Difference between revisions of "Part:BBa K2368026"
 
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<h1>Introduction</h1>
 
<h1>Introduction</h1>
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<partinfo>BBa_K2368026 short</partinfo>
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<p style="text-align: center"><partinfo>BBa_K2368026 short</partinfo></p>
 
<h3>General</h3>
 
<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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<p>The original <i>P<sub>fus</sub></i>  (BBa_K1154001) is 201bp long with three Ste12 binding sites, and the Ste12 is the transcriptional activator of <i>P<sub>fus</sub></i> in the endogenous GPCR pathways of yeast.</p>
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<img src="http://prts.igem.org/wiki/images/5/5d/T_BIT-China_2017part_K2368026.png">
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[[File:T_BIT-China_2017part_K2368026.png|center|500px|默认文字]]
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<p style="text-align: center">Fig.1 The sequence of original <i>P<sub>fus</sub></i></p>
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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>File:T-BIT-China-2017yhy-122.jpeg
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[[File:T-BIT-China-2017yhy-122.jpeg|center|500px|默认文字]]
  
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<p>fig.1 The sequence of original Pfus</p>
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<p style="text-align: center">Fig.2 Sequencing result of original <i>P<sub>fus</sub></i> and mutant <i>P<sub>fus</sub></i></p>
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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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<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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<p>And we add 5umol/L α pheromone to detect the modified promoter, and the result is shown in fig.2.</p>
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[[File:T-BIT-China-2017part-33.png|center|500px|默认文字]]
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<p>http://prts.igem.org/wiki/images/c/ca/T_BIT-China_2017part2_K2368026.png</p>
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<p style="text-align: center">Fig.3 The <i>mRFP</i> intensity of two kinds of promoters</p>
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<p>fig.2 Sequencing result of original Pfus and mutant Pfus</p>
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<p>After the modified promoter assembling into the detection circuit(加个链接), we added 5umol/L α pheromone to detect it, and the result is shown in fig.3.</p>
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<p>http://prts.igem.org/wiki/images/8/8c/T_BIT-China_2017part3_K2368026.png</p>
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<p>fig.3 The 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 <i>P<sub>fus</sub></i>, the steric hindrance of Ste12 may increases and Ste12 couldn’t combine the promoter as smoothly as before. </p>
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<p>The intensity of modified promoter is about 1/3 of the wild type, which doesn’t match with our expectation. According to Ting-Cheng Su’s[1] result, there are serious limitations in STE12 multimers identifying STE12 binding sites. So when we change the structure of Pfus, the steric hindrance of STE12 may increases and STE12 can't combine the promoter as smoothly as before. </p>
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<p>But from another point of view, still, we got the new <i>P<sub>fus</sub></i> 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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<p>But from another point of view, still, we get the new Pfus with different transcription initiation activity.</p>
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<h3>Reference</h3>
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[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
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<p>This part is an improvement of [https://parts.igem.org/Part:BBa_K1154001 BBa_K1154001] of 2013 RHIT team.</p>
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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 <i>Saccharomyces cerevisiae</i>. FEBS Journal, 277: 3235–3248. doi:10.1111/j.1742-4658.2010.07728.x </p>
  
  
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<partinfo>BBa_K2368026 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2368026 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_K2368026

Latest revision as of 16:24, 28 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 the transcriptional activator of Pfus in the endogenous GPCR pathways of yeast.



默认文字

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.

File:T-BIT-China-2017yhy-122.jpeg
默认文字

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.

默认文字

Fig.3 The mRFP 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]