Difference between revisions of "Part:BBa K1692002"
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<h2>Introduction</h2> | <h2>Introduction</h2> | ||
− | Ferulic acid decarboxylase (FDC) catalyzes the conversion of trans-cinnamic acid to styrene. We codon-optimized S. cerevisiae’s FDC gene for expression in E. coli. | + | Ferulic acid decarboxylase (FDC) catalyzes the conversion of trans-cinnamic acid to styrene. We codon-optimized S. cerevisiae’s FDC gene for expression in E. coli. This construct includes 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 FDC enzyme. |
[[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> | [[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> | ||
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<h2>Background</h2> | <h2>Background</h2> | ||
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[[File:SB2015_styrene_enzymes_SDS_PAGE.png|thumbnail|center|700px|This is a <b>SDS PAGE gel</b> with purified PAL, FDC and UbiX protein. We ran a Mark 12 protein ladder to verify that our proteins were the correct molecular weight.]]<br><br> | [[File:SB2015_styrene_enzymes_SDS_PAGE.png|thumbnail|center|700px|This is a <b>SDS PAGE gel</b> with purified PAL, FDC and UbiX protein. We ran a Mark 12 protein ladder to verify that our proteins were the correct molecular weight.]]<br><br> | ||
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Revision as of 02:07, 18 September 2015
codon optimized FDC with T7 promoter and Flag Tag
Introduction
Ferulic acid decarboxylase (FDC) catalyzes the conversion of trans-cinnamic acid to styrene. We codon-optimized S. cerevisiae’s FDC gene for expression in E. coli. This construct includes 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 FDC enzyme.
Background
Reference
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 233
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]