Part:BBa_K3256444

chpBK*

Introduction

ChpBK, one of the TA modules in E. coli, is a toxic gene that will suppress cell growth and may cause cell death. Its amino acid sequence holds 35% identity with MazF and 52% similar to it, a famous mRNA interferase in E. coli that can cause cell death. The cognate antitoxin of ChpBK is ChpBI, which can deactivate ChpBK by binding with it. Synthesis of protein can be processed normally once ChpBI is expressed. The modified amino acid sequence includes V83C, R84A. And the protein was truncated at 85^th amino acid with a stop codon(shown below). After these modifications, the toxin retains the function of suppressing the growth of E. coli, and makes the E. coli more stable from being killed easily.


Result

Cloning

  We conducted colony PCR to verify that our target gene was correctly cloned into the E. coli BL21 (DE3).

Figure 1:Colony PCR result of toxin genes after cloning into chpBK* BBa_K3256444.

  Figure 1 was the electrophoresis results of the colony PCR with a marker on the left side and the target gene on the right side. The lengths are labeled beside each band. As a result, we successfully cloned five target genes, respectively, into E. coli.

Functional Test

   After confirming the cloning of target genes, we tested their function by measuring the O.D. value after IPTG induction when the O.D. value reached 0.3 and compared with the O.D. value of the control. The O.D. values were documented in every five minutes for seven hours in total.
Figure 2:Growth curve of E. coli BL21(DE3) with 500uM IPTG induction chpBK toxin gene (blue), and control (orange)

Calculating Toxicity of Toxin Genes

  We first fit the control’s experiment data to the following equation, dBT/dt=g⋅BT(1−BT/BMax), and we fit the induced data to another equation, dBT/dt=g⋅BT(1−BT/BMax)−Ttoxin⋅BN⋅[toxin]. Next, we compared the two equations to calculate Ttoxin, the toxicity of the toxin gene (Figure 3). In the end, we chose the ydfD gene, which had the most significant toxicity, and the ccdB gene, which had relatively weak toxicity, moving on to the Functional test with mutagens.

Figure 3:Toxicity of different toxin genes. Ttoxin: The toxicity of toxin gene.

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]

References

Zhang, Y., et al. (2005). "Characterization of ChpBK, an mRNA Interferase from Escherichia coli." Journal of Biological Chemistry 280(28): 26080-26088.