Part:BBa_K4494001

A. caldus csoR

csoR is an important family of copper-sensing operon repressors. The part presented here is a kind of csoR identified from Acidithiobacillus caldus, as indicated in Hou et al. (2021). csoR effectively binds to Cu(I) as a copper toxicity buffer in A. caldus, and it could therefore be used as a copper biosensor.

Structure of A. caldus csoR as predicted by AlphaFold.

Characterization

In order to characterize csoR, we first placed it in a transcriptional unit (composite part K4494198). The transcriptional unit functions with PIII-RBS units (composite part K4494196) via the following mechanism: in regular conditions, csoR is released by the constant promoter, and represses PIII (K4494002) so that mApple (our reporter, K4494000) is not released; when copper is present, csoR binds to copper instead and so mApple is released by PIII.

The result of our characterization is as follows:

Characterization Results

As could be seen, the concentration constantly decreases due to survival pressure from copper, so it could be concluded that the original PIII-RBS combination does not respond properly to csoR and is not a copper-sensitive part.

Improvement

We did not alter the csoR sequence during the improvement. Instead, we altered the PIII-RBS combination and characterized all 96 systems of csoR and PIII-RBS variants. The variant sequences are registered as parts K4494003-K4494098, respectively labeled 1-96. A summary of the characterization results is as follows:

Heatmap of Characterization Results with 96 Variants

Reference

Hou, S., Tong, Y., Yang, H., & Feng, S. (2021). "Molecular Insights into the Copper-Sensitive Operon Repressor in Acidithiobacillus caldus". Applied and Environmental Microbiology: 87(16).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]