Difference between revisions of "Part:BBa K2582000"

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