Difference between revisions of "Part:BBa K4390051"

Revision as of 22:24, 12 September 2022

Lead biosensor

The Edinburgh-UHAS_Ghana team for 2022 designed a construct to detect lead 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 lead promotor which acts as the binding site for the pbrR 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 should be used with a cell-free lysate which contains the pbrR repressor T7 RNA polymerase, chemical energy (ATP), NTPs and DFHBI or DFHO to induce fluorescence in the presence of arsenic ions. The pbrR repressor will bind to the pbrR binding site on the lead promotor when Pb(II) salts are 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 lead ions are present in the reaction the pbrR repressor will then bind to the Pb(II) salts and it will allow the transcription of the RNA aptamer, this aptamer can then bind to the DFHBI or DFHO which will induce fluorescence. Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal SpeI site found at 258
12
INCOMPATIBLE WITH RFC[12]
Illegal SpeI site found at 258
21
COMPATIBLE WITH RFC[21]
23
INCOMPATIBLE WITH RFC[23]
Illegal SpeI site found at 258
25
INCOMPATIBLE WITH RFC[25]
Illegal SpeI site found at 258
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 134

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 ===Usage and Biology===
-This biosensor should be used with a cell-free lysate which contains the pbrR repressor T7 RNA polymerase, chemical energy (ATP), NTPs and DFHBI or DFHO to induce fluorescence in the presence of arsenic ions. The pbrR repressor will bind to the pbrR binding site on the lead promotor when Pb(II) salts are 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 arsenic ions are present in the reaction the pbrR repressor will then bind to the Pb(II) salts and it will allow the transcription of the RNA aptamer, this aptamer can then bind to the DFHBI or DFHO which will induce fluorescence.
+This biosensor should be used with a cell-free lysate which contains the pbrR repressor T7 RNA polymerase, chemical energy (ATP), NTPs and DFHBI or DFHO to induce fluorescence in the presence of arsenic ions. The pbrR repressor will bind to the pbrR binding site on the lead promotor when Pb(II) salts are 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 lead ions are present in the reaction the pbrR repressor will then bind to the Pb(II) salts and it will allow the transcription of the RNA aptamer, this aptamer can then bind to the DFHBI or DFHO which will induce fluorescence.
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 <span class='h3bb'>Sequence and Features</span>