Difference between revisions of "Part:BBa K1692004"
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<h2>Background</h2> | <h2>Background</h2> | ||
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+ | 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. | ||
[[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> | ||
+ | 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 PAL enzyme. With the solution resulting from our extraction, we were able to perform kinetic time course assays on PAL at various concentrations of its substrate phenylalanine. These results constitute functional evidence that our PAL works as expected. | ||
[[File:SB2015_PAL_initial test.png|thumbnail|center|900px|<b>In an initial assay</b>, we took absorbance spectra of the following reactions using a Nanodrop 2000 machine. <b>[Left]</b> Phenylalanine and PAL alone: a negative control to show the absorbance of pure phenylalanine and pure PAL in tris buffer (ph 8.0). | [[File:SB2015_PAL_initial test.png|thumbnail|center|900px|<b>In an initial assay</b>, we took absorbance spectra of the following reactions using a Nanodrop 2000 machine. <b>[Left]</b> Phenylalanine and PAL alone: a negative control to show the absorbance of pure phenylalanine and pure PAL in tris buffer (ph 8.0). |
Revision as of 02:03, 18 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, the first step in our styrene synthesis pathway. 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 [1].
Background
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 PAL enzyme. With the solution resulting from our extraction, we were able to perform kinetic time course assays on PAL at various concentrations of its substrate phenylalanine. These results constitute functional evidence that our PAL works as expected.
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1315
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1533
- 1000COMPATIBLE WITH RFC[1000]