Part:BBa_K4012003

pPGK1

A Saccharomyces cerevisiae promoter. It is used to initiate MsCHI sequence to express

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 1105
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 3
Illegal BsaI.rc site found at 1115

Obtaining the pPGK1 fragment and BsaI digested verification

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

We construct pPGK1 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-tPGK1(238bp) matched with previous expectations by compared with Marker MK8000 ladder, confirming the successful assembly of Toolkit plasmids and availability in subsequent construction.

pPGK1 in Level1 plasmid assembly

Figure 2: [2] A: the construction strategy; B: results of colony PCR; C: results of sequencing analysis of coding sequence Pc4CL

The construction schematic of Pc4CL sequence demonstrated as Fig.2 The initiation of the Pc4CL sequence is done by promoter pPGK1, with termination done by tSSA1. The sequence ConL1 and ConR2 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.4 The band length 3000 bp match with expectations. The sequence analysis results show no significant mutations or deletions, representing the success of the assembly.

pPGK1 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

pPGK1 is also involved in assembly of synthesis pathway of naringenin, shown by Fig.3.