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     <title>Protein Description</title>
 
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         <img src="https://static.igem.wiki/teams/5143/bba-k5143021-mruby-microscpoe.png" width="400" alt="Test of the Promoter activation">
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         <img src="https://static.igem.wiki/teams/5143/bba-k5143021-mruby-microscope.png" width="400" alt="Test of the Promoter activation">
 
         <figcaption><i><u>Figure 3</u>: Test of the promoter activation by measuring the fluorescence</i></figcaption>
 
         <figcaption><i><u>Figure 3</u>: Test of the promoter activation by measuring the fluorescence</i></figcaption>
 
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     <h1>Construction</h1>
 
     <h1>Construction</h1>

Revision as of 11:31, 29 September 2024


pUC57-pGAP-AF_V1-Venus-ENO1_term-p_ADH1-AGA2-mRuby2-6xHis-TDH1_term

Protein Description

Description

Our team has nicknamed this plasmid “Venus/mRuby plasmid”. Venus/mRuby 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.

mRuby2
Figure 1: Use of mRuby2
Venus
Figure 2: Use of Venus
Test of the Promoter activation
Figure 3: Test of the promoter activation by measuring the fluorescence

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


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal suffix found in sequence at 4360
    Illegal EcoRI site found at 7799
    Illegal EcoRI site found at 8326
  • 12
    INCOMPATIBLE 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
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 7799
    Illegal EcoRI site found at 8326
    Illegal XhoI site found at 5964
    Illegal XhoI site found at 7805
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal suffix found in sequence at 4361
    Illegal EcoRI site found at 7799
    Illegal EcoRI site found at 8326
  • 25
    INCOMPATIBLE 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
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 2727
    Illegal SapI.rc site found at 5073