Part:BBa_K1976027

Colicin E2 immunity protein

This part encodes for the wild-type immunity protein inhibiting the nicking DNase Colicin E2. According to molecular dynamics simulations this protein inhibits also miniColicin (BBa_K1976048 and BBa_K1976049).

Figure 1:Structure of Colicin E2 imminity protein with (Y8OMT) mutation. The mutated amino acid is highlighted. The structure is derived from an molecular dynamics modeling calculation.

Usage

The Colicin E2 immunity protein can be used in combination with the Colicin E2 DNase or its engineered truncated variant miniColicin (BBa_K1976048 and BBa_K1976049).

Characteristics

Figure 2: SDS-PAGE of the Colicin E2 immunity protein. The arrow marks the band of the expression. The expression of protein variants, wild-type (BBa_K1976026) and with amber mutation (BBa_K1976027) were tested under control of the T7 promoter (BBa_K1976033 and BBa_K1976031) in BL21 without an supressor orthogonal pair. Each sample was applied before and after induction with IPTG. It can be seen that the immunity protein with a molecular weight if ~10 kDa is only present after induction with the wild-type variant.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 150
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization from Igem Team UNILausanne 2020

Functional kill switch assay with IPTG and aTc gradients on agar plate

E.coli Nissle 1917 were transformed with a plasmid containing the part BBa K3482004 and the part BBa_K3482014 (IM2 antitoxin part) and plated with a gradient of aTc and IPTG on agar plate. The plate shows strong activity of the IM2 antitoxin with aTc induction, whereas IPTG induction promotes production of the MiniColicin E2 toxin, resulting in a number of surviving cells (probable mutants) proportional to the dilution of the plated culture.

We also tested our pKC1 plasmid encoding for the miniColicin E2 toxin and IM2 antitoxin in E. coli Nissle 1917 ΔclbA. Again, we used different concentrations of IPTG and aTc to study the effect of differential expression of the toxin and antitoxin on the growth of our strain. We compared this to the strain transformed with the empty vector pAND

Dose-response growth curve of E. coli Nissle 1917 ΔclbA harboring kill switch plasmid pKC1 at 37°C. E. coli Nissle with pAND (red line), E. coli Nissle with pKC1 (green line). The lines and shade represent the mean ± standard error.

With E. coli Nissle 1917 with pKA1, we observed desired growth inhibition of the strain with pKC1 at high IPTG and low aTc concentrations, while the pAND strain showed no alteration in growth in any of the tested conditions. Contrastingly to E. coli Nissle 1917 transformed with pKA1, the induction of aTc was clearly able to rescue cell growth. For increasing concentrations of IPTG, also increasing concentrations of aTc were necessary to rescue cell growth. Similar to E. coli Nissle 1917 pKA1, cell growth could be completely inhibited for 6-8 h (IPTG: 100 µM, aTc: 0 ng/mL) before observing rapid growth. Also similarly to pKA1, the reduction in IPTG concentration gave a lesser but more constant growth inhibition, which also persisted to at least 10 h. Overall, we demonstrated that the miniColicin E2/IM2 encoding pKC1 plasmid could efficiently inhibit the growth of E. coli Nissle 1917 at 100 µM IPTG for up to 6 h and provided even longer growth inhibition at 5 µM IPTG.

References
[1] Cascales et al, Colicin Biology, Microbiology and Molecular Biology Reviews, vol. 71, pp. 158-229, 2007