Coding

Part:BBa_K1692004

Designed by: Daniel Kunin   Group: iGEM15_Stanford-Brown   (2015-08-03)
Revision as of 22:59, 17 September 2015 by Daniel.kunin (Talk | contribs)

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 pathwayThis is where we write


Background

SDS PAGE Gel of Enzymes for Styrene SynthesisThis is a SDS PAGE gel with purified FDC, UbiX and PAL 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 separate: 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]


[edit]
Categories
//awards/part_collection/2015
//chassis/prokaryote/ecoli
//collections/probiotics/production
Parameters
None