Part:BBa_K4012016

MsCHI

MsCHI is the coding sequence that codes for Medicago sativa chalcone isomerase in Medicago sativa. It is the Isomerase involved in the flavonoid biosynthesis. In nature, it has highest expression in young root tips. Medicago sativa chalcone isomerase catalyzes the intramolecular cyclization of bicyclic chalcones into tricyclic (S)-flavanones. Responsible for the isomerization of 4,2',4',6'-tetrahydroxychalcone (also termed chalcone) into naringenin. Optimum pH is 7-8.5 with 4,2',4'-trihydroxychalcone as substrate, at 25 degrees Celsius. In terms of catalytic activity, a chalcone = a flavanone. EC:5.5.1.6 In our project, the sequence will express chalcone isomerase to convert p-Coumaroyl-CoA into naringenin.

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
  COMPATIBLE WITH RFC[1000]

MsCHI in Level1 plasmid assembly

Figure 2: [1] A: the construction strategy; B: results of colony PCR; C: results of sequencing analysis of coding sequence GbCHS

The construction schematic of MsCHI sequence demonstrated in Fig.7. The initiation of the MsCHI sequence is done by promoter pAOX1, with termination done by tTDH1. The sequence ConL3 and ConRE 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.7 B The band length 2021bp match with expectations. The sequence analysis results show no significant mutations or deletions, representing the success of the assembly.

MsCHI in Level2 plasmid assembly

Figure 3: [2] 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

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