Team:KULeuven/Lab/Vanillin Production

From 2009.igem.org

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=VANILLIN SYNTHESIS=
=VANILLIN SYNTHESIS=
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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]]
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==required==
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==Required==
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*biobricks:  
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Biobricks:  
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    SAM8: BBa_ I742142: coding sequence without RBS
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*SAM8: {{kulpart|BBa_I742142}}: coding sequence without RBS
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    SAM5: no number: RBS site + PstI restriction site removed
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*SAM5: {{kulpart|BBa_K238007}}: RBS site + PstI restriction site removed
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    COMT: BBa_I742107: coding sequence without RBS
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*COMT: {{kulpart|BBa_I742107}}: coding sequence without RBS
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    FCS: BBa_I742115: RBS + fcs in pSB1A2  
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*FCS: {{kulpart|BBa_I742115}}: RBS + fcs in pSB1A2  
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    ECH: BBa_I742113: RBS + ech in pSB1A2
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*ECH: {{kulpart|BBa_I742113}}: RBS + ech in pSB1A2
==Where from==
==Where from==
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*1. COMT is available in 2009 spring distribution plate 1 well 19D
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* SAM8, SAM5, COMT, FCS, ECH will be sent to us from the French Lab from the university Edinburgh
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DNA is compatible
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*2. SAM8, SAM5, COMT, FCS, ECH will be sent to us from the French Lab from the university Edinburgh
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==Steps==
==Steps==
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*we will be working in three different stages
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*We will be working in three different stages
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1.Testing the transformation of tyrosin to ferulic acid. The enzymes are put under a constitutive promoter and transcribed. Ferulic acid concentrations are then measured with GC  
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#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  
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2.Testing the transformation of ferulic acid to vanillin. The enzymes are put under a constitutive promoter and transcribed. Ferulic acid concentrations are then measured with GC   
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#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   
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3.If both are tested and ok, they can be combined into one plasmid and again tested.
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#If both are tested and ok, they can be combined into one plasmid and again tested.
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4.Then, the locks need to be added.
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#Then, the locks need to be added.
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*testing the enzymes: by detecting the endproducts through gaschromatography.
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*Testing the enzymes by detecting the endproducts through gaschromatography.
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*One problem might be that the tyrosin of E.Coli might be depleted to fast
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*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.

Making vanillin from tyrosine

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
  1. 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
  2. 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
  3. If both are tested and ok, they can be combined into one plasmid and again tested.
  4. 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
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