Team:KULeuven/Wetlab/Vanillin Receptor

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__NOTOC__
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=VANILLIN SYNTHESIS=
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=Vanillin Receptor: Planning=
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==Goal==
==Goal==
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Making vanillin from tyrosine in a five-step pathway.
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Besides producing vanillin, we would also like to control the concentration of vanillin outside of the bacteria. To do this, we need a way to detect the vanillin extracellular and create a proper response intracellular. The vanillin sensing system receptor we propose, consists of the VirA/G system normally found in the Agrobacterium Tumefaciens. VirA is a receptor that responses to monosaccharides and phenols. Hence, it is able to sense vanillin. When binding vanillin, VirA is activated and will activate in its turn VirG. VirG is a transcription activator that will bind to a Vir box sequence in order to activate gene expression. This Vir box sequence can be found in the VirB promoter region. RpoA is a alfa subunit polymerase that is needed to help VirG work in E. coli. For extra literature see: Y.W Lee, S.Jin, W.S.Sim and E.W.Nester, ''Genetic evidence for direct sensing of phenolic compounds by the vira protein of agrobacterium tumefaciens''. Proc. Natl. Acad. Sci. U.S.A, 1995 december 19; 92(26): 12245–12249.
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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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Biobricks:  
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*Strains/isolated DNA of Agrobacterium tumefaciens: pTiBo542 and pTiA6NC/A1011
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*SAM8: {{kulpart|BBa_I742142}}: coding sequence without RBS
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*Biobricks:
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*SAM5: {{kulpart|BBa_K238007}}: RBS site + PstI restriction site removed
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**{{kulpart|BBa_K238008}}: VirA
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*COMT: {{kulpart|BBa_I742107}}: coding sequence without RBS
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**{{kulpart|BBa_K238009}}: VirG
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*FCS:  {{kulpart|BBa_I742115}}: RBS + fcs in pSB1A2
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**{{kulpart|BBa_K238010}}: RpoA
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*ECH: {{kulpart|BBa_I742113}}: RBS + ech in pSB1A2
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**{{kulpart|BBa_K238011}}: VirB promoter region
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*Primers:
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For VirA, VirG, RpoA, VirB promoter region and for the site directed mutagenesis to remove unwanted restriction sites in VirA (PstI 2x) and RpoA (EcoRI 2x)
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*TOPO-Vector or iGEM vector
==Where from==
==Where from==
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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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*The primers were ordered
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*The plasmids: from the lab
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*One strain was ordered and another was available in the lab
==Steps==
==Steps==
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*We will be working in three different stages
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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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#The Agrobacterium strain is plated out on agar from -80°C. From the other strain, DNA is expected to be sent.
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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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#The primers are used to perform a PCR on the bacteria or on the isolated DNA to amplify the different genes or fragments (VirA, VirG,  RpoA and virB promoter region). An agarose is performed as a control.
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#If both are tested and ok, they can be combined into one plasmid and again tested.
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#The PCR fragments are ligated in a TOPO vector and electroporated into E. coli.
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#Then, the locks need to be added.
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#The plasmids are isolated via miniprep and again verified through restriction digest and agarose gel.
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#Site directed mutagenesis (according to stratagene) is performed on RpoA and VirA to get the EcoRI and PstI restriction sites out.
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*Testing the enzymes by detecting the endproducts through gaschromatography.
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#All parts are sequenced.
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*One problem might be that the tyrosine of E. coli is depleted too fast
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#The different plasmids (consideration: put in registry as separate part) are combined into one standard iGEM plasmid, with the appropriate promoter, RBS and terminators.  
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#The system’s function is checked by linking a GFP to the virB-promoter region and subsequent induction with vanillin and Ferulic Acid
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{{Team:KULeuven/Common2/PageFooter}}

Latest revision as of 07:35, 12 October 2009

Vanillin Receptor: Planning

Goal

Besides producing vanillin, we would also like to control the concentration of vanillin outside of the bacteria. To do this, we need a way to detect the vanillin extracellular and create a proper response intracellular. The vanillin sensing system receptor we propose, consists of the VirA/G system normally found in the Agrobacterium Tumefaciens. VirA is a receptor that responses to monosaccharides and phenols. Hence, it is able to sense vanillin. When binding vanillin, VirA is activated and will activate in its turn VirG. VirG is a transcription activator that will bind to a Vir box sequence in order to activate gene expression. This Vir box sequence can be found in the VirB promoter region. RpoA is a alfa subunit polymerase that is needed to help VirG work in E. coli. For extra literature see: Y.W Lee, S.Jin, W.S.Sim and E.W.Nester, Genetic evidence for direct sensing of phenolic compounds by the vira protein of agrobacterium tumefaciens. Proc. Natl. Acad. Sci. U.S.A, 1995 december 19; 92(26): 12245–12249.

Required

  • Strains/isolated DNA of Agrobacterium tumefaciens: pTiBo542 and pTiA6NC/A1011
  • Biobricks:
    • VirA
      VirG
      RpoA
      VirB promoter region
  • Primers:

For VirA, VirG, RpoA, VirB promoter region and for the site directed mutagenesis to remove unwanted restriction sites in VirA (PstI 2x) and RpoA (EcoRI 2x)

  • TOPO-Vector or iGEM vector

Where from

  • The primers were ordered
  • The plasmids: from the lab
  • One strain was ordered and another was available in the lab

Steps

  1. The Agrobacterium strain is plated out on agar from -80°C. From the other strain, DNA is expected to be sent.
  2. The primers are used to perform a PCR on the bacteria or on the isolated DNA to amplify the different genes or fragments (VirA, VirG, RpoA and virB promoter region). An agarose is performed as a control.
  3. The PCR fragments are ligated in a TOPO vector and electroporated into E. coli.
  4. The plasmids are isolated via miniprep and again verified through restriction digest and agarose gel.
  5. Site directed mutagenesis (according to stratagene) is performed on RpoA and VirA to get the EcoRI and PstI restriction sites out.
  6. All parts are sequenced.
  7. The different plasmids (consideration: put in registry as separate part) are combined into one standard iGEM plasmid, with the appropriate promoter, RBS and terminators.
  8. The system’s function is checked by linking a GFP to the virB-promoter region and subsequent induction with vanillin and Ferulic Acid

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