Part:BBa_K2368008
Introduction
PGal 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 PGal1/PGal10+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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[2] Neumoin A, Cohen L S, Arshava B, et al. Biophysical Journal, 2009, 96(8):3187-3196.
[3] Bohlmann L, Tredwell G D, Yu X, et al. Nature Chemical Biology, 2015, 11(12):955.
[4] Dubois G E. Molecular mechanism of sweetness sensation.[J]. Physiology & Behavior, 2016, 164(Pt B):453.
[5] Kofahl B, Klipp E. Modelling the dynamics of the yeast pheromone pathway.[J]. Yeast, 2004, 21(10):831.
[6] Richardson, Kathryn. Mechanisms of GPCR signal regulation in fission yeast[J]. University of Warwick, 2014.
[7] Nie Y, Vigues S, Hobbs J R, et al. Distinct contributions of T1R2 and T1R3 taste receptor subunits to the detection of sweet stimuli.[J]. Current Biology Cb, 2005, 15(21):1948-52.
[8] Audet M, Bouvier M. Restructuring G-Protein- Coupled Receptor Activation[J]. Cell, 2012, 151(1):14-23.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 563
Illegal BglII site found at 2466
Illegal BglII site found at 2490
Illegal BglII site found at 2991
Illegal BglII site found at 3072
Illegal BamHI site found at 674 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 291
Illegal AgeI site found at 747
Illegal AgeI site found at 1149
Illegal AgeI site found at 2751
Illegal AgeI site found at 3351 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2455
Illegal BsaI.rc site found at 3185
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