Part:BBa_K1980007

pCusC mKate2

Description

pCusC, a copper responsive promoter (activated by the CusS/R two component system), with a downstream RFP variant (mKate).

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]

Usage and Biology

A system that E. coli uses to respond to external copper is the CusS/CusR two-component system. This consists of the transmembrane histidine kinase enzyme CusS in the bacterial cytoplasmic membrane and a cytoplasmic response regulator, CusR. When CusS binds periplasmic copper it transfers a phosphate group from ATP to CusR aspartate residue 51 via CusS histidine residue 271. Phosphorylated CusR can bind to DNA inverted repeat CusR boxes (AAAATGACAANNTTGTCATTTT) found in the pCusC promoter and activate gene expression. In E. coli this box is present between the promoters for CusCFBA operon which encodes a multi-protein pump that exports cytoplasmic and periplasmic copper from the cell and the CusRS operon which encodes the two component system (therefore acting as a positive feedback loop in vivo)⁽¹⁾.

Experience

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. This promoter is sensitive and responsive over the whole range of the copper solutions tested (0-2mM).

References

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