• Vanillin synthesis: DNA not yet complete/in library [http://partsregistry.org/wiki/index.php/Part:BBa_I742140]
  • Combination of 5 genes; 3 are available
    • Edinburgh Vanilla production (Pathway synthesis, Edinburgh iGEM 2007)
  • [http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=17437627#id538997 Vanillin production using metabolically engineered Escherichia coli under non-growing conditions], Ferulic accid --> Vanillin, 5k bp

• Biobricks for vanillin synthesis
  • Send mail to University of Tuscia [Done]
  • Order:
    • (sam8 (tyrosine-ammonia lyase) coding sequence) [ordered]
    • (sam5 (coumarate hydroxylase) coding sequence) [ordered]
    • (COMT gene with ribosome binding site) [Available in kit]

Extra info on vanillin synthesis

• http://www.ncbi.nlm.nih.gov/pubmed/16232583 1 B C Okeke and V Venturi ,Construction of recombinants Pseudomonas putida BO14 and Escherichia coli QEFCA8 for ferulic acid biotransformation to vanillin, J Biosci Bioeng. 1999; 88(1): 103–106.
  
• http://www.ncbi.nlm.nih.gov/pubmed/14602615 2 Jörg Overhage, Alexander Steinbüchel, and Horst Priefert,Highly Efficient Biotransformation of Eugenol to Ferulic Acid and Further Conversion to Vanillin in Recombinant Strains of Escherichia coli, Appl Environ Microbiol. 2003 November; 69(11): 6569–6576. doi: 10.1128/AEM.69.11.6569-6576.2003.
  
• http://www.ncbi.nlm.nih.gov/pubmed/10616715 3 J Overhage, H Priefert, J Rabenhorst, and A Steinbüchel,Biotransformation of eugenol to vanillin by a mutant of Pseudomonas sp. strain HR199 constructed by disruption of the vanillin dehydrogenase (vdh) gene, Appl Microbiol Biotechnol. 1999 November; 52(6): 820–828.
  
• http://www.labmeeting.com/paper/2776259/yoon-enhanced-vanillin-production-from-recombinant-e-coli-using-ntg-mutagenesis-and-adsorbent-resin 4 Yoon SH, Lee EG, Das A, Lee SH, Li C, Ryu HK, Choi MS, Seo WT, and Kim SW, Enhanced vanillin production from recombinant E. coli using NTG mutagenesis and adsorbent resin, Biotechnology progress 23(5):1143.
  
• http://www.scielo.br/scielo.php?pid=S1517-83822003000500037&script=sci_arttext 5 Attilio ConvertiI; Danilo de FaveriI; Patrizia PeregoI; Paolo BarghiniII; Maurizio RuzziII; Luciane SeneIII, Vanillin production by recombinant strains of Escherichia coli, Braz. J. Microbiol. vol.34 suppl.1 São Paulo Nov. 2003
  
• http://www.springerlink.com/content/bj47xnqn5kr65wvp/fulltext.pdf 6 S. Achterholt, H. Priefert, and A. Steinbüchel, Identification of Amycolatopsis sp. strain HR167 genes, involved in the bioconversion of ferulic acid to vanillin, Appl Microbiol Biotechnol. 2000 December; 54(6): 799–807
  

• Focus on the last two parts in the synthesis of vanillin since that pathway has been proven
  • Can always add additional 3 paths

• Send mail to U. Edinburgh for the proteins for vanillin synthesis [Done]
  • Received mail back from Edinburgh, biobricking of the parts should still be done. Will receive answer next week

• sam5; no part number, Pst1 site removed
• sam8; coding sequence without RBS
• COMT; coding sequence without RBS
• ech; RBS + ech, in ,
• fcs; RBS + fcs, in ,

• Modeling:
  • Searching for reaction kinetics parameters: degradation of enzymes.

• RBS + ech, in ,
• RBS + fcs, in ,

They were put in the freezer and will be incorporated into competent cells, so they can be multiplied and stored.

• RBS + ech, in ,
• RBS + fcs, in ,

The cells were then transferred to liquid LBmedium to recuperate from the procedure. After a few hours they were transferred to LB/amp plates and grown overnight at 37°C.