Difference between revisions of "Part:BBa K2368009"
 
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<h1>Introduction</h1>
 
<h1>Introduction</h1>
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<partinfo>BBa_K2368009 short</partinfo>
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<p style="text-align: center"><partinfo>BBa_K2368009 short</partinfo></p>
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<p> This part sweetness receptor T1R2 fusion with the His tag, just like the picture showed below. </p>
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[[File:T-BIT-China-2017yhy-111.png|center|500px|默认文字]]
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<p style="text-align: center">Fig. 1 The schematic diagram of T1R2-HIS</p>
  
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In order to confirm the expression and location of sweetness receptor, T1R2 and T1R3, we decide to construct T1R2-6*His tag. This tag can be recognized by specific monoclonal antibodies then we use fluorescence second antibody to interact with monoclonal antibodies to show the T1R2’s expression and location.
 
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Because the T1R2 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 T1R2 and His tag. And hope to show the location of T1R2 through immunofluorescence for detecting His tag.
 
  
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<h1>Design</h1>
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<p> In order to show whether the sweet receptor T1R2 are translated and located correctly, we fusion the His tag to N-terminal of T1R2. The reason fused tag to N-terminal is that the C-terminal of T1R2 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 T1R2 is located at the right position. </p>
  
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[[File:T-BIT-China-2017parts-39.png|center|500px|默认文字]]
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<p style="text-align: center">Fig. 2 The schematic diagram of making T1R2-HIS</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 HIS tag. Then, fused T1R2 with the His 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===
 
===Functional Parameters===
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<partinfo>BBa_K2368009 parameters</partinfo>
 
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Latest revision as of 16:00, 28 October 2017


Introduction

His+T1R2 overlap

This part sweetness receptor T1R2 fusion with the His tag, just like the picture showed below.

默认文字

Fig. 1 The schematic diagram of T1R2-HIS


Design

In order to show whether the sweet receptor T1R2 are translated and located correctly, we fusion the His tag to N-terminal of T1R2. The reason fused tag to N-terminal is that the C-terminal of T1R2 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 T1R2 is located at the right position.


默认文字

Fig. 2 The schematic diagram of making T1R2-HIS

Experiment

At the beginning, we construct the specific primer that consists of the gene sequence of HIS tag. Then, fused T1R2 with the His 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]