Part:BBa_K4012019

tTDH1 It is a terminator that ends the expression of the sequence that is responsible for the production of chalcone isomerase.

Secondary Structure

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Measurement

• [http://openwetware.org/wiki/Cconboy:Terminator_Characterization/Results How these parts were measured]

Obtaining the tTDH1 fragment and BsaI digested verification

Figure 1: [1] Results of yeast toolkit plasmids enzyme-digested verification

We construct tTDH1 with vector type8-pSB1K3-GFP and proceed digested verification using BsaI. According to Fig.1, we obtain two clear bands after BsaI digested, the length of the vector(1622bp) and the inserted fragments Type-4-tTDH1(237bp) matched with previous expectations by compared with Marker MK8000 ladder, confirming the successful assembly of Toolkit plasmids and availability in subsequent construction.

tTDH1 in Level1 plasmid assembly

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

The construction schematic of MsCHI sequence demonstrated as Fig.2. 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, 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.

Figure 3: [3] 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.3. The initiation of the GbCHS sequence is done by promoter pTEF1, with termination done by tTDH1. The sequence ConL3 and ConRE 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.

tTDH1 in Level2 plasmid assembly

Figure 4: [4] 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

tTDH1 is also involved in assembly of synthesis pathway of naringenin, shown by Fig.4.