Coding

Part:BBa_K1692007

Designed by: Daniel Kunin   Group: iGEM15_Stanford-Brown   (2015-09-17)
Revision as of 03:12, 19 September 2015 by Daniel xiang (Talk | contribs)

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

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.



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]


[edit]
Categories
//awards/part_collection/2015
Parameters
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