Part:BBa_K1692008

Styrene Synthesis Operon

Overview

This operon is a composite of three enzymes in the following order: FDC (BBa_K1692002), UbiX (BBa_K1692007), and PAL (BBa_K1692004).

PAL converts phenylalanine to cinnamic acid. FDC converts cinnamic acid to styrene. UbiX modifies flavin mononucleotide to produce a cofactor that is required for FDC activity.

The entire operon is controlled via an inducible T7 promoter. Each protein-coding sequence is preceded by a ribosome binding site and followed by a FLAG-tag peptide, enabling easy and efficient extraction.

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.

The decision to place FDC first, PAL last, and UbiX in between was based on results from our mathematical model. Specifically, our model suggested that FDC exerts greatest control on the flux of biomaterials through the pathway; in other words, changes in FDC concentration result in the greatest changes in final styrene production. We combined this finding with the fact that earlier genes in an operon are expressed at higher levels. Taken together, these results implied that we should place FDC first.

Additionally, since UbiX’s cofactor product is essential for FDC activity, we decided to place UbiX immediately after FDC.

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 233
  Illegal BglII site found at 2154
  Illegal BglII site found at 3528
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 3746
  
• 1000
  COMPATIBLE WITH RFC[1000]