Difference between revisions of "Part:BBa K4907032"
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===Biology===
 
===Biology===
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<i>ccdA</i>is another gene found within the <i>ccd>/i> operon, encoding the antidote protein (CcdA) that protects cells from the toxic effects of CcdB.
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<i>ccdA</i> is the gene found within the <i>ccd</i> operon, encoding the antidote protein (CcdA) that protects cells from the toxic effects of CcdB. CcdA protein is easily degraded by Lonprotease.The cell loses the <i>ccdA</i> gene due to the loss of the F plasmid, causing the cell to succumb to the toxicity of CcdB.
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===Usage and design===
 
===Usage and design===
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To verify the role of CcdA in resisting the CcdB toxin, we designed composite part: <partinfo>BBa_K4907138</partinfo> to characterize <i>ccdA</i>. The constructed circuit was transformed into <i>E. coli</i> DH10&beta;, followed by chloramphenicol selection of positive transformants, and confirmation was carried out through colony PCR and sequencing.  
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The <i>ccdA</i> gene is used in our biosafety section. In our design, the <i>ccdA</i> gene will be introduced into the <i>E. coli</i> DH10&beta;genome to prevent HGT. See our [https://2023.igem.wiki/xmu-china/design Design] page for more information.To verify the neutralization of antitoxin CcdA, we designed a composite part: <partinfo>BBa_K4907138</partinfo> to characterize <i>ccdA</i>. The constructed circuit was transformed into <i>E. coli</i> DH10&beta;, followed by chloramphenicol selection of positive transformants.  
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<center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/yxy/parts/parts/i13453-b0034-ccda-b0015.png" width="400px"></html></center>
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<center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/yxy/parts/parts/i13453-b0034-ccda-b0015-1.png" width="400px"></html></center>
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<center><b>Fig. 1: Gene Circuit of BBa_K4907138</b></center>
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<center><b>Fig. 1 Gene Circuit of BBa_K4907138</b></center>
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===Characterization===
 
===Characterization===
 
====Verification of dual-plasmid transformation====
 
====Verification of dual-plasmid transformation====
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To validate the resistance of CcdA to CcdB, we performed a dual-plasmid transformation.
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To verify the neutralization of antitoxin CcdA, we performed a dual-plasmid transformation.
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<div class="myPage-paragraph-table-div" style="text-align: center">
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    <table class="myPage-paragraph-table" border = "1">
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        <tr>
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            <th style="width:20%;text-align: center;">Experiment</th>
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            <th style="width:25%;text-align: center;">Dual-plasmid system</th>
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            <th style="width:50% text align: center;">Strain</th>
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            <th style="width:30%;text-align: center;">Result</th>
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            <th style="width:10%;text-align: center;">Colonies</th>
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        </tr>
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        <tr>
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            <td rowspan="2">Verfication of <i>ccdA</i> in plasmid</td>
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            <td><partinfo>BBa_K4907139</partinfo>_pSB4K5<br><partinfo>BBa_K4907138</partinfo>_pSB1C3</td>
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            <td rowspan="2">in DH10&beta;</td>
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            <td>
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                <div class="td-img-container" style="width:100%;text-align: center;">
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                  <center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/yxy/032-ban1.jpg" width="100px"></html></center>
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                </div>
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            </td>
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            <td>✔</td>
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        </tr>
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        <tr>
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            <td><partinfo>BBa_K4907131</partinfo>_pSB4K5<br><partinfo>BBa_K4907138</partinfo>_pSB1C3</td>
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            <td>
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                <div class="td-img-container" style="width:100%;text-align: center;">
 +
                    <center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/yxy/032-ban2.jpg" width="100px"></html></center>
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                </div>
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            </td>
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            <td>✖️</td>
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        </tr>
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    </table>
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</div>
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<center><b>Table. 1 Performance of different dual-plasmid systems in <i>E. coli</i> DH10&beta; </b></center>
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The results showed that <i>E. coli</i> DH10&beta; transformed with both toxins and antitoxins also exhibited good growth. This confirmed the killing effect of the toxin CcdB again and the neutralization of antitoxin CcdA. For more specific details, please refer to [https://2023.igem.wiki/xmu-china/results Results].
  
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Table. 1 Performance of different dual-plasmid systems in <i>E. coli</i> DH10&beta;
 
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The results showed that <i>E. coli</i> DH10&beta; transformed with both toxins and antitoxins also exhibited good growth. This confirmed the killing effect of the toxin CcdB again and the neutralization of antitoxin CcdA.
 
 
===<span class='h3bb'>Sequence and Features</span>===
 
===<span class='h3bb'>Sequence and Features</span>===
 
<partinfo>BBa_K4907032 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4907032 SequenceAndFeatures</partinfo>

Latest revision as of 12:06, 12 October 2023


ccdA

Biology

ccdA is the gene found within the ccd operon, encoding the antidote protein (CcdA) that protects cells from the toxic effects of CcdB. CcdA protein is easily degraded by Lonprotease.The cell loses the ccdA gene due to the loss of the F plasmid, causing the cell to succumb to the toxicity of CcdB.

Usage and design

The ccdA gene is used in our biosafety section. In our design, the ccdA gene will be introduced into the E. coli DH10βgenome to prevent HGT. See our Design page for more information.To verify the neutralization of antitoxin CcdA, we designed a composite part: BBa_K4907138 to characterize ccdA. The constructed circuit was transformed into E. coli DH10β, followed by chloramphenicol selection of positive transformants.

Fig. 1 Gene Circuit of BBa_K4907138

Characterization

Verification of dual-plasmid transformation

To verify the neutralization of antitoxin CcdA, we performed a dual-plasmid transformation.

Experiment Dual-plasmid system Strain Result Colonies
Verfication of ccdA in plasmid BBa_K4907139_pSB4K5
BBa_K4907138_pSB1C3		 in DH10β
✔
BBa_K4907131_pSB4K5
BBa_K4907138_pSB1C3
✖️
Table. 1 Performance of different dual-plasmid systems in E. coli DH10β

The results showed that E. coli DH10β transformed with both toxins and antitoxins also exhibited good growth. This confirmed the killing effect of the toxin CcdB again and the neutralization of antitoxin CcdA. For more specific details, please refer to Results.

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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]