Part:BBa_K4012012

CrCPR(Catharanthus roseus NADPH--cytochrome P450 reductase sequence)

CrCPR is a coding sequence that codes for NADPH--cytochrome P450 reductase, an oxidoreductase which has FAD, Flavoprotein, FMN, NADP as ligand in Catharanthus roseus. In nature, the enzyme is This enzyme is required for electron transfer from NADP to cytochrome P450 in microsomes. It can also provide electron transfer to heme oxygenase and cytochrome B5. Involved in ergosterol biosynthesis. It also has NADPH-dependent ferrireductase activity on the plasma membrane. In term of its catalytic activity, NADPH + 2 oxidized [cytochrome P450] = H+ + NADP+ + 2 reduced cytochrome P450 In our project, the sequence is used to amplify the expression of the sequences in lower course.

Figure 1: [1] The schematic of catalytic activity

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 211
  Illegal BglII site found at 946
  Illegal BglII site found at 994
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

CrCPR in Level1 plasmid assembly

Figure 2: [2] A: the construction strategy; B: results of colony PCR; C: results of sequencing analysis of coding sequence CrCPR

The construction schematic of CrCPR sequence demonstrated in Fig.8. The initiation of the CrCPR sequence is done by promoter pGAL1, with termination done by tTDH1. The sequence ConLS and ConR1 are connector sequences within the Level 1 plasmid assembly. Furthermore, type9-KVF and type9-VR are imposed to enact selection through colonies PCR, results shown in Fig.2 The band length 3327bp match with expectations. The sequence analysis results show no significant mutations or deletions, representing the success of the assembly.

CrCPR in Level2 plasmid assembly

Figure 3: [3] A: The results of colony PCR of upstream transformant; B: The results of colony PCR of downstream transformant; C: Construction of catechin metabolic pathway; D：Construction of naringenin metabolic pathway

GbCHS is successfully inserted into the vector type9 C-mTurquoise-Ura.