Part:BBa_K1129046
4-coumarate 3-hydroxylase under pTET constitutive promoter
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 100
Illegal NgoMIV site found at 108
Illegal NgoMIV site found at 858
Illegal NgoMIV site found at 1031
Illegal NgoMIV site found at 1433
Illegal NgoMIV site found at 1437 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 632
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 constitutive promoter (BBa_J23118) were transformed into E.coli 10G cells. An overnight culture of these cells harboring (BBa_K1129046) and were harvested for GC-MS after 7 hours of growth.
Ech and fcs genes( BBa_K1129022 and BBa_K1129024), both of which are under constitutive promoters (BBa_J23118), were ligated into one pSB1C3 plasmid. An overnight culture of E.coli 10G cells harboring this plasmid was inoculated into a fresh culture of 5mL minimal media containing ferulic acid. Cells were then harvested for GC-MS after 7 hours of growth.
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 constitutive expressed 4-CMH. C) Possible conversion of p-coumaric acid to caffeic acid by inducibly expressed 4CMH. The mass spectrum however is confounded by another compound.
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