Difference between revisions of "Part:BBa K4907138"
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===Usage and design=== | ===Usage and design=== | ||
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> DH10β, followed by chloramphenicol selection of positive transformants, and confirmation was carried out through colony PCR and sequencing. | 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> DH10β, followed by chloramphenicol selection of positive transformants, and confirmation was carried out through colony PCR and sequencing. | ||
| − | <center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/yxy/parts/parts/i13453-b0034-ccda-b0015.png" width="400px"></html></center> | + | <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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====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. | ||
| + | <div class="myPage-paragraph-table-div" style="text-align: center"> | ||
| + | <table class="myPage-paragraph-table" border = "1"> | ||
| + | <tr> | ||
| + | <th style="width:20%;text-align: center;">Experiment</th> | ||
| + | <th style="width:25%;text-align: center;">Dual-plasmid system</th> | ||
| + | <th style="width:50% text align: center;">Strain</th> | ||
| + | <th style="width:30%;text-align: center;">Result</th> | ||
| + | <th style="width:10%;text-align: center;">Colonies</th> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td rowspan="2">Verfication of <i>ccdA</i> in plasmid</td> | ||
| + | <td><partinfo>BBa_K4907139</partinfo>_pSB4K5<br><partinfo>BBa_K4907138</partinfo>_pSB1C3</td> | ||
| + | <td rowspan="2">in DH10β</td> | ||
| + | <td> | ||
| + | <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-ban1.jpg" width="100px"></html></center> | ||
| + | </div> | ||
| + | </td> | ||
| + | <td>✔</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td><partinfo>BBa_K4907131</partinfo>_pSB4K5<br><partinfo>BBa_K4907138</partinfo>_pSB1C3</td> | ||
| + | <td> | ||
| + | <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> | ||
| + | </div> | ||
| + | </td> | ||
| + | <td>✖️</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
<center><b>Table. 1 Performance of different dual-plasmid systems in <i>E. coli</i> DH10β </b></center> | <center><b>Table. 1 Performance of different dual-plasmid systems in <i>E. coli</i> DH10β </b></center> | ||
The results show that <i>E. coli</i> 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. | The results show that <i>E. coli</i> 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. | ||
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.
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).
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 |
 |
✖️ |
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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 125
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 65
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]