Difference between revisions of "Part:BBa K537009:Experience"
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===Applications of BBa_K537009===
 
===Applications of BBa_K537009===
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ZJU-China 2012 Characterizes BioBrick Part K537009
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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.
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Competent E. coli (strain DH5a) cells were transformed with plasmid vectors containing the “Promoter-Theophylline riboswitch -Venus-Double terminator”. The bacterial colony appeal pink.
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[[Image:ZJU colony.jpg]]
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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.
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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.)
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“Fluorescence intensity / OD” increases greatly with theophylline concentration.
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[[Image:ZJU sheet.jpg]]
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Fluorescence Microscope could also show this part work beautifully.
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Except for the difference that though K537009 is an YFP, we excite it at 532nm (green light) and it glow red.
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[[Image:ZJU fluorescent.jpg]]
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We improve this part, embed an RNA scaffold after the theophylline aptamer in K539003 in K537009 to become riboscaffold [https://parts.igem.org/Part:BBa_K738002 K738002], thus empower K537003 & K537009 with new application.
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We use split GFP to test how theophylline affect the 3D structure of whole RNA.
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[http://2012.igem.org/Team:ZJU-China/project.htm ZJU-China 2012 wiki]
  
 
===User Reviews===
 
===User Reviews===
 
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<I>Experience by Wits-CSIR_SA iGEM2011</I>
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<I>Username</I>
 
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<center><img src="https://static.igem.org/mediawiki/2011/7/73/Datasheet1.JPG" width="750" vspace="15"></center>
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<p>For the pdf version of this datasheet, click <a href="https://static.igem.org/mediawiki/2011/2/27/Datasheet1.pdf">here</a></p>
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Latest revision as of 12:51, 10 October 2012

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

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. ZJU colony.jpg


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.

ZJU sheet.jpg



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.

ZJU fluorescent.jpg


We improve this part, embed an RNA scaffold after the theophylline aptamer in K539003 in K537009 to become riboscaffold K738002, thus empower K537003 & K537009 with new application. We 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]

User Reviews

UNIQ606b5762b54610e0-partinfo-00000000-QINU UNIQ606b5762b54610e0-partinfo-00000001-QINU