Part:BBa_K4390050
Mercury biosensor
Biosensor for mercury The Edinburgh-UHAS_Ghana team for 2022 designed a construct to detect mercury and cadmium in contaminated water. This construct is composed of a fluorescent RNA aptamer Spinach2 (BBa_K3773013) which is flanked by the F30 tRNA scaffolds (BBa_K3380101 and BBa_K3380102) which under a strong T7 RNA promoter (BBa_I712074), downstream of the promotor there is the PmerT promotor which acts as the binding site for the merR repressor (BBa_K346002) as to allow transcriptional repression of the T7 promotor. This construct is designed to be cell-free and only requires transcription of the RNA aptamer to produce fluorescence. It should be noted that T7 RNA polymerase, chemical energy (ATP), NTPs and DFHBI or DFHO are also required in the cell-free reaction so that fluorescence is observed.
Usage and Biology
This biosensor is able to detect both mercury and cadmium depending on which transcription factor you use in the cell-free reaction. For mercury detection, you would use a cell lysate expressing the merR repressor, however, for cadmium, you would use the mutant merR which has stronger binding to cadmium. You can get cell-free lysates expressing these transcription factors by transforming cells with the two expression constructs we designed (BBa_K4390004 and BBa_K4390003). To produce a fluorescent output you will need to use a cell-free lysate expressing either merR or mut_merR depending on what metal you want to detect. You will also need T7 RNA polymerase, chemical energy (ATP), NTPs and DFHBI or DFHO to induce fluorescence in the presence of mercury or cadmium ions. The merR or mut_merR repressor will bind to the merR binding site on the PmerT promotor when either metal is not present in the reaction. This causes transcriptional repression of the RNA aptamer as the T7 polymerase is physically occluded from reading the linear construct. When cadmium ions are present in the reaction the mut_merR repressor will then bind to their respective metal ions and it will allow the transcription of the RNA aptamer, however, merR will bind to the mercury ions and remain bound to the linear construct and will then act as an activator of transcription, this aptamer can then bind to the DFHBI or DFHO which will induce fluorescence. Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal SpeI site found at 221
- 12INCOMPATIBLE WITH RFC[12]Illegal SpeI site found at 221
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal SpeI site found at 221
- 25INCOMPATIBLE WITH RFC[25]Illegal SpeI site found at 221
- 1000COMPATIBLE WITH RFC[1000]
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