggesting that trans-cinnamic acid was produced. [Right] Pure trans-Cinnamic Acid: a positive control to confirm the absorbance peak of pure trans-cinnamic acid.

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 on our enzymes kinetic parameters. 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 in real time. We created reaction mixtures of PAL along with 8 different concentrations of its substrate, phenylalanine, and tracked the reaction in real time over the course of about 4 hours using a 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.

PAL Kinetic time course assay Using a Spectramax Pro spectrophotometer we tracked, in real time, the absorbance of trans-cinnamic acid at 268 nm. We ran a total of 24 reactions with varying concentrations of phenylalanine so that we could determine the kinetic parameters of our enzyme. Shown here is a time profile of trans-cinnamic acid production starting with 0.6 mM phenylalanine over a four hour period. Sample points were taken every two minutes.

Reference

[1] 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.

[2] 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. Sequence and Features

Assembly Compatibility:

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