Composite

Part:BBa_K4138005

Designed by: Carlos Classen   Group: iGEM22_Aachen   (2022-09-28)


VVD-CMI

Profile

Fusion construct made of the photoreceptor VVD (BBa_K4138002) and the colicin M immunity protein CMI-4AEQ (BBa_K4138003). VVD is a fungal blue light photoreceptor from Neurospora crassa. CMI is found in Escherichia coli . Both proteins, VVD and CMI, are linked by a linker (BBa_K4138006).

Base pairs VVD: 447 pb

Base pairs linker: 36 pb

Base pairs CMI: 294 bp

Usage and Biology

The homodimeric CMI immunity protein (BBa_K4138003) binds phosphate as a ligand. [1] The goal of the fusion with the homodimeric photoreceptor VVD (BBa_K4138002) was to enable the reversible binding and release of phosphate by light irradiation. [2] The protein construct was irradiated with blue light, which induces the dimerization of the VVD subunits within seconds through a covalent bond between a cysteine residue and a flavin group. [3,4] In absence of blue light, the VVD transitions within several hours to its monomeric form and thus causes the conformational change of CMI and the release of phosphate.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 226
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 226
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 226
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 226
    Illegal AgeI site found at 90
  • 1000
    COMPATIBLE WITH RFC[1000]

Expression

The protein fusion construct was insert in the pET-28a(+) vector. After transformation, the VVD-CMI construct was expressed in E. coli BL21(DE3) gold. We used an autoinduction medium for the expression. First we incubated VVD-CMI for three hours at 37 °C, after that then at 18 °C for three days.


References

[1] RCSB PDB - 4AEQ: Crystal structure of the dimeric immunity protein Cmi solved by direct methods (Arcimboldo)

[2] Conformational Switching in the Fungal Light Sensor Vivid DOI: 10.1126/science.1137128

[3] https://www.optobase.org/switches/LOV-domains/VVD/ (source last opened 30.09.2022)

[4] Mechanism Based Tuning of a LOV Domain Photoreceptor, doi:10.1038/nchembio.210

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