Revision as of 06:34, 24 October 2017

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 Pfus, a mRFP reporter, and a yeast terminator. In CEN.PK2-1C (a mating type yeast) Pfus is activated by α pheromone, thereby initiating the expression of the reporter gene.

Fig.1 The detection circuit

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

Fig.2 The result of cPCR

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.

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 RFP 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