Part:BBa_K2207021

Fused promoter consisting of calcineurin-dependent response element and CYC1 promoter

CDRE，calcineurin-dependent response element，is a segment of DNA sequence which can be regulated by specific transcription factor. This TF is activated by calcineurin in the present of calcium ion. To create a promoter can be up-regulated by calcium influx，we replaced the upstream activating sequence（UAS）of CYC1 promoter with 4 CDREs. This promoter is designed for Saccharomyces cerevisiae and it depends on S.cerevisiae's endogenous calcineurin and calmodulin. We choose mRFP as the report gene.We cultured the yeast in calcium-inducing medium and uninduced medium which contains a relatively low concentration of calcium ion (1xYPD medium).

Obviously, the transgenic yeast cultured in the calcium-inducing medium(200mM Ca2+) is turning red while the control shows no significant changes. Then we detected the fluorescence of this two cultures. To eliminate the influence of the concentration of the yeast, we calculated the value of Fluorescent Intensity/OD600 to evaluate these two groups.

The value indicated that the fluorescence intensity of the calcium-induced group has improved about 147%. It’s not quite a huge change and the uninduced group has already shown a great intensity of fluorescence. Then we detected the intracellular calcium ion concentration with Fluo 4-AM whose fluorescent intensity can represent the relative concentration of calcium ion.

As the result shows, 8% change of the intracellular ion concentration can contribute to 147% higher expression level of the downstream gene. It seems that our CDRE promoter is much more sensitive than we expected.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 214
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]