Part:BBa_K3256005

Plac+ccdB*

This composite part contains lac promoter, RBS and ccdB*. More detail about the toxin is shown below.

Introduction

The original part is from BBa_P1010. The modified amino acid sequence includes N95T(shown below). And the protein was truncated at 96^th amino acid with a stop codon. 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 E. coli BL21(DE3) ccdB* BBa_K3256440

  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. All experiments were completed in triplicates. (Figure 2~6)

Figure 2:Growth curve of E. coli BL21(DE3) with 500uM IPTG induction ccdB 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

Bernard P, Gabant P, Bahassi EM, Couturier M, Université Libre de Bruxelles. Positive-selection vectors using the F plasmid ccdB killer gene." Gene 1994 Oct 11;148(1):71-4. pmid:7926841