Difference between revisions of "Part:BBa K1692008"

Revision as of 16:08, 19 September 2015

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.
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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. 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 one of the correct combo plasmids into T7 expressing cells to extract our protein as before. Again, we ran our proteins sample on an SDS PAGE and confirmed that not only were all of our proteins expressing correctly, but that we had the most FDC and least PAL, as expected.

We are currently performing in vivo experiments to determine whether cells expressing our operon are fully capable of expressing 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