Difference between revisions of "Part:BBa K5508007"

 
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K5508007 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5508007 SequenceAndFeatures</partinfo>
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==Results==
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===(1)Plasmid Construction===
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The construction process used the following primers mCherry-Gibson-F (CACTATAGGGCCCGGGCGTCGACatggtgagcaagggcgaggag), and mCherry-Gibson-R (GCTAGCCGCGGTACCAAGCTTttacttgtacagctcgtcc) to amplify the mCherry gene fragments, which were later assembled using Gibson. SalI and HindIII were used to linearise the carrier. The vectors were linearised with SalI and HindIII, followed by Gibson assembly to ligate the vectors to the target genes.
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<html>
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<style>
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    .bild {max-width: 35% ; height: auto;}
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</style>
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<p>
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  <img class="bild" src="https://static.igem.wiki/teams/5508/hangzhoumedx-parts/23.png">
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  <div class="unterschrift"><b> Electrophoretic detection of PCR results</b>
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  </div>
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</p>
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</html>
  
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<html>
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<style>
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    .bild {max-width: 35% ; height: auto;}
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</style>
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<p>
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  <img class="bild" src="https://static.igem.wiki/teams/5508/hangzhoumedx-parts/24.png">
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  <div class="unterschrift"><b> Results of E. coli plasmid transformation</b>
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  </div>
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</p>
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</html>
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<html>
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<style>
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    .bild {max-width: 35% ; height: auto;}
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</style>
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<p>
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  <img class="bild" src="https://static.igem.wiki/teams/5508/hangzhoumedx-parts/25.png">
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  <div class="unterschrift"><b> Sequencing results</b>
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  </div>
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</p>
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</html>
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===(2)Fluorescence detection===
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After the correctness of the recombinant plasmid was verified, it was transferred into BY4741 for culture. After 48 hours of adding the inducer galactose, the results are shown. The bacterial fluid appeared a light pink color, proving that our expression system was successful.
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<html>
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<style>
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    .bild {max-width: 35% ; height: auto;}
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</style>
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<p>
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  <img class="bild" src="https://static.igem.wiki/teams/5508/hangzhoumedx-parts/26.png">
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  <div class="unterschrift"><b> Results of plasmid transformation in Saccharomyces cerevisiae </b>
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  </div>
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</p>
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</html>
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<html>
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<style>
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    .bild {max-width: 35% ; height: auto;}
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</style>
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<p>
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  <img class="bild" src="https://static.igem.wiki/teams/5508/hangzhoumedx-parts/27.png">
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  <div class="unterschrift"><b> Expression of fluorescent protein</b>
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  </div>
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</p>
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</html>
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 06:58, 1 October 2024


Gal1,10-mCherry-CYC1

The fluorescent protein gene BBa_K4335004 (mCherry) was placed under the control of the BBa_K5477005 promoter (Gal1, 10), and its transcription was terminated by BBa_K3384311(CYC1) to validate the availability of theSaccharomyces cerevisiae's protein expression system.

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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 374
  • 1000
    COMPATIBLE WITH RFC[1000]

Results

(1)Plasmid Construction

The construction process used the following primers mCherry-Gibson-F (CACTATAGGGCCCGGGCGTCGACatggtgagcaagggcgaggag), and mCherry-Gibson-R (GCTAGCCGCGGTACCAAGCTTttacttgtacagctcgtcc) to amplify the mCherry gene fragments, which were later assembled using Gibson. SalI and HindIII were used to linearise the carrier. The vectors were linearised with SalI and HindIII, followed by Gibson assembly to ligate the vectors to the target genes.

Electrophoretic detection of PCR results

Results of E. coli plasmid transformation

Sequencing results

(2)Fluorescence detection

After the correctness of the recombinant plasmid was verified, it was transferred into BY4741 for culture. After 48 hours of adding the inducer galactose, the results are shown. The bacterial fluid appeared a light pink color, proving that our expression system was successful.

Results of plasmid transformation in Saccharomyces cerevisiae

Expression of fluorescent protein