Difference between revisions of "Part:BBa K4907139"

Revision as of 08:47, 12 October 2023

K206001-B0034-ccdB

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. BBa_K206001 is a variant pBAD promoter with a modified AraI1 site that has been shown to be less responsive at low concentrations of arabinose.

Usage and design

To validate the interaction between ccdA and ccdB, we constructed a composite part: BBa_K4907139.

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 505bp, a target band of approximately 500bp was observed (Fig. 2).

Verification of double 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_K4907139_pSB4K5 BBa_I13453_pSB1C3			✖️
Verfication of ccdA in genome (ccdB is controlled by pBAD)	BBa_K4907139_pSB4K5 BBa_K4907138_pSB1C3	in DB3.1		✔
		in DH10β		✖️

The results showed that E. coli DB3.1 transformed with toxin genes and E. coli DH10β transformed with pBAD (BBa_K206001) 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 pBAD 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