Part:BBa_K3762007

Reactive sulfur species sensor with GFP

SqrR is a repressor protein that is involved in sulfide-dependent gene expression in the bacterium Rhodobacter capsulatus.[1] When no sulfide is present, SqrR binds to the Psqr promoter, inhibiting the expression of the gene encoding Sulfide:quinone oxidoreductase (SQR). SQR is an enzyme that oxidises sulfide to zero-valent sulfur (S0), which can then conjugate with multiple different nucleophiles or low molecular weight thiols to form reactive sulfur species (RSS).[1] Upon reaction with a RSS, a di-, tri-, or tetrasulfide bond forms between two cysteine residues of SqrR, resulting in a conformational change that reduces its DNA binding affinity. RSS thereby functions as inducers of the genes under control of the Psqr promoter that is repressed by SqrR.[1]

This composite part takes advantage of the sulfide-binding properties of SqrR, as well as its repressor function, so as to function as a sensor for hydrogen sulfide. When RSS are present, reaction with SqrR induces the expression of the reporter gene. The part consists of the fluorescent reporter gene GFP placed under control of the Psqr promoter, sqrR, the gene that encodes SqrR, placed under control of a constitutive promoter, as well as biobrick ribosomal binding sites and terminators.

In order to function as a hydrogen sulifede, the part must be used in conjugation with a component that oxidises sulfide to S0, such as SQR. In our project, oxidised glutathione (GSSG) was reacted with sulfide to form glutathione persulfide (GSSH), a RSS.

This part is very similar to Part:BBa_K3762005, and Part:BBa_K3762006, but different reporters (fluorescent proteins) are used.

[1] Shimizu, T., Shen, J., Fang, M., Zhang, Y., Hori, K., Trinidad, J. C., Bauer, C. E., Giedroc, D. P., & Masuda, S. (2017). Sulfide-responsive transcriptional repressor SqrR functions as a master regulator of sulfide-dependent photosynthesis. Proceedings of the National Academy of Sciences of the United States of America, 114(9), 2355–2360. https://doi.org/10.1073/pnas.1614133114

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 893
  Illegal NheI site found at 916
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 848
  Illegal XhoI site found at 1192
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 725