Part:BBa_K1758324:Experience

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Use by Oxford iGEM 2016

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 from part. However it can be seen from the sequence that the part is actually very different from the subparts suggested above which suggests that CueR is expressed divergent from the sfGFP (on the opposite strand and transcribed in the opposite direction).

If you look at the sequence level above 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:

How BBa_K1758324 should be labelled in the registry based on its underlying sequence

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, because it was too AT rich to be synthesised. This formed our part: BBa_K1980006 which we used to control the expression of our copper chelators

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