Difference between revisions of "Part:BBa K1129025"
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<partinfo>BBa_K1129025 short</partinfo> | <partinfo>BBa_K1129025 short</partinfo> | ||
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<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
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<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
<partinfo>BBa_K1129025 SequenceAndFeatures</partinfo> | <partinfo>BBa_K1129025 SequenceAndFeatures</partinfo> | ||
| + | ===Background=== | ||
| + | ---- | ||
| − | Phenylalanine Ammonia Lyase (PAL) is an enzyme responsible for the conversion of the amino acid phenylalanine to trans-cinnamic acid. PAL is the first committed enzyme in the Phenylpropanoid pathway and is therefore involved in lignin degradation. PAL’s can be found in plants, fungi, and bacteria and it has a molecular mass in the range of 270-330 kDa. The deaminating action of PAL is conserved among the ammonia Lyase family | + | Phenylalanine Ammonia Lyase (PAL) is an enzyme responsible for the conversion of the amino acid phenylalanine to trans-cinnamic acid (1). PAL is the first committed enzyme in the Phenylpropanoid pathway and is therefore involved in lignin degradation(1,2). PAL’s can be found in plants, fungi, and bacteria and it has a molecular mass in the range of 270-330 kDa(1,3). The deaminating action of PAL is conserved among the ammonia Lyase family which includes Histadine Ammonia Lyase, and Tyrosine Ammonia Lyase(1). |
| − | ''' | + | In the figure below figure we coupled the activity of PAL with a cinnamic acid ligase and a Cinnamoyl Co A reductase to yield cinnamaldehyde from the aromatic amino acid phenylalanine. Cinnamaldehyde is a common flavouring compound used in cooking and in some countries as a therapeutic: cinnamaldehyde comes from the bark of the cinnamon tree. |
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| + | <html><center><IMG id="emp" SRC="https://static.igem.org/mediawiki/2013/a/ad/UBC-Cinnamic-Acid-structure.png" USEMAP="#cinnamaldehyde"> | ||
| + | <map name="cinnamaldehyde" id="cinnamaldehyde"> | ||
| + | <area shape="rect" coords="127,33,267,109" href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1129026" target="" /> | ||
| + | <area shape="rect" coords="337,56,474,108" href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1129042" target="" /> | ||
| + | <area shape="rect" coords="560,55,695,106" href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1129039" target="" /> | ||
| + | </map> | ||
| + | </center></html> | ||
| + | |||
| + | |||
| + | ===Experimental Data=== | ||
| + | ---- | ||
| + | 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/2013/0/03/EncP_data.jpg</p> | ||
| + | |||
| + | <p align=center>https://static.igem.org/mediawiki/2013/0/03/Const.EncP.jpg</p> | ||
| + | |||
| + | '''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://en.wikipedia.org/wiki/Phenylalanine_ammonia-lyase | 1) http://en.wikipedia.org/wiki/Phenylalanine_ammonia-lyase | ||
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3) http://www.uniprot.org/uniprot/P25872 | 3) http://www.uniprot.org/uniprot/P25872 | ||
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Latest revision as of 02:23, 29 October 2013
Phenylalanine Ammonia Lyase (Enc-P) under arabinose promoter
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 125
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 65
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 691
Illegal NgoMIV site found at 794 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1390
Illegal BsaI.rc site found at 1645
Background
Phenylalanine Ammonia Lyase (PAL) is an enzyme responsible for the conversion of the amino acid phenylalanine to trans-cinnamic acid (1). PAL is the first committed enzyme in the Phenylpropanoid pathway and is therefore involved in lignin degradation(1,2). PAL’s can be found in plants, fungi, and bacteria and it has a molecular mass in the range of 270-330 kDa(1,3). The deaminating action of PAL is conserved among the ammonia Lyase family which includes Histadine Ammonia Lyase, and Tyrosine Ammonia Lyase(1).
In the figure below figure we coupled the activity of PAL with a cinnamic acid ligase and a Cinnamoyl Co A reductase to yield cinnamaldehyde from the aromatic amino acid phenylalanine. Cinnamaldehyde is a common flavouring compound used in cooking and in some countries as a therapeutic: cinnamaldehyde comes from the bark of the cinnamon tree.
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.
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://en.wikipedia.org/wiki/Phenylalanine_ammonia-lyase
2) http://www.genome.jp/kegg-bin/show_pathway?map00940
3) http://www.uniprot.org/uniprot/P25872