Part:BBa_K4907140

I0500-B0034-ccdB

Biology

ccdB gene is located on the Escherichia coli F-factor plasmid and is part of the toxin-antitoxin system encoded by the ccd operon, responsible for plasmid maintenance during cell division. ccdB encodes a toxic protein that acts as a DNA gyrase poison. It can bind DNA gyrase to the broken double-stranded DNA, leading to cell death.

Usage and design

We took ccdB/ccdA toxin/antitoxin genes as the core of our HGT prevention, combining it with an inducible promotor system, terminator, and a series of necessary elements. The whole part was inserted into a plasmid backbone and finally transformed into E.coli to gain enough correct and functional circuits. we designed a composite part: BBa_K4907140 to characterize ccdB. The constructed circuit was transformed into E.coli DH10β, followed by kanamycin selection of positive transformants, and confirmation was carried out through colony PCR and sequencing.To validate the toxicity of the ccdB protein in E. coil DH10β, we conducted a colony-forming unit (CFU) counting experiment.

Fig. 1 Gene Circuit of BBa_K4907140

Characterization

Agarose gel electrophoresis (AGE)

During the construction of this circuit, colony PCR and gene sequencing were used to verify the correctness of the transformants. Around 1868bp, a target band of approximately 2000bp was observed (Fig. 2).

Fig. 2 DNA gel electrophoresis of the colony PCR products of BBa_K4907140_pSB4k5

CFU measurement

The experimental group (bacteria harboring BBa_K4907140_pSB4K5; BBa_I13453_pSB1C3) and the control group (bacteria harboring BBa_K206001_pSB4K5; BBa_I13453_pSB1C3) were separately induced with L-arabinose, with 3 replicates set for each. Then measure OD₆₀₀ and count CFU at 0, 2, 4, 6, and 8 hours.

Fig. 3 Growth curve and survival assay for characterizing the function of ccdB
a. The value of OD₆₀₀ against time (h) for different groups. b. CFUs /mL calculated of different groups are plotted against time (h).

After induction by L-arabinose, OD₆₀₀ was measured every two hours. The bacteria that had no ccdB expressed proliferated, while the one evpressing ccdB showed a significant growth defect, as the optical density (at 600nm) of which increased very slightly (Fig. 1a). At each time, the spot assay was also performed, then the cell viability was measured by CFU count (Fig. 2a). Consistent with the trend of OD₆₀₀ value against time, only the absence of ccdB allowed the host cells to survive. All these results indicated that ccdB is toxic enough to the engineered bacteria so that this toxin could be applied to cases when the suicide of genetically engineered microorganisms (GEMs) is strongly needed.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1205
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1452
  Illegal SapI site found at 961