Difference between revisions of "Part:BBa K4907138"

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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 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, and confirmation was carried out through colony PCR and sequencing.  
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We took <i>ccdB</i>/<i>ccdA</i> toxin/antitoxin genes as the core of our HGT prevention.To verify the role of CcdA in resisting the CcdB toxin, 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, and confirmation was carried out through colony PCR and sequencing.  
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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>
 
<center><b>Fig. 1 Gene Circuit of BBa_K4907138</b></center>
 
<center><b>Fig. 1 Gene Circuit of BBa_K4907138</b></center>
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===Characterization===
 
===Characterization===
 
====Agarose gel electrophoresis (AGE)====
 
====Agarose gel electrophoresis (AGE)====
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====Verification of dual-plasmid transformation====
 
====Verification of dual-plasmid transformation====
 
To validate the resistance of CcdA to ccdB, we performed a dual-plasmid transformation.
 
To validate the resistance of CcdA to ccdB, 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;">
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                    <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>
 
<center><b>Table. 1 Performance of different dual-plasmid systems in <i>E. coli</i> DH10&beta; </b></center>
 
<center><b>Table. 1 Performance of different dual-plasmid systems in <i>E. coli</i> DH10&beta; </b></center>
 
The results show 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.
 
The results show 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.

Latest revision as of 09:03, 12 October 2023


I13453-B0034-ccdA-B0015

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

We took ccdB/ccdA toxin/antitoxin genes as the core of our HGT prevention.To verify the role of CcdA in resisting the CcdB toxin, 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, and confirmation was carried out through colony PCR and sequencing.

Fig. 1 Gene Circuit of BBa_K4907138

Characterization

Agarose gel electrophoresis (AGE)

In the construction of this circuit, colony PCR and gene sequencing were used to verify the correctness of the transformants. At around 689bp, a target band of approximately 750bp was observed (Fig. 2).

Fig. 2 DNA gel electrophoresis of the colony PCR products of BBa_K4907138_pSB1C3

Verification of dual-plasmid transformation

To validate the resistance of CcdA to ccdB, 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 show 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.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 125
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 65
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]