Technology

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Bioprocess

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Discussion

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Challenges

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Alternative methods

Our final system was of course not the only possibility. There were some points where we had to decide...

Reporter protein

Finally we decided for GFP protein because of it's versality, simplicity and fact that GFP does not require any additional reagents (eg. IPTG).
We could have used other fluorescent proteins, for instance superfolder fluorescent proteins constructed by iGEM Warsaw 2013 Team, but regular GFP was the simpliest choice.

Binding agent

Although, unfortunately, we could not have implemented lanthanide binding system because of lack of time, we had several ideas how to accomplish this.
Poly-LBT peptide
First was to construct a poly-LBT peptide which would be then anchored in the outer membrane of E. coli or transported to periplasm. We discarded this idea due to problems with modelling of behaviour of this polymer and problems with wet-lab design.
PmrB over-expression
Another idea consisted of PmrB dependent over-expression of PmrB(LBT) protein. In such system we would have pmr^C promoter - some logical device to boost the signal - PmrB(LBT), so in presence of lanthanides amount of PmrB(LBT) protein per cell would rise sharply, which should allow effective binding of lanthanides.
Small peptide fused with LBT
Our final and probably best idea was to create a construct peptide of such composition:
BBa_J32015(E. coli periplasm signal peptide)-structure peptide(ubiquitin or 1L2Y [BBa_K1459015]) - lanthanide binding tag.
The plan was to create a small, 'rubbish' protein which would only bind lanthanides without having any physiological function in cell (since we were afraid whether over-expression of PmrB would be cytotoxic).

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