• Main Components
• Design
• Modeling
• Wetlab Planning
• Notebook

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

• We will be working in three different stages

1. Testing the transformation of tyrosine to ferulic acid. The enzymes are put under a constitutive promoter and transcribed. Ferulic acid concentrations are then measured with GC
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
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