Part:BBa_K1980011

pCopA MymT with divergent expressed CueR

Description

This composite part contains the cytoplasmic chelator MymT from M. tuberculosis behind the pCopA promoter with divergently expressed CueR (a copper sensitive transcription factor) in the biobrick.

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
  COMPATIBLE WITH RFC[21]
• 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⁾

Experience

We cloned pCopA MymT with divergent expressed CueR from a gBlock into the shipping plasmid pSB1C3. E. coli strain MG1655 was transformed using the specific recombinant plasmid and a 5ml culture of a transformed colony was grown overnight.

This is how the part is intended to work in vivo:

schematic of the construct and how it works

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

Author: Andreas Hadjicharalambous