Part:BBa_K4012002

pGAL1

It is a lactose induced promoter, which is used to activate the CrCPR sequnece in recombined plasmids.

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 547
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 163
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 3
  Illegal BsaI.rc site found at 557
  

Obtaining the pGAL1 fragment and BsaI digested verification

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

We construct pGAL1 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 pGAL1(542bp) matched with previous expectations by compared with Marker MK8000 ladder, confirming the successful assembly of Toolkit plasmids and availability in subsequent construction.

pTEF1 in Level1 plasmid assembly

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

The construction schematic of CrCPR sequence demonstrated as Fig.2. The initiation of the CrCPR sequence is done by promoter pGAL1, with termination done by tTDH1. The sequence ConLS and ConR1 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.7 The band length 3300bp match with expectations. The sequence analysis results show no significant mutations or deletions, representing the success of the assembly.

pTEF1 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

pGAL1 is also involved in assembly of synthesis pathway of naringenin, shown by Fig.3.