Difference between revisions of "Part:BBa K2582017"

 
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<partinfo>BBa_K2582017 short</partinfo>
 
<partinfo>BBa_K2582017 short</partinfo>
  
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This part is a constitutive expression cassette of a RNA reporter that can be used to measure promoter activity directly. It contains the light-up RNA Aptamer [http://doi.org/10.1021/ja508478x dimeric Broccoli]in the human tRNALys3 scaffold.
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This biobrick is an improvement from [https://parts.igem.org/wiki/index.php?title=Part:BBa_K1330000 K1330000], which is brighter than the original design.
  
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===Usage and Biology===
 
===Usage and Biology===
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The Spinach aptamer and its derivatives allow the docking of fluorogen 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) and increases its quantum yields by hundreds of folds. By surrounding it with a tRNA scaffold, its folding in cell can be enhanced and its expression level can be stabilized.
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===Characterization===
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This aptamer can be expressed in <i>E. coli</i> DH5a under a constitutive lpp promoter. Comparing the 3 RNA reporters we constructed and the pre-existed Spinach2.1, miniSpinach is moderately brighter.
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[[File:Improve a previous bobrick.jpg|400px|thumb|left|Fluorescence data obtained from plate reader. DH5a transformed with pSB1C3-lpp-Spinach2.1 or other Spinach variants were incubated overnight at 37 degrees Celsius. Overnight culture was normalized to OD=0.2 and incubated in 200uM DFHBI for 45 minutes. N=1. More replicates are needed.]]
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2582017 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2582017 SequenceAndFeatures</partinfo>

Revision as of 19:37, 16 October 2018

lpp promoter-tdBroccoli

This part is a constitutive expression cassette of a RNA reporter that can be used to measure promoter activity directly. It contains the light-up RNA Aptamer [http://doi.org/10.1021/ja508478x dimeric Broccoli]in the human tRNALys3 scaffold. This biobrick is an improvement from K1330000, which is brighter than the original design.

Usage and Biology

The Spinach aptamer and its derivatives allow the docking of fluorogen 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) and increases its quantum yields by hundreds of folds. By surrounding it with a tRNA scaffold, its folding in cell can be enhanced and its expression level can be stabilized.

Characterization

This aptamer can be expressed in E. coli DH5a under a constitutive lpp promoter. Comparing the 3 RNA reporters we constructed and the pre-existed Spinach2.1, miniSpinach is moderately brighter.

Fluorescence data obtained from plate reader. DH5a transformed with pSB1C3-lpp-Spinach2.1 or other Spinach variants were incubated overnight at 37 degrees Celsius. Overnight culture was normalized to OD=0.2 and incubated in 200uM DFHBI for 45 minutes. N=1. More replicates are needed.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]