Difference between revisions of "Part:BBa K1129039"
(→‎Experimental Data)
 
Line 26: Line 26:
 
The genes encoding PAL ([https://parts.igem.org/Part:BBa_K1129003 BBa_K1129003]), 4-Cinnamic acid ligase([https://parts.igem.org/wiki/index.php?title=Part:BBa_K1129042 BBa_K1129042]) and a Cinnamoyl Co-A reductase([https://parts.igem.org/wiki/index.php?title=Part:BBa_K1129005 BBa_1129005]) were assembled into one PSB1C3 plasmid under constitutive promoters and transformed into E. ''coli''. E.''coli'' 10G cells were grown over night before being incubated with L-Phenylalanine for 7 hours, the sample was extracted before it was run on GC-MS.  
 
The genes encoding PAL ([https://parts.igem.org/Part:BBa_K1129003 BBa_K1129003]), 4-Cinnamic acid ligase([https://parts.igem.org/wiki/index.php?title=Part:BBa_K1129042 BBa_K1129042]) and a Cinnamoyl Co-A reductase([https://parts.igem.org/wiki/index.php?title=Part:BBa_K1129005 BBa_1129005]) were assembled into one PSB1C3 plasmid under constitutive promoters and transformed into E. ''coli''. E.''coli'' 10G cells were grown over night before being incubated with L-Phenylalanine for 7 hours, the sample was extracted before it was run on GC-MS.  
  
−
The first two panels show experimental data for our PAL (Enc-P) expressed from PSB1C3, under constitutive promoters alone. The last panel shows experimental data for the entire biosynthetic pathway for the generation of cinnamaldehyde.
 
  
 
<p align=center>https://static.igem.org/mediawiki/2013/2/2a/Const._EncP_%2B_AtCCR1.jpg</p>
 
<p align=center>https://static.igem.org/mediawiki/2013/2/2a/Const._EncP_%2B_AtCCR1.jpg</p>

Latest revision as of 02:37, 29 October 2013

Cinnamoyl-CoA reductase from Arabidopsis thaliana under pTET constitutive promoter


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 BglII site found at 357
    Illegal BamHI site found at 65
    Illegal XhoI site found at 247
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 459
  • 1000
    COMPATIBLE WITH RFC[1000]

Background

ATCCR1 is a Cinnamoyl CoA reductase found in Artabidopsis thaliana it functions to reduce the cinnamoyl coA molecule into cinnamaldehyde by utilizing the favourable kinetics offered by releasing the coA group. This enzyme belongs to the oxioreductase family and is involved in both the phenylpropanoid pathway and thus in the degradation of lignin.

Experimental Data

The genes encoding PAL (BBa_K1129003), 4-Cinnamic acid ligase(BBa_K1129042) and a Cinnamoyl Co-A reductase(BBa_1129005) were assembled into one PSB1C3 plasmid under constitutive promoters and transformed into E. coli. E.coli 10G cells were grown over night before being incubated with L-Phenylalanine for 7 hours, the sample was extracted before it was run on GC-MS.


Const._EncP_%2B_AtCCR1.jpg

Figure 1. Compound generation identification by GC-MS. Chromatograms (left) and mass spectra (right) for select peaks are shown. Structures represent predictions based on library matching or comparison to standards. Controls represent plasmids missing the gene of interest. Top) GC-MS of a Cinnamic acid (10.580) control. Bottom) Conversion of Phenylalanine to Cinnamaldehyde (11.819) through the cinnamic acid intermediate(10.580) via the EncP, 4CL, and ATCCR1 gene construct under a constitutive promoter. The mass spec data shows a highly oxidized cinnamaldehyde that may have resulted from poor sample preparation

Resources

http://www.uniprot.org/uniprot/Q1H8P3

http://www.arabidopsis.org/servlets/TairObject?id=137093&type=locus

http://www.genome.jp/kegg-bin/show_pathway?map00940