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Team:KULeuven/Lab/Vanillin Production
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[[Image:Vanillin_Biosynthesis_Pathway.jpg|center|thumb|365px|Making vanillin from tyrosine]] | [[Image:Vanillin_Biosynthesis_Pathway.jpg|center|thumb|365px|Making vanillin from tyrosine]] | ||
| - | == | + | ==Required== |
| - | + | Biobricks: | |
*SAM8: {{kulpart|BBa_I742142}}: coding sequence without RBS | *SAM8: {{kulpart|BBa_I742142}}: coding sequence without RBS | ||
*SAM5: {{kulpart|BBa_K238007}}: RBS site + PstI restriction site removed | *SAM5: {{kulpart|BBa_K238007}}: RBS site + PstI restriction site removed | ||
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==Steps== | ==Steps== | ||
| - | * | + | *We will be working in three different stages |
| - | #Testing the transformation of tyrosin to | + | #Testing the transformation of tyrosin to ferrulic acid. The enzymes are put under a constitutive promoter and transcribed. Ferrulic acid concentrations are then measured with GC |
| - | #Testing the transformation of | + | #Testing the transformation of ferrulic acid to vanillin. The enzymes are put under a constitutive promoter and transcribed. Ferrulic acid concentrations are then measured with GC |
#If both are tested and ok, they can be combined into one plasmid and again tested. | #If both are tested and ok, they can be combined into one plasmid and again tested. | ||
#Then, the locks need to be added. | #Then, the locks need to be added. | ||
| - | * | + | *Testing the enzymes by detecting the endproducts through gaschromatography. |
| - | *One problem might be that the | + | *One problem might be that the tyrosine of E. coli is depleted too fast |
Latest revision as of 06:25, 18 August 2009
Contents |
VANILLIN SYNTHESIS
Goal
Making vanillin from tyrosine in a five-step pathway.
Required
Biobricks:
- SAM8: : coding sequence without RBS
- SAM5: : RBS site + PstI restriction site removed
- COMT: : coding sequence without RBS
- FCS: : RBS + fcs in pSB1A2
- ECH: : RBS + ech in pSB1A2
Where from
- SAM8, SAM5, COMT, FCS, ECH will be sent to us from the French Lab from the university Edinburgh
Steps
- We will be working in three different stages
- Testing the transformation of tyrosin to ferrulic acid. The enzymes are put under a constitutive promoter and transcribed. Ferrulic acid concentrations are then measured with GC
- Testing the transformation of ferrulic acid to vanillin. The enzymes are put under a constitutive promoter and transcribed. Ferrulic acid concentrations are then measured with GC
- If both are tested and ok, they can be combined into one plasmid and again tested.
- Then, the locks need to be added.
- Testing the enzymes by detecting the endproducts through gaschromatography.
- One problem might be that the tyrosine of E. coli is depleted too fast
