Composite

Part:BBa_K4907139

Designed by: Xinyi Yang   Group: iGEM23_XMU-China   (2023-09-16)


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.

Fig. 1 Gene Circuit of 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).

Fig. 2 DNA gel electrophoresis of the colony PCR products of BBa_K4907139_pSB4k5

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
✖️
Table. 1 Performance of different dual-plasmid systems in E. coli DH10β and DB3.1

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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 125
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 65
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 372


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