Difference between revisions of "Part:BBa K2582018"

 
 
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<partinfo>BBa_K2582018 short</partinfo>
 
<partinfo>BBa_K2582018 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 [https://parts.igem.org/wiki/index.php?title=Part:BBa_K1330000 K1330000]in the human tRNALys3 scaffold.
  
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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, Spinach2.1 is moderately less bright.
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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_K2582018 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2582018 SequenceAndFeatures</partinfo>

Latest revision as of 19:40, 16 October 2018

lpp promoter-tRNALys3-Spinach2.1

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 K1330000in the human tRNALys3 scaffold.

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, Spinach2.1 is moderately less bright.

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]