Difference between revisions of "Part:BBa K2368026"
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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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| − | <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> | + | <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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<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> | ||
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<h2>Sequence and Features</h2> | <h2>Sequence and Features</h2> | ||
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===Functional Parameters=== | ===Functional Parameters=== | ||
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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.
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
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 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]