Team:KULeuven/Wetlab/Vanillin Production

From 2009.igem.org

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__NOTOC__
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=VANILLIN SYNTHESIS=
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=Vanillin Production: Planning=
==Goal==
==Goal==
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Making vanillin from tyrosine in a five-step pathway.
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Making vanillin from tyrosine in a five-step pathway, implement a key lock function and add the LVA tag on the Sam8 and ComT enzymes.
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[[Image:Vanillin_Biosynthesis_Pathway.jpg‎|left|thumb|365px|Making vanillin from tyrosine]]
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[[Image:Vanillin_Biosynthesis_Pathway.jpg‎|center|thumb|365px|Making vanillin from tyrosine]]
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==Required==
==Required==
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==Steps==
==Steps==
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*We will be working in three different stages
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# Test the transformation of tyrosine to ferulic acid. The enzymes are put under a constitutive promoter and transcribed. Ferulic acid concentrations are then measured with HPLC
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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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# Test the transformation of ferulic acid to vanillin. The enzymes are put under a constitutive promoter and transcribed. Ferulic acid concentrations are then measured with HPLC  
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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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# add LVA tag to ComT (and Sam8)
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#If both are tested and ok, they can be combined into one plasmid and again tested.
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# If both constructs are tested and are working, they can be combined into one plasmid and again tested.
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#Then, the locks need to be added.
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# Then, the locks need to be added.
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'''The experimental setup for transformation by both biobrick constructs'''
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Electrocompetent Top10 cells were electroporated with {{kulpart|K238022}} and {{kulpart|K238018}}, additionally for blanc measurement cultures with {{kulpart|K238021}} and {{kulpart|K238017}} were electroporated. These cells were plated out on agar plates with the correct AntiBiotic and grown overnight (37°C). From these, a 5ml liquid culture supplied with AB was prepared and also grown overnight at 37°C. Subsequently 1:100th was transfered to a new liquid culture containing AB and tyrosine (5mg/L or 50 mg/L) for the first 3 enzymes(Sam8, Sam5 and ComT, {{kulpart|K238022}}), Ferulic acid (5mg/L or 50mg/L) for the second 2 enzymes (ech and fcs, {{kulpart|K238018}}) And vanillin (5mg/L or 50 mg/L) to determine the degradation or transformation of vanillin by E.Coli. After certain time intervals ranging between 30 minutes and many hours samples (1-5ml) were taken and centrifuged at 13000RPM for 1 minute. The supernatant was then transfered to a new tube and placed in -20°C until it was ready for analysis. Analysis was done on HPLC (High pressure liquid chromatography) using a standard LB culture for blanco and the measurements from the experiments.
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*Testing the enzymes by detecting the endproducts through gaschromatography.
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{{Team:KULeuven/Common2/PageFooter}}
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*One problem might be that the tyrosine of E. coli is depleted too fast
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Latest revision as of 22:49, 20 October 2009

Vanillin Production: Planning

Goal

Making vanillin from tyrosine in a five-step pathway, implement a key lock function and add the LVA tag on the Sam8 and ComT enzymes.

Making vanillin from tyrosine

Required

Biobricks:

  • SAM8: : coding sequence without RBS
  • SAM5: : RBS site + PstI restriction site removed
  • COMT: : coding sequence without RBS
    • LVA tag (AANDENYALAA) attached to COMT (nucleotide code: gctgcaaacgacgaaaactacgctttagtagct)
  • 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

  1. Test the transformation of tyrosine to ferulic acid. The enzymes are put under a constitutive promoter and transcribed. Ferulic acid concentrations are then measured with HPLC
  2. Test the transformation of ferulic acid to vanillin. The enzymes are put under a constitutive promoter and transcribed. Ferulic acid concentrations are then measured with HPLC
  3. add LVA tag to ComT (and Sam8)
  4. If both constructs are tested and are working, they can be combined into one plasmid and again tested.
  5. Then, the locks need to be added.


The experimental setup for transformation by both biobrick constructs

Electrocompetent Top10 cells were electroporated with and , additionally for blanc measurement cultures with and were electroporated. These cells were plated out on agar plates with the correct AntiBiotic and grown overnight (37°C). From these, a 5ml liquid culture supplied with AB was prepared and also grown overnight at 37°C. Subsequently 1:100th was transfered to a new liquid culture containing AB and tyrosine (5mg/L or 50 mg/L) for the first 3 enzymes(Sam8, Sam5 and ComT, ), Ferulic acid (5mg/L or 50mg/L) for the second 2 enzymes (ech and fcs, ) And vanillin (5mg/L or 50 mg/L) to determine the degradation or transformation of vanillin by E.Coli. After certain time intervals ranging between 30 minutes and many hours samples (1-5ml) were taken and centrifuged at 13000RPM for 1 minute. The supernatant was then transfered to a new tube and placed in -20°C until it was ready for analysis. Analysis was done on HPLC (High pressure liquid chromatography) using a standard LB culture for blanco and the measurements from the experiments.

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