Difference between revisions of "Part:BBa K2368026"

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__NOTOC__
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<h1>Introduction</h1>
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<partinfo>BBa_K2368026 short</partinfo>
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<h3>General</h3>
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<p>The original Pfus (BBa_K1154001) is 201bp long with three STE12 binding sites, and the STE12 is Pfus’s transcriptional activator in the endogenous GPCR pathways of yeast.</p>
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[[File:T_BIT-China_2017part_K2368026.png|945px|加载失败时候的说明文字]]
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<p style="text-align: center">fig.1 The sequence of original Pfus</p>
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<br />
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<br />
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<p>In order to enhance the transcription initiation activity of the promoter, we add additional one binding site(gatgaaacaa) in the front of the promoter.</p>
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[[File:T_BIT-China_2017part_K2368027-1.png|945px|加载失败时候的说明文字]]
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<p style="text-align: center">fig.2 Sequencing result of original Pfus and mutant Pfus</p>
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<p>After the modified promoter assembling into the [http://2017.igem.org/Team:BIT-China/Project/#Detection detection circuit], we added 5umol/L α pheromone to detect it, and the result is shown in fig.3.</p>
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[[File:T_BIT-China_2017part_K2368027666.png|945px|加载失败时候的说明文字]]
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<p style="text-align: center">fig.3 The RFP intensity of two kinds of promoters</p>
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<p>The intensity of modified promoter was about 1/3 of the wild type, which didn’t match with our expectation. According to Ting-Cheng Su’s[1] result, there were serious limitations in Ste12 multimers identifying Ste12 binding sites. So when we changed the structure of Pfus, the steric hindrance of Ste12 may increases and Ste12 couldn’t combine the promoter as smoothly as before. </p>
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<br />
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<p>But from another point of view, still, we got the new Pfus with different transcription initiation activity. With different promoters, we can apply them in the regulation subsystem according to various situations or wide-ranging needs.</p>
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<br />
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<br />
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<p>This part is an improvement of BBa_K1154001 of 2013 RHIT team.</p>
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<br />
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<br />
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<br />
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<br />
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<br />
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<p>[1] Su, T.-C., Tamarkina, E. and Sadowski, I. (2010), Organizational constraints on Ste12 cis-elements for a pheromone response in Saccharomyces cerevisiae. FEBS Journal, 277: 3235–3248. doi:10.1111/j.1742-4658.2010.07728.x </p>
  
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<!-- Add more about the biology of this part here
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===Usage and Biology===
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<h2>Sequence and Features</h2>
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<partinfo>BBa_K2368027 SequenceAndFeatures</partinfo>
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<!-- Uncomment this to enable Functional Parameter display
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===Functional Parameters===
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<partinfo>BBa_K2368027

Revision as of 03:34, 24 October 2017

Introduction

Pfus(4 Ste12 binding sites)

General

The original Pfus (BBa_K1154001) is 201bp long with three STE12 binding sites, and the STE12 is Pfus’s transcriptional activator in the endogenous GPCR pathways of yeast.



加载失败时候的说明文字

fig.1 The sequence of original Pfus



In order to enhance the transcription initiation activity of the promoter, we add additional one binding site(gatgaaacaa) in the front of the promoter.

加载失败时候的说明文字

fig.2 Sequencing result of original Pfus and mutant Pfus

After the modified promoter assembling into the [http://2017.igem.org/Team:BIT-China/Project/#Detection detection circuit], we added 5umol/L α pheromone to detect it, and the result is shown in fig.3.

加载失败时候的说明文字

fig.3 The RFP intensity of two kinds of promoters




The intensity of modified promoter was about 1/3 of the wild type, which didn’t match with our expectation. According to Ting-Cheng Su’s[1] result, there were serious limitations in Ste12 multimers identifying Ste12 binding sites. So when we changed the structure of Pfus, the steric hindrance of Ste12 may increases and Ste12 couldn’t combine the promoter as smoothly as before.


But from another point of view, still, we got the new Pfus with different transcription initiation activity. With different promoters, we can apply them in the regulation subsystem according to various situations or wide-ranging needs.



This part is an improvement of BBa_K1154001 of 2013 RHIT team.






[1] Su, T.-C., Tamarkina, E. and Sadowski, I. (2010), Organizational constraints on Ste12 cis-elements for a pheromone response in Saccharomyces cerevisiae. FEBS Journal, 277: 3235–3248. doi:10.1111/j.1742-4658.2010.07728.x


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]