Difference between revisions of "Part:BBa K4907032"
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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> DH10βgenome 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> DH10β, followed by chloramphenicol selection of positive transformants. | 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> DH10βgenome 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> DH10β, followed by chloramphenicol selection of positive transformants. | ||
<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><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 | + | <center><b>Fig. 1 Gene Circuit of BBa_K4907138</b></center> |
===Characterization=== | ===Characterization=== | ||
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.
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 |
 |
✖️ |
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]