Part:BBa_K1692008

Styrene Synthesis Operon

Overview

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

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 [2]. 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 [3].

Experiments and Results

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 [1]. 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.

Once we determined the gene order for our FDC-UbiX-PAL operon, we needed to make it. To do this we used the New England Biolabs Gibson Assembly kit. We designed eight primers according to a primer design protocol (also from NEB) for FDC, UbiX, PAL and RFP plasmids. We then ran a PCR extension on all four of the resulting segments. We ran these four products on a gel and gel extracted the bands that appeared at the desired migration distances. We used this purified product for the Gibson assembly and transformed our resultant plasmid into NEB 5-alpha competent cells (NEB). We ran a colony PCR and found three colonies with the expected band length of over 4,000 base pairs. We grew up liquid cultures of these three colonies, miniprepped, and ran sequencing using five internal primers designed specifically for the combo plasmid. We found that two of the three colonies had the correct sequence!

We transformed the combo plasmid with FDC-UbiX-PAL operon (sequence confirmed) into T7 expressing competent cells (T7 Express, NEB) and performed a western blot using FITC-conjugated anti-flag antibody (Sigma) to confirm that all three genes were actually expressing the recombinant proteins with expected sizes.

Western blot analysis of in vivo expressed FDC-PAL-Ubix proteins

Confirming the clear over-expression of FDC over PAL protein from the FDC-UbiX-PAL operon in vivo, we are currently performing GC-MS analysis to see if the cells are fully capable of producing styrene at detectable and useful levels.

Reference

[1] Lim, H. N., Y. Lee, and R. Hussein. "Fundamental Relationship between Operon Organization and Gene Expression." Proceedings of the National Academy of Sciences (2011): 10626-0631. Print.

[2] 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.

[3] 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]