Difference between revisions of "Part:BBa K1692002"

Revision as of 02:04, 18 September 2015

codon optimized FDC with T7 promoter and Flag Tag

Introduction

Ferulic acid decarboxylase (FDC) catalyzes the conversion of trans-cinnamic acid to styrene. We codon-optimized S. cerevisiae’s FDC gene for expression in E. coli.

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. 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.

Background

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.

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.

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 FDC enzyme.

Styrene AbsorbanceThe absorbance spectrum of pure styrene (1 mM) measured on a Spectramax Pro spectrophotometer

trans-Cinnamic Acid AbsorbanceThe absorbance spectrum of pure trans-Cinnamic Acid (1 mM) measured on a Spectramax Pro spectrophotometer

Flavin Mononucleotide AbsorbanceThe absorbance spectrum of pure Flavin Mononucleotide (1 mM) measured on a Spectramax Pro spectrophotometer

Reference

Sequence and Features