Part:BBa_K4012015

GhF3'H

GhF3’H is a sequence codes for Flavonoid 3'-monooxygenase, which catalyzes the 3'-hydroxylation of the flavonoid B-ring to the 3',4'-hydroxylated state, converting naringenin to eriodictyol and dihydrokaempferol to dihydroquercetin. Flavonoid 3'-monooxygenase is involved in Flavonoid biosynthesis, using Heme, Iron, Metal-binding, NADP. The catalytic activity: a 3'-unsubstituted flavone + O2 + reduced [NADPH—hemoprotein reductase] = a 3'-hydroxyflavone + H+ + H2O + oxidized [NADPH—hemoprotein reductase]

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 BamHI site found at 1200
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

GhF3'H in Level1 plasmid assembly

Figure 2: [2] A: the construction strategy; B: results of colony PCR; C: results of sequencing analysis of coding sequence F3'H

The construction schematic of GhF3’H sequence demonstrated in Fig.10. The initiation of the GnF3’H sequence is done by promoter pPGK1, with termination done by tADH1. The sequence ConL2 and ConR3 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 B The band length 2892bp match with expectations. The sequence analysis results show no significant mutations or deletions, representing the success of the assembly.

GhF3;H 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

GhF3'H is successfully inserted into the vector type9 C-mTurquoise-Ura.