Difference between revisions of "Part:BBa K3792006"
 
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__NOTOC__
 
__NOTOC__
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<partinfo>BBa_K3792003 short</partinfo>
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<partinfo>BBa_K3792006 short</partinfo>
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<p>The NodCIJ Generator I has a BIOFAB promoter with low strength. NodC is a chitin synthase that produces tetrameric chitooligomers. NodI and NodJ are transport proteins that usually secrete lipochitooligomers but there is evidence that they also transport chitooligomers.</p>
  
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apFAB98 is a weak promoter taken from the BIOFAB collection. This device uses apFAB98 to express mRFP1, a red fluorescent protein. The apFAB98 test device consists of apFAB98, an RBS, the coding sequence for mRFP1, and a terminator.  
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<p>This part consists of 2555 base-pair length. It includes a promoter, 3 ribosome binding sites, 3 protein-coding sequences, and a terminator. We used promoter K2832184, a weak promoter from the BIOFAB collection. In addition, we also used protein-coding sequences from Chitin synthase from Rhiozobium leguminosarum, Nodl a protein possibly involved in transporting chitin across the cell membrane, and NodJ, a protein also possibly involved in the transportation of chitin.</p>
  
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This test device and two others with BIOFAB promoters of different strengths were each assembled into the pSB1K3 plasmid and then transformed into E. coli NEB 5 alpha.  
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<p>This generator was assembled into the pSB1K3 plasmid, and then transformed into E.Coli NEB 5-Alpha. To test if the cells were secreting chitin, The FSU 2021 team used the Chitosan Assay Kit (XAN-5126) available from Cell Biolabs. The manual for this kit contains the protocols for converting chitin to chitosan, along with a protocol for detecting chitosan.</p>
  
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The fluorescence of the cells was measured using flow cytometry. By comparing the relative fluorescence, we can determine which promoters caused higher levels of expression of mRFP1.
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<p>Chitin from the cells was converted into chitosan by reacting with 12.5M NaOH at 95°C for 24 hours. The solution was then neutralized with acetic acid. The assay was then performed on the supernatant of the cell culture.</p>
  
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<div id="my-chart">
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<table id="modular-promoter-library-chart" class="charts-css column show-labels show-data show-primary-axis show-10-secondary-axes">
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      <caption>Chitosan Detection Assay</caption>
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      <tr>
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        <th scope="row">Cell Supernatant</th>
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        <td style="--size: calc(2.477/2.5)"><span class="tooltip">Chitin Secretion Cells Supernatant</span><span class="data">2.477</span></td>
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      </tr>
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      <tr>
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      <th scope="row">LB and Assay</th>
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      <td style="--size: calc(1.515/2.5)"><span class="tooltip">LB Growth Medium with the Chitosan Detection Assay</span><span class="data">1.515</span></td>
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      </tr>
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      <tr>
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        <th scope="row">DI Water and Assay</th>
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        <td style="--size: calc(0.084/2.5)"><span class="tooltip">Deionized Water with the Chitosan Assay</span><span class="data">0.084</span></td>
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      </tr>
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      <tr>
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        <th scope="row">LB and No Assay</th>     
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        <td style="--size: calc(0.08/2.5)"><span class="tooltip">LB Growth Medium with No Chitosan Assay</span><span class="data">0.08</span></td>
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    </tr>
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  </table>
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  <div class="data-axis">Absorbance of 540 nm Light</div>
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</div>
  
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This data reflects the absorbance of chitosan from the assay kit. From our data, we can see how the 400 ug/ml chitosan solution had an absorbance of 2.477 with the chitosan assay kit. However, the LB medium with the assay kit had a very high absorbance of 1.515. As a result, we suspect that our medium, LB, has chitosan. Furthermore, we can confirm there is chitosan in our chitosan solution, however, we cannot be sure if this is directly due to the cells or due to the medium LB. We hypothesize that there is chitosan in both our cells and the LB medium, as a result, we will further be testing our chitin secretion cells in a middle medium to confirm if our cells directly secret chitosan.
  
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===
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<!-- -->
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3792003 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K3792006 SequenceAndFeatures</partinfo>
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<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K3792003 parameters</partinfo>
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<partinfo>BBa_K3792006 parameters</partinfo>
 
<!-- -->
 
<!-- -->

Latest revision as of 04:00, 22 October 2021


NodCIJ Generator I

The NodCIJ Generator I has a BIOFAB promoter with low strength. NodC is a chitin synthase that produces tetrameric chitooligomers. NodI and NodJ are transport proteins that usually secrete lipochitooligomers but there is evidence that they also transport chitooligomers.

This part consists of 2555 base-pair length. It includes a promoter, 3 ribosome binding sites, 3 protein-coding sequences, and a terminator. We used promoter K2832184, a weak promoter from the BIOFAB collection. In addition, we also used protein-coding sequences from Chitin synthase from Rhiozobium leguminosarum, Nodl a protein possibly involved in transporting chitin across the cell membrane, and NodJ, a protein also possibly involved in the transportation of chitin.

This generator was assembled into the pSB1K3 plasmid, and then transformed into E.Coli NEB 5-Alpha. To test if the cells were secreting chitin, The FSU 2021 team used the Chitosan Assay Kit (XAN-5126) available from Cell Biolabs. The manual for this kit contains the protocols for converting chitin to chitosan, along with a protocol for detecting chitosan.

Chitin from the cells was converted into chitosan by reacting with 12.5M NaOH at 95°C for 24 hours. The solution was then neutralized with acetic acid. The assay was then performed on the supernatant of the cell culture.

Chitosan Detection Assay
Cell Supernatant Chitin Secretion Cells Supernatant2.477
LB and Assay LB Growth Medium with the Chitosan Detection Assay1.515
DI Water and Assay Deionized Water with the Chitosan Assay0.084
LB and No Assay LB Growth Medium with No Chitosan Assay0.08
Absorbance of 540 nm Light


This data reflects the absorbance of chitosan from the assay kit. From our data, we can see how the 400 ug/ml chitosan solution had an absorbance of 2.477 with the chitosan assay kit. However, the LB medium with the assay kit had a very high absorbance of 1.515. As a result, we suspect that our medium, LB, has chitosan. Furthermore, we can confirm there is chitosan in our chitosan solution, however, we cannot be sure if this is directly due to the cells or due to the medium LB. We hypothesize that there is chitosan in both our cells and the LB medium, as a result, we will further be testing our chitin secretion cells in a middle medium to confirm if our cells directly secret chitosan.


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 492
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 854