Part:BBa_K2368006

Introduction

Signal reporter device (P_fus-mRFP-CYC1t)

General

To measure the sweetness of sweeteners, we designed the detection circuit. This composite part is based on the Saccharomyces cerevisiae pheromone signaling pathway. It contains a yeast induced promoter P_fus , a mRFP reporter, and a yeast terminator. In CEN.PK2-1C (a mating type yeast) P_fus is activated by α pheromone, thereby initiating the expression of the reporter gene.

Fig.1 The detection circuit

We constructed P_fus-mRFP-cyc1t-PRS42K recombinant plasmid, and transformed it into CEN.PK2-1C (a type).

Fig.2 The result of colony PCR

Cells transformed with the signal reporter device were cultured to the exponential phase and treated with α pheromone.

We added 5umol/L α pheromone into the transformed yeast. And after 4 hours, it was fluorescent.

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Fig.3 Images of yeast fluorescence. a: Initial fluorescence image; b: Fluorescence image after 4 hours.

In order to characterize the function of detection circuit, we measured absorbance and fluorescence of the cells in the plate reader after treating them with varying concentrations of α pheromone.

Fig.4 mRFP intensity of CEN.PK2-1C induced by α factor

As shown in this figure, adding higher concentration of α pheromone, the fluorescence intensity of the yeast increases. After 20 hours, α pheromone gradually failed, and the fluorescence intensity also decreased.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 993
  Illegal AgeI site found at 1105
  
• 1000
  COMPATIBLE WITH RFC[1000]