Difference between revisions of "Part:BBa K2368011"
 
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<h1>Introduction</h1>
 
<h1>Introduction</h1>
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<partinfo>BBa_K2368011 short</partinfo>
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<p style="text-align: center"><partinfo>BBa_K2368011 short</partinfo></p>
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<p> This part sweetness receptor T1R3 fusion with the Flag tag, just like the picture showed below. </p>
  
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<p>In order to confirm the expression and location of sweetness receptor, T1R2 and T1R3, we decide to construct T1R3-flag tag. This tag can be recognized by specific monoclonal antibodies then we use fluorescence second antibody to interact with monoclonal antibodies to show the T1R3’s expression and location.</p>
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[[File:T-BIT-China-2017parts-45.png|center|500px|默认文字]]
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<p>Because the T1R3 is belonged to C-class family G protein coupling receptor (GPCR) and the carboxyl terminal of GPCR interacts with the G protein αsubunit ,which is a crucial factor during signal deliver through G protein signal pathway, we perform the fusion between the N-terminal of T1R3 and flag tag. And hope to show the location of T1R3 through immunofluorescence for detecting flag tag.</p>
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<p style="text-align: center">Fig. 1 The schematic diagram of T1R3-Flag</p>
  
  
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<h1>Design</h1>
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<p> In order to show whether the sweet receptor T1R3 are translated and located correctly, we fusion the Flag tag to N-terminal of T1R3. The reason fused tag to N-terminal is that the C-terminal of T1R3 interacts with the G protein α subunit, which is a crucial factor during signal deliver through G protein signal pathway. And the N-terminal is outside the cell, so it can be detected without killing it, using immunofluorescence can figure out whether T1R3 is located at the right position. </p>
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[[File:T-BIT-China-2017parts-46.png|center|500px|默认文字]]
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<p style="text-align: center"><span>Fig. 2 The schematic diagram of making T1R3-Flag</span></p>
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<h1>Experiment</h1>
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<p> At the beginning, we construct the specific primer that consists of the gene sequence of Flag tag. Then, fused T1R3 with the Flag using PCR. The length of sequence is 50bp. </p>
 
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===Usage and Biology===
 
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<h2>Sequence and Features</h2>
 
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===Functional Parameters===
 
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<partinfo>BBa_K2368011 parameters</partinfo>
 
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Latest revision as of 16:00, 28 October 2017


Introduction

Flag+T1R3 overlap

This part sweetness receptor T1R3 fusion with the Flag tag, just like the picture showed below.

默认文字

Fig. 1 The schematic diagram of T1R3-Flag


Design

In order to show whether the sweet receptor T1R3 are translated and located correctly, we fusion the Flag tag to N-terminal of T1R3. The reason fused tag to N-terminal is that the C-terminal of T1R3 interacts with the G protein α subunit, which is a crucial factor during signal deliver through G protein signal pathway. And the N-terminal is outside the cell, so it can be detected without killing it, using immunofluorescence can figure out whether T1R3 is located at the right position.


默认文字

Fig. 2 The schematic diagram of making T1R3-Flag

Experiment

At the beginning, we construct the specific primer that consists of the gene sequence of Flag tag. Then, fused T1R3 with the Flag using PCR. The length of sequence is 50bp.

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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]