Part:BBa_K5184020

pTDH3-ShZPO-tTDH1-pPGK1-AtCPR1-tPGK1

In order to achieve enhanced effects, we aimed to produce 9HZ and 9H10epoZ also, which have better repellent effects than 7epiZ. Due to difficulties of expressing the oxidase and reductases in E. coli, we decide to change the chassis to S. cerevisiae. This part presents the collection of promoters, genes coding for the oxidase and reductase and terminator. This enzyme collection may provide future iGEM teams with an insight into the expression of an oxidase and a reductase in S. cerevisiae.

Abstract

ShZPO is a cytochrome P450 oxygenase found in Solanum habrochaites. It carries out two successive oxidations to generate two sesquiterpenes from a monocyclic sesquiterpene as the susbtrate. AtCPR1 is a cytochrome P450 reductase found in Arabidopsis thaliana. AtCPR1 is co-localized with ShZPO on the ER membrane, ensuring efficient electron transfer. We used pTDH3 and tTDH3 for the expression of ShZPO; pPGK1 and tPGK1 for the expression of AtCPR1.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 3525
  Illegal XhoI site found at 2177
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2423
  Illegal BsaI.rc site found at 3535
  

Usage and Biology

ShZPO is a cytochrome P450 oxygenase found in Solanum habrochaites. It carries out two successive oxidations to generate two sesquiterpenes from a monocyclic sesquiterpene as the susbtrate. AtCPR1 is a cytochrome P450 reductase found in Arabidopsis thaliana. It contains two domains, one with binding sites for FAD, flavin adenine dinucleotide, and NADPH, nicotinamide adenine dinucleotide; the other with binding site for FMN, flavin mononucleotide. AtCPR1 functions through transferring electrons to ShZPO.