Part:BBa_K3945012
Elektra 2: An electrochemical- measurement system for rare earth elements
Usage and Biology
The Elektra system is compromised of the lanmodulin protein with the addition of a few of key amino acids on its terminal ends. Two cysteine amino acids on the C-terminal end will be used to covalently couple lanmodulin to a gold electrode via EDC coupling. On the N-terminal end of lanmodulin, there is a histidine residue which will allow a ruthenium signal molecule to be attached onto the other end of lanmodulin to create Elektra. Upon binding to lanthanides the N-terminal end of lanmodulin will be brought closer to the gold electrode and at a certain voltage, the ruthenium molecule redox potential will change, emitting an electrochemical signal that can be measured using a potentiostat. As such Elektra can quickly transform protein metal binding to a measurable electrochemical signal.
Design
The reason histidine was chosen to be the amino acid that would covalently attach to the ruthenium molecule was because there were no other histidine amino acids in lanmodulin. This will allow for selective binding as it is very important that there are no other ruthenium molecules that are attached to lanM except on the end (as it could affect REE binding in addition to creating false positives in Elektra).
In order to find the best construct for Elektra, we ordered three constructs each with a differing number of cysteines on the c-terminal end (one cysteine - Elektra 1, two cysteines- Elektra 2, or three cysteines - Elektra 3). This will allow us to determine which one will give us the best protein density that will not create too much background signal, allow us to easily attach lanM onto the gold electrode, and be selective and sensitive to lanthanides.
Experimental Workflow
In order to measure REE concentration using Elektra, there were several steps we would need to undergo. After successful protein production, we would need to determine background signal emittance. We would do this by measuring the redox potential of the protein in a solution with no lanthanides. Afterwards, a calibration curve using known REE concentrations will have to be calculated. This will be done by resuspending a constant amount of the protein in differing concentrations of REE solution and measuring the electrochemical output using a potentiostat. Once this calibration curve was completed, Elektra could then be used to measure REE concentration in a solution.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 89
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 89
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 89
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 89
- 1000COMPATIBLE WITH RFC[1000]
References
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