Part:BBa_K1692007

UbiX with T7 promoter and FLAG tag

Overview

UbiX is a flavin prenyltransferase that normally plays a role in ubiquinone biosynthesis in E. coli. UbiX transfers a prenyl group from dimethylallyl monophosphate (DMAP) to flavin mononucleotide (FMN), thereby creating a cofactor that happens to be essential to the functionality of FDC. Our genetic construct includes the FDC gene, 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 UbiX enzyme.

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.

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

[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:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 578
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]