Difference between revisions of "Part:BBa K1129042"
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<partinfo>BBa_K1129042 short</partinfo>
 
<partinfo>BBa_K1129042 short</partinfo>
  
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The 4-coumarate-CoA ligase (4CL) enzyme catalyzes the ligation of a CoA group to the end of the molecule. The addition of this CoA provides better kinetics for a variety of downstream reactions. 4CL is also a member of the phenylpropanoid pathway and is found in a wide variety of organisms that partake in the degradation of lignin. Here we harness the 4CL to provide better kinetics for reduction of cinnamic acid to cinnamaldehyde.
 
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'''Resources'''
 
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http://www.uniprot.org/uniprot/Q1H8P3
 
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http://en.wikipedia.org/wiki/4-Coumarate-CoA_ligase
 
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http://www.genome.jp/kegg-bin/show_pathway?map00940
 
  
 
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<partinfo>BBa_K1129042 parameters</partinfo>
 
<partinfo>BBa_K1129042 parameters</partinfo>
 
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===Background===
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----
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The 4-coumarate-CoA ligase (4CL) enzyme catalyzes the ligation of a CoA group to the end of the molecule. The addition of this CoA provides better kinetics for a variety of downstream reactions. 4CL is also a member of the phenylpropanoid pathway and is found in a wide variety of organisms that partake in the degradation of lignin. Here we harness the 4CL to provide better kinetics for reduction of cinnamic acid to cinnamaldehyde.
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===Experimental Data===
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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.
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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.
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<p align=center>https://static.igem.org/mediawiki/2013/0/03/EncP_data.jpg</p>
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<p align=center>https://static.igem.org/mediawiki/2013/0/03/Const.EncP.jpg</p>
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'''Figure 1.''' 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. '''Top''' Internal control using cinnamic acid. '''Middle''' cinnamic acid detection from culture containing phenylalanine and strains harbouring constitutive EncP.  '''Bottom''' Cinnamic acid and cinnamaldehyde detection from culture containing phenylalanine and strains harbouring constitutive EncP, 4CH and ATRCC1.
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===Resources===
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----
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1) http://www.uniprot.org/uniprot/Q1H8P3
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2) http://en.wikipedia.org/wiki/4-Coumarate-CoA_ligase
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3) http://www.genome.jp/kegg-bin/show_pathway?map00940

Revision as of 01:48, 29 October 2013

4-coumarate-CoA ligase (4CL) under pTET constitutive promoter


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
    Illegal NheI site found at 1147
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1714
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1346


Background

The 4-coumarate-CoA ligase (4CL) enzyme catalyzes the ligation of a CoA group to the end of the molecule. The addition of this CoA provides better kinetics for a variety of downstream reactions. 4CL is also a member of the phenylpropanoid pathway and is found in a wide variety of organisms that partake in the degradation of lignin. Here we harness the 4CL to provide better kinetics for reduction of cinnamic acid to cinnamaldehyde.

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.

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.

EncP_data.jpg

Const.EncP.jpg

Figure 1. 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. Top Internal control using cinnamic acid. Middle cinnamic acid detection from culture containing phenylalanine and strains harbouring constitutive EncP. Bottom Cinnamic acid and cinnamaldehyde detection from culture containing phenylalanine and strains harbouring constitutive EncP, 4CH and ATRCC1.

Resources

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

2) http://en.wikipedia.org/wiki/4-Coumarate-CoA_ligase

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