Revision as of 17:43, 26 October 2017

Introduction

P_{Gal 1/10}-T1R3-ADH1t

This part consists of the promoter Gal1/Gal10, sweetness receptor T1R3 and the terminator ADH1. The promoter Gal1/Gal10 is a bi-directional galactose-inducible promoter, which requires two percent of galactose to induce twelve hours to elicit protein expression. T1R3 is the sweetness receptor of the human tongue cells belonging to C type G protein coupled receptor.

Fig.1 The schematic diagram of Gal1/Gal10+T1R3+ADH1

Experiments

Part 1: Dry experiments of T1R2

1、T1R2/T1R3 receptor activation part

The part is about the sweeteners binding to the receptor and the GPCR produce the initial signal through changing structure. For T1R2/T1R3, we divide the process of inducing signal into four conditions of T1R2/T1R3 as show in Fig.2. And the reaction between different conditions of protein is used ODEs to describe it. The all ODEs are shown follow.

Fig.2 T1R2/T1R3 receptor activation reaction diagram

(T1R2/3: the T1R2/T1R3 heterodimer)

The parameters of this part are listed in the Table 1.

Table 1． The value of parameter in T1R2/T1R3 receptor activation part

The output of the T1R2/T1R3 receptor activation part is showed. (Fig.3)

Fig. 3 The output signal, activated state of T1R2/T1R3, induced by different concentration ligand

As we can see, the activated state of T1R2/T1R3 establishes clear distant under different concentration of ligand for binding. It can prove that this pathway can sense different strength of signal and the signal will decrease soon, which means our system will have less signal interfere.

2、The result of T1R3 expression

As the result of combination model established, we can see the curve of RFP intensity does not increase at first 5 hours, and then the RFP intensity increases rapidly among 5-15 hours. At the same time, the differences of each ligand concentration are displayed in the middle of culture time (around at 15 hours), which is similar to our experimental data. Finally, the RFP of all groups reduce slowly.

Fig. 4 The simulation result of RFP intensity in population level.

Part 2: Wet experiments of T1R3

Design

We construct the part by Overlap Extension PCR. And the Gal1/Gal10 promoter and the ADH1 terminator stem from the PESC-Ura plasmid.

Result

In our experiment, the T1R3 gene is used to accept the signal inputted in our whole pathway. We synthesized this part by OE PCR.

Fig.5 The electrophoresis of the positive result of T1R2. <p>Then the expression and location of the receptor are detected by immunofluorescence. The principle of immunofluorescence is that the antibody A binds to the tag, and then the fluorescent antibody B binds to the A. This is the positive result of our immunofluorescence.

Fig.6 Immunofluorescence of T1R2/T1R3 in yeast. The scale bars represent 10µm

As the result of combination model established, we can see the curve of RFP intensity does not increase at first 5 hours, and then the RFP intensity increases rapidly among 5-15 hours. At the same time, the differences of each ligand concentration are displayed in the middle of culture time (around at 15 hours), which is similar to our experimental data. Finally, the RFP of all groups reduce slowly after 15 hours.

Fig. 7 The fluorescence intensity of different sweeteners

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Sequence and Features