Difference between revisions of "Part:BBa K1692004"

Revision as of 02:14, 18 September 2015

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

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.

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.

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