Composite

Part:BBa_K4138004

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


SOPP3-2ABH

Profile

Fusion construct made of the photosensitizer SOPP3 (BBa_K4138001) and the phosphate-binding protein 2ABH (BBa_K4138000). SOPP3 is engineered synthetically. Its protein structure is based on a protein originating in Arabidopsis thaliana and is light-sensitive. 2ABH is a phosphate-binding protein found in Escherichia coli. Both proteins, SOPP3 and 2ABH, are linked by a spacer (BBa_K4138007).

Base pairs SOPP3: 381 bp

Base pairs Spacer: 72 bp

Base pairs 2ABH: 963 bp

Usage and Biology

The phosphate-binding protein 2ABH binds phosphate with high affinity and allows the release of the bound phosphate in vitro only under extreme conditions. The fusion with the photosensitizer SOPP3 enables the release of the phosphate by light irradiation. For this purpose, the protein constructs were irradiated with blue light. Blue light induces the production of reactive oxygen species (ROS). The synthesized ROS lead to chemical degradation of 2ABH and thus cause the release of the phosphate bound to 2ABH. Upon irradiation, the chemical degradation follows in minutes [1], [2], [3].

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 187
    Illegal PstI site found at 316
    Illegal PstI site found at 346
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 187
    Illegal PstI site found at 316
    Illegal PstI site found at 346
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 187
    Illegal PstI site found at 316
    Illegal PstI site found at 346
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 187
    Illegal PstI site found at 316
    Illegal PstI site found at 346
    Illegal AgeI site found at 1098
  • 1000
    COMPATIBLE WITH RFC[1000]


Transformation and Expression

The protein fusion construct inserted in the pET-28a(+) vector was transformed into chemically competent E. coli cells via heat shock. After transformation, the SOPP3-2ABH construct was expressed in E. coli BL21(DE3) gold.

Medium used for

Transformation: LB-medium with the addition of kanamycin

Expression: TB-medium with the addition of kanamycin, IPTG-induced


References

[1] Torra, J., Lafaye, C., Signor, L. et al. Tailing miniSOG: structural bases of the complex photophysics of a flavin-binding singlet oxygen photosensitizing protein. Sci Rep 9, 2428 (2019). https://doi.org/10.1038/s41598-019-38955-3

[2] Christie JM, Salomon M, Nozue K, Wada M, Briggs WR. LOV (light, oxygen, or voltage) domains of the blue-light photoreceptor phototropin (nph1): binding sites for the chromophore flavin mononucleotide. Proc Natl Acad Sci U S A. (1999). https://doi.org/10.1073/pnas.96.15.8779

[3] Z. Wang, A. Choudhary, P.S. Ledvina, F.A. Quiocho, Fine tuning the specificity of the periplasmic phosphate transport receptor. Site-directed mutagenesis, ligand binding, and crystallographic studies., Journal of Biological Chemistry, Volume 269, Issue 40, 1994, Pages 25091-25094, https://doi.org/10.1016/S0021-9258(17)31503-X.

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