Part:BBa_K4012010

GbCHS

GbCHS is a sequence code for Chalcone synthase in Anthurium andraeanum. The primary product of this enzyme is 4,2',4',6'-tetrahydroxychalcone (also termed naringenin-chalcone or chalcone) which can under specific conditions spontaneously isomerize into naringenin. It is a Acyltransferase involved in Flavonoid biosynthesis. The catalytic activity: 4-coumaroyl-CoA + 2 H+ + 3 malonyl-CoA = 2',4,4',6'-tetrahydroxychalcone + 3 CO2 + 4 CoA

Figure 1: [1] The schematic of catalytic activity

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 891
  Illegal SapI.rc site found at 840
  

GbCHS in Level1 plasmid assembly

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

The construction schematic of GbCHS sequence demonstrated as Fig .2. The initiation of the GbCHS sequence is done by promoter pTEF1, with termination done by tADH1. The sequence ConL2 and ConR3 are connector sequences within the Level 1 plasmid assembly. Furthermore, typr9-KVF and type9-VR are imposed to enact selection through colonies PCR, results shown in Fig.2 The band length 2500bp match with expectations. The sequence analysis results show no significant mutations or deletions, representing the success of the assembly.

GbCHS 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.