Part:BBa_K4012014

DuLAR(Desmodium uncinatum Leucoanthocyanidin reductase)

DuLAR is a coding sequence that codes for leucoanthocyanidin reductase, an oxidoreductase with ligand NADP, in Desmodium uncinatum. In nature, it catalyzes the synthesis of catechin from 3,4-cis-leucocyanidin. Also synthesizes afzelechin and gallocatechin. In terms of ots catalytic activity, (2R,3S)-catechin + H2O + NADP+ = (2R,3S,4S)-leucocyanidin + H+ + NADPH In our project, the sequence is used to express leucoanthocyanidin reductase to convert leucoanthocyanidin into catechin in saccharomyces cerevisiae.

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
  COMPATIBLE WITH RFC[1000]

DuLAR in Level1 plasmid assembly

File:LAR.jpg

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 DuLAR sequence demonstrated in Fig.12. The initiation of the DuLAR sequence is done by promoter pPOP6, with termination done by tSSA1. The sequence ConL4 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.12 B The band length 2502bp match with expectations. The sequence analysis results show no significant mutations or deletions, representing the success of the assembly.

DuLAR 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

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