Part:BBa_K2407000

Shortened CUP1 promoter without RFC sites

Design

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This is a shortened CUP1 promoter, improved from BBa_K2165004 (BBa_K2165004). This promoter is designed to be deleted with a few bases on the two ends. We retained 5 ACE1/CUP1 binding sites, 2 TATA boxes, and one initiation element in the promoter. The complex of ACE1 and copper ions will bind the promoter, which causes the activation of CUP1 promoter with TATA boxes' help. In this way, this promoter played its key role with less bases.

Fig 1.Structure of redesigned CUP1 promoter used in our project, based on BBa_K2165004

Characterization

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Strains of S. cerevisiae BY4742 containing either BBa_K2165004-yEmRFP and BBa_K2407000-yEmRFP with an initial OD₆₀₀ of 0.1 were grown for 24 hours in SC-URA medium at 30 degrees Celsius, and then were induced with 0.1 mM Cu²⁺. Samples were tested with fluorescent microplate reader after 1, 3, 6, 12, and 24 hours.

Fig 2.The characterization of both BBa_K2165004 and BBa_K2407000

Figure 2 shows the expression of yEmRFP with both the two promoters were very similar, so we can tell the deletion didn’t influence the core function of CUP1 promoter. Based on the new part, we carried out a further improvement.

Further Improvement

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The technology of Error-Prone PCR was taken into our experiment to reduce the leakage expression and increase the response rage. Visist BBa_K2407013, BBa_K2407014, BBa_K2407015, and BBa_K2407016.

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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]