Difference between revisions of "Part:BBa K2582000"

 
 
(6 intermediate revisions by the same user not shown)
Line 1: Line 1:
−
 
 
__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K2582000 short</partinfo>
 
<partinfo>BBa_K2582000 short</partinfo>
  
−
miniSpinach is a minimal version of the Spinach aptamer developed by Paige et. al. (2011). It is created previously by creating mutants of the Spinach aptamer by decreasing stem lengths (Ong et. al. 2017). When transcribed in vitro, it allows the docking of fluorogen 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) and increases its quantum yields by hundreds of folds. However, it does not fluoresce well in E. coli, possibly due to low folding efficiency.
+
This Biobrick contains the DNA sequence of the light-up RNA Aptamer, miniSpinach.
  
−
<!-- Add more about the biology of this part here
 
 
===Usage and Biology===
 
===Usage and Biology===
 +
miniSpinach is a minimal version of the Spinach aptamer developed by Paige et. al. (2011). It is created previously by generating mutants of the Spinach aptamer by decreasing stem lengths (Ong et. al. 2017). When transcribed in vitro, it allows the docking of fluorogen 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) and increases its quantum yields by hundreds of folds.
 +
 +
===Characterization===
 +
miniSpinach can be in vitro transcribed or expressed in E. coli.
 +
 +
Mixing the Spinach aptamer with excess DFHBI yielded a 200-fold increase in the fluorescence level of the reaction mixture.
 +
 +
[[File:BBa K2582000-Fig1.png|800px|thumb|left|Fluorescence of miniSpinach under (a) Chemidoc Blue Light Excitation (b) Microplate Reader]]
 +
 +
<p style="clear:left">
 +
However, its fluorescence in E. coli is hard to visualize and quite close to the E. coli autofluorescence level. Upon comparison between in-cell fluorescence and total RNA, it is found that miniSpinach does not fold well in E. coli.
 +
</p>
 +
 +
[[File:BBa K2582000-Fig2.png|500px|thumb|left|Comparison of miniSpinach fluorescence between in-cell and extracted conditions. For total RNA fluorescence assay, the same RNA amount corresponding to OD600 for in-cell assay was used.]]
 +
 +
<p style="clear:left"></p>
  
−
<!-- -->
 
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2582000 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2582000 SequenceAndFeatures</partinfo>

Latest revision as of 03:15, 15 October 2018

miniSpinach (P1-a5-b3)

This Biobrick contains the DNA sequence of the light-up RNA Aptamer, miniSpinach.

Usage and Biology

miniSpinach is a minimal version of the Spinach aptamer developed by Paige et. al. (2011). It is created previously by generating mutants of the Spinach aptamer by decreasing stem lengths (Ong et. al. 2017). When transcribed in vitro, it allows the docking of fluorogen 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) and increases its quantum yields by hundreds of folds.

Characterization

miniSpinach can be in vitro transcribed or expressed in E. coli.

Mixing the Spinach aptamer with excess DFHBI yielded a 200-fold increase in the fluorescence level of the reaction mixture.

Fluorescence of miniSpinach under (a) Chemidoc Blue Light Excitation (b) Microplate Reader

However, its fluorescence in E. coli is hard to visualize and quite close to the E. coli autofluorescence level. Upon comparison between in-cell fluorescence and total RNA, it is found that miniSpinach does not fold well in E. coli.

Comparison of miniSpinach fluorescence between in-cell and extracted conditions. For total RNA fluorescence assay, the same RNA amount corresponding to OD600 for in-cell assay was used.

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]