Part:BBa_K4907032

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]