Difference between revisions of "Part:BBa K1129043"
 
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<partinfo>BBa_K1129043 short</partinfo>
 
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Arab.+rbs+4-CMH+term
 
  
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4-coumarate 3-hydroxylase is an enzyme in the vanillin pathway. 4-coumarate 3-hydroxylase (4-CMH) hydrolyses p-coumaric acid to caffeic acid. It is the 2nd enyzme the biosynthetic pathway of vanillin from the amino acid tyrosine. The enzyme is under arabinose inducible promoter and a terminator.<!-- Add more about the biology of this part here
 
 
===Usage and Biology===
 
===Usage and Biology===
  
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<partinfo>BBa_K1129043 parameters</partinfo>
 
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<h2>Background</h2>
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<p> 4-coumarate 3-hydroxylase is an enzyme in the vanillin pathway. 4-coumarate 3-hydroxylase (4-CMH) hydrolyses p-coumaric acid to caffeic acid. It is the 2nd enzyme in the biosynthetic pathway of vanillin bioproduction from the amino acid tyrosine. 4-CMH is biobrick (<html><b><a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K238007">BBa_K238007</b></a></html>) from <html><b><a href="http://2009.igem.org/Team:KULeuven/Design/Vanillin_Production">KULeuven iGEM 2009</b></a></html> </p>
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<html><center><IMG id="emp" SRC="https://static.igem.org/mediawiki/2013/b/bf/UBC-Vanillin-structure.png" USEMAP="#vin">
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<map id="vin" name="vin"><area shape="rect" alt="" title="" coords="104,101,242,184" href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1129000" target="" /><area shape="rect" alt="" title="" coords="353,103,491,189" href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1129046" target="" /><area shape="poly" alt="" title="" coords="595,76,750,77,750,150,696,151,699,263,647,263,644,164,595,164" href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1129041" target="" /><area shape="rect" alt="" title="" coords="489,387,630,487" href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1129024" target="" /><area shape="rect" alt="" title="" coords="205,384,351,487" href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1129022" target="" /><!-- Created by Online Image Map Editor (http://www.maschek.hu/imagemap/index) --></map>
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</center></html>
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<h2>Experimental Data</h2>
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4-CMH under the arabinose promoter (<html><b><a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_J23118">BBa_J23118</b></a></html>) were transformed into ''E.coli 10G'' cells. An overnight culture of these cells harboring (<html><b><a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_I13453">BBa_I13453</b></a></html>)was inoculated into a fresh culture of 5mL minimal media containing tyrsoine.  Cells  and were harvested for GC-MS after 7 hours of growth.
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<p align=center>https://static.igem.org/mediawiki/2013/5/5f/PhpGF9BoJPM%281%29.jpg</p>
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Figure 3. Compound generation identification by GC-MS. Chromatograms and mass spectra for select peaks are shown. Structures represent predictions based on library matching or comparison to standards. Controls represent plasmids missing the gene of interest. A) Internal control using caffeic acid. B) Conversion of p-coumaric acid to caffeic acid by constitutively expressed 4-CMH. C) Possible conversion of p-coumaric acid to caffeic acid after induction by arabinose on 4CMH under the control by the arabinose promoter. The mass spectrum however is confounded by another compound.
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Latest revision as of 02:33, 29 October 2013

4-coumarate 3-hydroxylase under arabinose promoter


Usage and Biology

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 125
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 65
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 195
    Illegal NgoMIV site found at 203
    Illegal NgoMIV site found at 953
    Illegal NgoMIV site found at 1126
    Illegal NgoMIV site found at 1528
    Illegal NgoMIV site found at 1532
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 727


Background

4-coumarate 3-hydroxylase is an enzyme in the vanillin pathway. 4-coumarate 3-hydroxylase (4-CMH) hydrolyses p-coumaric acid to caffeic acid. It is the 2nd enzyme in the biosynthetic pathway of vanillin bioproduction from the amino acid tyrosine. 4-CMH is biobrick (BBa_K238007) from KULeuven iGEM 2009



Experimental Data

4-CMH under the arabinose promoter (BBa_J23118) were transformed into E.coli 10G cells. An overnight culture of these cells harboring (BBa_I13453)was inoculated into a fresh culture of 5mL minimal media containing tyrsoine. Cells and were harvested for GC-MS after 7 hours of growth.


PhpGF9BoJPM%281%29.jpg


Figure 3. Compound generation identification by GC-MS. Chromatograms and mass spectra for select peaks are shown. Structures represent predictions based on library matching or comparison to standards. Controls represent plasmids missing the gene of interest. A) Internal control using caffeic acid. B) Conversion of p-coumaric acid to caffeic acid by constitutively expressed 4-CMH. C) Possible conversion of p-coumaric acid to caffeic acid after induction by arabinose on 4CMH under the control by the arabinose promoter. The mass spectrum however is confounded by another compound. ,