Difference between revisions of "Part:BBa K2368029"
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<h1>Introduction</h1> | <h1>Introduction</h1> | ||
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<h3>General</h3> | <h3>General</h3> | ||
| − | <p>The original <i>P<sub>fus</sub></i> (BBa_K1154001) is 201bp long with three Ste12 binding sites, and the Ste12 is <i>P<sub>fus</sub></i> | + | <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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[[File:T_BIT-China_2017part_K2368026.png|center|500px|默认文字]] | [[File:T_BIT-China_2017part_K2368026.png|center|500px|默认文字]] | ||
<p style="text-align: center">Fig.1 The sequence of original <i>P<sub>fus</sub></i></p> | <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 decrease the transcription initiation activity of the promoter, we used the method of one-step mutation to remove a binding sites. (from tgaaaca to ggacac)</p> | <p>In order to decrease the transcription initiation activity of the promoter, we used the method of one-step mutation to remove a binding sites. (from tgaaaca to ggacac)</p> | ||
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[[File:T-BIT-China-2017part-22.png|center|500px|默认文字]] | [[File:T-BIT-China-2017part-22.png|center|500px|默认文字]] | ||
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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> | <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> | ||
<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> | <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> | ||
| − | [[File: | + | [[File:T-BIT-China-2017part-33.png|center|500px|默认文字]] |
<p style="text-align: center">Fig.3 The <i>mRFP</i> intensity of two kinds of promoters</p> | <p style="text-align: center">Fig.3 The <i>mRFP</i> intensity of two kinds of promoters</p> | ||
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| − | <p>The intensity of modified promoter was about 35% of the wild type, which did match with our expectation. We got the new | + | <p>The intensity of modified promoter was about 35% of the wild type, which did match with our expectation. 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 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> | <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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<h2>Sequence and Features</h2> | <h2>Sequence and Features</h2> | ||
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<!-- Uncomment this to enable Functional Parameter display | <!-- Uncomment this to enable Functional Parameter display | ||
===Functional Parameters=== | ===Functional Parameters=== | ||
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Latest revision as of 19:29, 26 October 2017
Introduction
Pfus(2 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.
Fig.1 The sequence of original Pfus
In order to decrease the transcription initiation activity of the promoter, we used the method of one-step mutation to remove a binding sites. (from tgaaaca to ggacac)
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.
Fig.3 The mRFP intensity of two kinds of promoters
The intensity of modified promoter was about 35% of the wild type, which did match with our expectation. 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.
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]