Difference between revisions of "Part:BBa K4390103"
 
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This part is an improvement of [[Part:BBa_K3380600]], as we have swapped the T7 promoter to one no longer containing a BsaI site. This means it can be used in Type IIS assembly techniques such as JUMP assembly, which was our primary assembly technique for the iGEM project.  
 
This part is an improvement of [[Part:BBa_K3380600]], as we have swapped the T7 promoter to one no longer containing a BsaI site. This means it can be used in Type IIS assembly techniques such as JUMP assembly, which was our primary assembly technique for the iGEM project.  
  
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The Edinburgh-UHAS_Ghana team for 2022 designed a construct to detect arsenic in contaminated water. This construct is composed of a fluorescent RNA aptamer iSpinach flanked by the F30 tRNA scaffolds (BBa_K4390094) which under a strong T7 RNA promoter (BBa_I712074), downstream of the promotor there is the arsenic promotor which acts as the binding site for the arsR 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.
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===Usage and Biology===
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This biosensor should be used with a cell-free lysate which contains the arsR repressor T7 RNA polymerase, chemical energy (ATP), NTPs and DFHBI or DFHO to induce fluorescence in the presence of arsenic ions. The arsR repressor will bind to the arsR binding site on the arsenic promotor when arsenic ions 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 arsenic ions 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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Latest revision as of 13:52, 12 October 2022


arsR controlled iSpinach biosensor

Improvement of part BBa_K3380600

This part is an improvement of Part:BBa_K3380600, as we have swapped the T7 promoter to one no longer containing a BsaI site. This means it can be used in Type IIS assembly techniques such as JUMP assembly, which was our primary assembly technique for the iGEM project.

The Edinburgh-UHAS_Ghana team for 2022 designed a construct to detect arsenic in contaminated water. This construct is composed of a fluorescent RNA aptamer iSpinach flanked by the F30 tRNA scaffolds (BBa_K4390094) which under a strong T7 RNA promoter (BBa_I712074), downstream of the promotor there is the arsenic promotor which acts as the binding site for the arsR 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 arsR repressor T7 RNA polymerase, chemical energy (ATP), NTPs and DFHBI or DFHO to induce fluorescence in the presence of arsenic ions. The arsR repressor will bind to the arsR binding site on the arsenic promotor when arsenic ions 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 arsenic ions 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 52
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 52
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 52
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 52
  • 1000
    COMPATIBLE WITH RFC[1000]