Difference between revisions of "Part:BBa K5143027"
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<p> | <p> | ||
Our team has nicknamed this plasmid “<b>Venus/Ruby plasmid</b>”. Venus/Ruby plasmid is composed of the composite part Venus <a href="https://parts.igem.org/Part:BBa_K5143026" target="_blank">BBa_K5143026</a>, the composite part mruby2 <a href="https://parts.igem.org/Part:BBa_K5143021" target="_blank">BBa_K5143021</a> and the pUC57 backbone <a href="https://parts.igem.org/Part:BBa_K5143005" target="_blank">BBa_K5143005</a>. <br> | Our team has nicknamed this plasmid “<b>Venus/Ruby plasmid</b>”. Venus/Ruby plasmid is composed of the composite part Venus <a href="https://parts.igem.org/Part:BBa_K5143026" target="_blank">BBa_K5143026</a>, the composite part mruby2 <a href="https://parts.igem.org/Part:BBa_K5143021" target="_blank">BBa_K5143021</a> and the pUC57 backbone <a href="https://parts.igem.org/Part:BBa_K5143005" target="_blank">BBa_K5143005</a>. <br> | ||
| − | <b>Venus/Ruby plasmid</b> has been digest by XhoI restriction enzyme and the Venus/Ruby fragment has been transformed into the BY4741 <i> S. cerevisiae </i> strain. | + | <b>Venus/Ruby plasmid</b> has been digest by XhoI restriction enzyme and then the Venus/Ruby fragment formed has been transformed into the BY4741 <i> S. cerevisiae </i> strain. Next, this fragment has recombinated with BY4741 <i> S. cerevisiae </i> genome at the URA3 locus. This new strain has been tested for the expression of Venus and mRuby2 under the control of GAP promoter and ADH1 promoter respectively, by measuring fluorescence. It has also been tested for the secretion of the two different protein thanks to the alphafactor secretion signal peptide and the AGA2 pre signal peptide. <br> |
| − | These tests are essential to know in our project if the | + | These tests are essential to know in our project if the GAP promoter works in our final construct <a href="https://parts.igem.org/Part:BBa_K5143025" target="_blank">BBa_K5143025</a> and if the fused peptides can be secreted thanks to the alphafactor. |
</p> | </p> | ||
<div class="image-container"> | <div class="image-container"> | ||
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<h1>Construction</h1> | <h1>Construction</h1> | ||
<p> | <p> | ||
| − | + | The Venus/Ruby fragment sequence was optimised for the expression and the synthesis in <i> S. cerevisiae </i>. Next, this fragment has been synthesized as for pUC57 backbone. Then, a TLTC cloning with the Venus/Ruby fragment and the linearised pUC57 backbone has been performed in order to build <b>Venus/Ruby plasmid</b>. | |
</p> | </p> | ||
<h1>References</h1> | <h1>References</h1> | ||
<p> | <p> | ||
| − | + | 1. Lam, A. et al. Improving FRET dynamic range with bright green and red fluorescent proteins. Nat Methods 9, 1005–1012 (2012).<br> | |
| + | 2. Lee, M. E., DeLoache, W. C., Cervantes, B. & Dueber, J. E. A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synth. Biol. 4, 975–986 (2015).<br> | ||
| + | 3. Qin, X. et al. GAP Promoter Library for Fine-Tuning of Gene Expression in Pichia pastoris. Appl Environ Microbiol 77, 3600–3608 (2011). | ||
Revision as of 09:20, 28 September 2024
pUC57-pGAP-AF_V1-Venus-ENO1_term-p_ADH1-AGA2-mRuby2-6xHis-TDH1_term
Description
Our team has nicknamed this plasmid “Venus/Ruby plasmid”. Venus/Ruby plasmid is composed of the composite part Venus BBa_K5143026, the composite part mruby2 BBa_K5143021 and the pUC57 backbone BBa_K5143005.
Venus/Ruby plasmid has been digest by XhoI restriction enzyme and then the Venus/Ruby fragment formed has been transformed into the BY4741 S. cerevisiae strain. Next, this fragment has recombinated with BY4741 S. cerevisiae genome at the URA3 locus. This new strain has been tested for the expression of Venus and mRuby2 under the control of GAP promoter and ADH1 promoter respectively, by measuring fluorescence. It has also been tested for the secretion of the two different protein thanks to the alphafactor secretion signal peptide and the AGA2 pre signal peptide.
These tests are essential to know in our project if the GAP promoter works in our final construct BBa_K5143025 and if the fused peptides can be secreted thanks to the alphafactor.
Construction
The Venus/Ruby fragment sequence was optimised for the expression and the synthesis in S. cerevisiae . Next, this fragment has been synthesized as for pUC57 backbone. Then, a TLTC cloning with the Venus/Ruby fragment and the linearised pUC57 backbone has been performed in order to build Venus/Ruby plasmid.
References
1. Lam, A. et al. Improving FRET dynamic range with bright green and red fluorescent proteins. Nat Methods 9, 1005–1012 (2012).
2. Lee, M. E., DeLoache, W. C., Cervantes, B. & Dueber, J. E. A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synth. Biol. 4, 975–986 (2015).
3. Qin, X. et al. GAP Promoter Library for Fine-Tuning of Gene Expression in Pichia pastoris. Appl Environ Microbiol 77, 3600–3608 (2011).
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal suffix found in sequence at 4360
Illegal EcoRI site found at 7799
Illegal EcoRI site found at 8326 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 7799
Illegal EcoRI site found at 8326
Illegal SpeI site found at 4361
Illegal PstI site found at 4375
Illegal NotI site found at 4368
Illegal NotI site found at 8332 - 21INCOMPATIBLE WITH RFC[21]Unknown
- 23INCOMPATIBLE WITH RFC[23]Illegal suffix found in sequence at 4361
Illegal EcoRI site found at 7799
Illegal EcoRI site found at 8326 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found in sequence at 8326
Illegal EcoRI site found at 7799
Illegal SpeI site found at 4361
Illegal PstI site found at 4375
Illegal NgoMIV site found at 2077 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2727
Illegal SapI.rc site found at 5073