Part:BBa_K1980006

pCopA with divergent expressed CueR

Description

This part contains the promoter pCopA with an RBS with the regulator CueR expressed divergently. We used this design in our composite parts consisting of this promoter (and regulator) upstream of our copper chelating parts (BBa K1980010, BBa K1980011 and BBa K1980012). This design worked better than a part relying on genomic CueR expression (BBa K1980005)

To facilitate making any protein under control of a copper-responsive promoter with the correct RBS-ATG distance, we designed this part so that any protein with the ATG biobrick prefix and universal suffix can be inserted with biobrick standard assembly.

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]

Our project was to investigate a probiotic treatment for the copper-accumulation disorder: Wilson's disease. This required a system able to detect dietary copper, ideally in the range over which copper concentration changes after a meal (around 5-10μM) and produce a copper chelating protein.

In the course of the project we investigated the E coli. CueR-linked copper sensing system and started from a part we found in the registry from iGEM Bielefeld-CeBiTec 2015: BBa K1758324

The team assembled this copper biosensor from two subparts which they then joined together:

The first part is a pCopA-RBS-sfGFP and the second part is the regulator CueR expressed from a constitutive promoter. The part is deposited in the registry and labelled to suggest the CueR is expressed divergent from the sfGFP (on the opposite strand and transcribed in the opposite direction):

However if you look at the sequence level this is clearly not the case. The constitutive promoter and the CueR start codon are at the 5’ end of the sfGFP coding strand and the CueR stop codon just upstream of pCopA. The part in fact has the constitutive promoter on the same strand as pCopA and sfGFP facing in the same direction and would be better represented like this:

As the two coding regions are not separated by a transcription terminator, there would be read through from the constitutive promoter to the sfGFP and sfGFP would be expressed even in the absence of copper. As no negative control is included in the plate reader graph they provide and no settings provided for their BioLector experiments in their protocols it is unclear just how high the expression level at 0mM copper was for this part compared to a negative control strain.

The CueR subpart BBa_K1758320 making up BBa_K1758324 is also incorrectly labelled.

We flipped the CueR and the constitutive promoter to face the opposite direction on the opposite strand i.e. so they were actually divergent. We also had to remove the 5'UTR, which Bielefeld found to increase expression, because it was too AT rich to be synthesised.

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</a>(2)</sup>.

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⁾

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