Part:BBa_K1980010

pCopA TAT Csp1 sfGFP with divergent CueR

Description

This part contains the bacterial copper chelator Csp1 (Copper Storage Protein 1) with a C-terminal sfGFP tag (connected with short hydrophilic flexible linker) behind the pCopA promoter with constitutively expressed CueR (a bacterial copper sensitive repressor/activator) in the biobrick.

schematic of the construct and how it works

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 1466
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1028
  
• 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 TAT Csp1 sfGFP 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. A plate with four repeats at 10 different copper concentrations (ranging from 0mM to 2mM) plus a negative control was made. The absorbance and fluorescence of each well was measured over time in order to assess the absolute fluorescence of the construct. We also used flow cytometry to measure fluorescence at the same copper concentration ranges, as well as microscopy to view fluorescence by eye

Absolute fluorescence as measured by the pate reader at 4 hours after copper induction

Flow cytometry measurements at the same copper concentration range

Microscopy pictures after no, intermediate and maximal copper induction

Likely due to the expression problems of the Csp1 chelator in E. coli, it responded worse than the similar part with our MymTsfGFP fusion protein (https://parts.igem.org/Part:BBa_K1980012)

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.