Part:BBa_K4907131
pRha-B0034-ccdB-B0015
Biology
ccdB encodes a toxic protein (CcdB) that, as a DNA gyrase poison, locks DNA gyrase together with broken double-stranded DNA, ultimately leading to cell death.
Usage and design
To validate the interaction between ccdA and ccdB, we constructed a composite part: BBa_K4907149.
Characterization
Colony PCR
In the construction of this circuit, colony PCR and gene sequencing were used to verify the correctness of the transformants. At around 871bp, a target band of approximately 1000bp was observed (Fig. 2).
Verification of double plasmid transformation
Taking into consideration the possibility of significant leakage from BBa_K206001, we constructed BBa_K4907131_pSB4K5. Then,we performed a double-plasmid transformation.
| Experiment | Dual-plasmid system | Strain | Result | Colonies |
|---|---|---|---|---|
| Verfication of ccdA in plasmid | BBa_K4907131_pSB4K5 BBa_K4907138_pSB1C3 |
in DH10β |
 |
✔ |
| BBa_K4907131_pSB4K5 BBa_I13453_pSB1C3 |
 |
✖️ | ||
| Verfication of ccdA in genome (ccdB is controlled by pRha) | BBa_K4907131_pSB4K5 BBa_I13453_pSB1C3 |
in DB3.1 |
 |
✔ |
| in DH10β |
 |
✖️ |
The results showed that E. coli DB3.1 transformed with toxin genes and E. coli DH10β transformed with pRha(BBa_K914003) promoter grew well. E. coli DH10β transformed with both toxins and antitoxins also exhibited good growth. However, E. coli DH10β with toxins did not grow. This confirmed the killing effect of the toxin CcdB again and the neutralization of antitoxin CcdA. Besides, the E. coli DB3.1 transformed with toxin controlled by pRha promoter without antitoxin both grew better compared with E. coli DH10β. From these results, we can draw the conclusion that whether the CcdA is in plasmid or genome can play the role of neutralization to CcdB.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 364
| None |