Part:BBa_K1980012

pCopA MymT sfGFP with divergent CueR

This part is our chelator MymTsfGFP expressed from this copper-sensitive promoter pCopA with the regulator of pCopA (CueR) expressed divergent from the promotor.

Sequence and Features:

Assembly Compatibility:

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

  Illegal NheI site found at 438
  Illegal NheI site found at 461
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 1175
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

E. coli cells use a protein called CueR to regulate the cytoplasmic copper concentration. CueR is a MerR-type regulator with an interesting mechanism of action whereby it can behave as a net activator or a net repressor under different copper concentrations through interaction with RNA polymerase⁽¹⁾. CueR forms dimers consisting of three functional domains (a DNA-binding, a dimerisation and a metal-binding domain). The DNA binding domains bind to DNA inverted repeats called CueR boxes with the sequence:

CCTTCCNNNNNNGGAAGG

This box is present at the promoter regions of the copper exporting ATPase CopA, some molybdenum cofactor synthesis genes and the periplasmic copper oxidase protein CueO.⁽²⁾

Schematic of the construct and how it works

Experience

The results of a plate reader experiment with different copper concentrations after four hours. Error bars show standard deviation of four repeats

Flow cytometry of this part at different copper concentrations

Microscopy performed on this part at different copper concentrations. Fluorescent, differential interference contrast channels and a composite shown

References

(1) Danya J. Martell, Chandra P. Joshi, Ahmed Gaballa, Ace George Santiago, Tai-Yen Chen, Won Jung, John D. Helmann, and Peng Chen (2015) “Metalloregulator CueR biases RNA polymerase’s kinetic sampling of dead-end or open complex to repress or activate transcription” Proc Natl Acad Sci U S A. 2015 Nov 3; 112(44): 13467–13472

(2) Yamamoto K, Ishihama A. (2005) “Transcriptional response of Escherichia coli to external copper.” Mol Microbiol. 2005 Apr;56(1):215-27