Difference between revisions of "Part:BBa K5269022"
 
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This sequence contains SD-sequence(BBa_K5269034), SpyTag, GH19Chitinase (BBa_K5269015) and His-Tag sequence. In addition, the BsmB I cleavage sites at both ends of the sequence allow for Golden Gate Assembly to the Golden Gate Assembly Site (BBa_K5269020) and T7 promoter-shRNA-T7 terminator-Golden Gate Assembly Site(BBa_K5269025). The SpyCatcher-SpyTag system works when used with Lpp-OmpA-SpyCatcher(BBa_K5269026) or INPNC-SpyCatcher(BBa_K5269027). Therefore, GH19Chitinase, which hydrolyzes chitin contained in the target bacterial membrane, is expected to be stably present on the Membrane Vesicle, which will facilitate the introduction of shRNA into the target fungus cells.
 
This sequence contains SD-sequence(BBa_K5269034), SpyTag, GH19Chitinase (BBa_K5269015) and His-Tag sequence. In addition, the BsmB I cleavage sites at both ends of the sequence allow for Golden Gate Assembly to the Golden Gate Assembly Site (BBa_K5269020) and T7 promoter-shRNA-T7 terminator-Golden Gate Assembly Site(BBa_K5269025). The SpyCatcher-SpyTag system works when used with Lpp-OmpA-SpyCatcher(BBa_K5269026) or INPNC-SpyCatcher(BBa_K5269027). Therefore, GH19Chitinase, which hydrolyzes chitin contained in the target bacterial membrane, is expected to be stably present on the Membrane Vesicle, which will facilitate the introduction of shRNA into the target fungus cells.
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Here are the results of SDS-PAGE of OmpA-GH19Chitinase and INP-GH19Chitinase bound by SpyCatcher-SpyTag. See our results page (https://2024.igem.wiki/tokyotech/results/) for details.
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<table border="1">
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  <thead>
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    <tr>
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      <th>Serial</th>
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      <th>Stock Name</th>
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      <th>Scaffolding Protein - Display Protein</th>
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      <th>Molecular Weight (kDa)</th>
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      <th>Corresponding SDS-PAGE Lanes (Periplasmic/Cytoplasmic/Membrane Fractions)</th>
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      <th>Presence of Bands (Periplasmic/Cytoplasmic/Membrane Fractions)</th>
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    </tr>
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  </thead>
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  <tbody>
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    <tr>
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      <td>O_1</td>
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      <td>O_Mgfp5</td>
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      <td>OmpA-Mgfp5</td>
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      <td>36.8</td>
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      <td>1/2/3</td>
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      <td>Yes/Yes/Yes</td>
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    </tr>
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    <tr>
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      <td>I_1</td>
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      <td>I_Mgfp5</td>
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      <td>INP-Mgfp5</td>
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      <td>54.2</td>
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      <td>4/5/6</td>
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      <td>Yes/Yes/Yes</td>
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    </tr>
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    <tr>
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      <td>O_2</td>
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      <td>O_Glucanases</td>
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      <td>OmpA-Glucanases</td>
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      <td>78.5</td>
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      <td>7/8/9</td>
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      <td>Yes/Yes/Yes</td>
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    </tr>
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    <tr>
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      <td>I_2</td>
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      <td>I_Glucanases</td>
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      <td>INP-Glucanases</td>
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      <td>95.9</td>
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      <td>10/11/12</td>
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      <td>Yes/Yes/Yes</td>
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    </tr>
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    <tr>
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      <td>O_3</td>
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      <td>O_Mgfp5-Glucanases</td>
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      <td>OmpA-Glucanases, OmpA-Mgfp5</td>
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      <td>78.5, 36.8</td>
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      <td>13/14/15</td>
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      <td>No/Yes/Yes, Yes/Yes/Yes</td>
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    </tr>
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    <tr>
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      <td>I_3</td>
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      <td>I_Mgfp5-Glucanases</td>
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      <td>INP-Glucanases, INP-Mgfp5</td>
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      <td>95.9, 54.2</td>
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      <td>16/17/18</td>
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      <td>No/No/No, No/Yes/Yes</td>
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    </tr>
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    <tr>
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      <td>O_4</td>
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      <td>O_Glucanases-GH19Chitinase</td>
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      <td>OmpA-Glucanases, OmpA-GH19Chitinase</td>
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      <td>78.5, 56.5</td>
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      <td>19/20/21</td>
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      <td>No/Yes/Yes, No/No/No</td>
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    </tr>
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    <tr>
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      <td>I_4</td>
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      <td>I_Glucanases-GH19Chitinase</td>
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      <td>INP-Glucanases, INP-GH19Chitinase</td>
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      <td>95.9, 73.9</td>
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      <td>22/23/24</td>
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      <td>Yes/Yes/No, No/No/No</td>
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    </tr>
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  </tbody>
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</table>
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<html>
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<img src="https://static.igem.wiki/teams/5269/parts-registry/sdspage.webp" alt="SDS-PAGE Image" style="width:80%;>
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<br>
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</html>
  
  

Latest revision as of 12:50, 2 October 2024


SpyTag-GH19Chitinase-His-Tag and BsmB I cut site(for GGA)

This sequence contains SD-sequence(BBa_K5269034), SpyTag, GH19Chitinase (BBa_K5269015) and His-Tag sequence. In addition, the BsmB I cleavage sites at both ends of the sequence allow for Golden Gate Assembly to the Golden Gate Assembly Site (BBa_K5269020) and T7 promoter-shRNA-T7 terminator-Golden Gate Assembly Site(BBa_K5269025). The SpyCatcher-SpyTag system works when used with Lpp-OmpA-SpyCatcher(BBa_K5269026) or INPNC-SpyCatcher(BBa_K5269027). Therefore, GH19Chitinase, which hydrolyzes chitin contained in the target bacterial membrane, is expected to be stably present on the Membrane Vesicle, which will facilitate the introduction of shRNA into the target fungus cells.

Here are the results of SDS-PAGE of OmpA-GH19Chitinase and INP-GH19Chitinase bound by SpyCatcher-SpyTag. See our results page (https://2024.igem.wiki/tokyotech/results/) for details.

<thead> </thead> <tbody> </tbody>
Serial Stock Name Scaffolding Protein - Display Protein Molecular Weight (kDa) Corresponding SDS-PAGE Lanes (Periplasmic/Cytoplasmic/Membrane Fractions) Presence of Bands (Periplasmic/Cytoplasmic/Membrane Fractions)
O_1 O_Mgfp5 OmpA-Mgfp5 36.8 1/2/3 Yes/Yes/Yes
I_1 I_Mgfp5 INP-Mgfp5 54.2 4/5/6 Yes/Yes/Yes
O_2 O_Glucanases OmpA-Glucanases 78.5 7/8/9 Yes/Yes/Yes
I_2 I_Glucanases INP-Glucanases 95.9 10/11/12 Yes/Yes/Yes
O_3 O_Mgfp5-Glucanases OmpA-Glucanases, OmpA-Mgfp5 78.5, 36.8 13/14/15 No/Yes/Yes, Yes/Yes/Yes
I_3 I_Mgfp5-Glucanases INP-Glucanases, INP-Mgfp5 95.9, 54.2 16/17/18 No/No/No, No/Yes/Yes
O_4 O_Glucanases-GH19Chitinase OmpA-Glucanases, OmpA-GH19Chitinase 78.5, 56.5 19/20/21 No/Yes/Yes, No/No/No
I_4 I_Glucanases-GH19Chitinase INP-Glucanases, INP-GH19Chitinase 95.9, 73.9 22/23/24 Yes/Yes/No, No/No/No


SDS-PAGE ImageSequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 20
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 356
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 885
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 20
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 20
    Illegal NgoMIV site found at 686
    Illegal AgeI site found at 641
  • 1000
    COMPATIBLE WITH RFC[1000]