Part:BBa_K537009:Experience

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Applications of BBa_K537009

ZJU-China 2012 Characterizes BioBrick Part K537009

To characterise the theophylline riboswitches (part K537009, iGEM11_WITS_CSIR_SA), we quantified their activation at different theophylline concentrations (0 mM, 1 mM, 5 mM, 10 mM and 20 mM) over 2 hours using fluorometry. Competent E. coli (strain DH5a) cells were transformed with plasmid vectors containing the “Promoter-Theophylline riboswitch -Venus-Double terminator”. The bacterial colony appeal pink.

Cultured until the mid-log phase of growth, a different concentration of theophylline was added to each culture for induction. The activation of the riboswitch was detected as a fluorescent response as a result of increased translation of the fluorescent protein Venus, in the presence of the activator. A Synergy hybrid reader was used to excite the cultures at 505 nm and the intensity of the emission was detected at 535 nm. Empty bacteria were used to correct for autofluorescence (IGEM11_WITS_CSIR_SA offered exciting at 514nm and emission at 528nm, but 514&528 is too close for our machine to detect.) “Fluorescence intensity / OD” increases greatly with theophylline concentration.

Fluorescence Microscope could also show this part work beautifully. Except for the difference that though K537009 is an YFP, we excite it at 532nm (green light) and it glow red.

We improve this kind of part, embed an RNA scaffold after similar theophylline aptamer and use split GFP to test how theophylline affect the 3D structure of whole RNA. [http://2012.igem.org/Team:ZJU-China/project.htm ZJU-China 2012 wiki]

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