Difference between revisions of "Part:BBa K1692004"

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Phenylalanine ammonia lyase (PAL) catalyzes the conversion of L-phenylalanine to trans-cinnamic acid. Our PAL construct is codon-optimized for expression in E. coli. The original sequence is derived from Anabaena variabilis. We chose the A. variabilis variant of PAL because the literature has characterized it as functioning well, in contrast to University of British Columbia’s 2013 PAL biobrick part (BBa_K1129003) from Streptomyces maritimus, which has much lower activity.
 
Phenylalanine ammonia lyase (PAL) catalyzes the conversion of L-phenylalanine to trans-cinnamic acid. Our PAL construct is codon-optimized for expression in E. coli. The original sequence is derived from Anabaena variabilis. We chose the A. variabilis variant of PAL because the literature has characterized it as functioning well, in contrast to University of British Columbia’s 2013 PAL biobrick part (BBa_K1129003) from Streptomyces maritimus, which has much lower activity.
  
[[File:SB2015_styrene_pathway.png|thumbnail|center|500px|<b>Styrene synthesis pathway</b>This is where we write]]<br><br>
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[[File:SB2015_styrene_pathway.png|thumbnail|center|500px|<b>Styrene synthesis pathway</b>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. 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.]]<br><br>
  
 
<h2>Background</h2>
 
<h2>Background</h2>

Revision as of 23:11, 17 September 2015

codon optimized PAL with T7 promoter and Flag Tag

Introduction

Phenylalanine ammonia lyase (PAL) catalyzes the conversion of L-phenylalanine to trans-cinnamic acid. Our PAL construct is codon-optimized for expression in E. coli. The original sequence is derived from Anabaena variabilis. We chose the A. variabilis variant of PAL because the literature has characterized it as functioning well, in contrast to University of British Columbia’s 2013 PAL biobrick part (BBa_K1129003) from Streptomyces maritimus, which has much lower activity.

Styrene synthesis pathwayThe 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. 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.


Background

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.



In an initial assay, we took absorbance spectra of the following reactions using a Nanodrop 2000 machine. [Left] Phenylalanine and PAL alone: a negative control to show the absorbance of pure phenylalanine and pure PAL in tris buffer (ph 8.0). [Center] Phenylalanine and PAL together: we observed a very large absorbance peak at 268 nm, suggesting that trans-cinnamic acid was produced. [Right] Pure trans-Cinnamic Acid: a positive control to confirm the absorbance peak of pure trans-cinnamic acid.


PAL FunctionalityThis is where we write


Reference

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 1315
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1533
  • 1000
    COMPATIBLE WITH RFC[1000]