Coding

Part:BBa_K1976026

Designed by: Sonja Elbrich, Claudia Kreher, Patrick Kunzmann, Nina Kuschik-Maczollek, Bianca Reisinger, Sabine Schäfer, Inka Schröter, Viktoria Schuster   Group: iGEM16_TU_Darmstadt   (2016-10-13)

Colicin E2 immunity protein (+amber codon)

This part encodes for the immunity protein inhibiting the nicking DNase Colicin E2. According to molecular dynamics simulations this protein inhibits also miniColicin (BBa_K1976048 and BBa_K1976049). This variant includes an amber stop codon near the start of the ORF (Y8OMT) for usage in amber supression. When using an orthogonal amber supressor pair, the immunity protein is only functionally produced when the respective non-natural amino acid is in the medium.


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 3: 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]


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

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