Team:Goettingen/Team/Reporter
From 2013.igem.org
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Since it was unclear, where the exact beginning and ending of the ''ydaO'' promoter, the ''ydaO'' riboswitch and the ''ydaO'' RBS were, we created four different constructs (Fig. 2.; [http://parts.igem.org/Part:BBa_K1045004 BBa_K1045004], [http://parts.igem.org/Part:BBa_K1045005 BBa_K1045005], [http://parts.igem.org/Part:BBa_K1045006 BBa_K1045006], [http://parts.igem.org/Part:BBa_K1045007 BBa_K1045007]). The sequence of [http://parts.igem.org/Part:BBa_K1045004 BBa_K1045004] was cloned in front of the reporter gene CFP ([http://parts.igem.org/Part:BBa_E0020 BBa_E0020]). For the termination of transcription, we exploited the terminator on pSB1C3. In principle, the riboswitch reporter system might respond in exactly the same way to c-di-AMP, as the DarR reporter system does: Without c-di-AMP, E. coli cells harboring [http://parts.igem.org/Part:BBa_K1045002 BBa_K1045002] will fluoresce blue. In contrast, when c-di-AMP is present, the ''ydaO'' riboswitch will terminate transcription of ''cfp'' leading to non-fluorescent cells (Fig. 2.2). This way, we can scan substances which can act on the riboswitch as c-di-AMP does. This might lead to the identification of competitors or inhibitors for future antibiotics, as well. | Since it was unclear, where the exact beginning and ending of the ''ydaO'' promoter, the ''ydaO'' riboswitch and the ''ydaO'' RBS were, we created four different constructs (Fig. 2.; [http://parts.igem.org/Part:BBa_K1045004 BBa_K1045004], [http://parts.igem.org/Part:BBa_K1045005 BBa_K1045005], [http://parts.igem.org/Part:BBa_K1045006 BBa_K1045006], [http://parts.igem.org/Part:BBa_K1045007 BBa_K1045007]). The sequence of [http://parts.igem.org/Part:BBa_K1045004 BBa_K1045004] was cloned in front of the reporter gene CFP ([http://parts.igem.org/Part:BBa_E0020 BBa_E0020]). For the termination of transcription, we exploited the terminator on pSB1C3. In principle, the riboswitch reporter system might respond in exactly the same way to c-di-AMP, as the DarR reporter system does: Without c-di-AMP, E. coli cells harboring [http://parts.igem.org/Part:BBa_K1045002 BBa_K1045002] will fluoresce blue. In contrast, when c-di-AMP is present, the ''ydaO'' riboswitch will terminate transcription of ''cfp'' leading to non-fluorescent cells (Fig. 2.2). This way, we can scan substances which can act on the riboswitch as c-di-AMP does. This might lead to the identification of competitors or inhibitors for future antibiotics, as well. | ||
+ | <html><a href="https://static.igem.org/mediawiki/2013/4/44/Goe-rt-riboswitchsystem.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/4/44/Goe-rt-riboswitchsystem.png" width="750"></a></html> | ||
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+ | ''Figure 2.1. The riboswitch reporter system is composed of the ydaO riboswitch and cfp as a reporter gene.'' | ||
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+ | <html><a href="https://static.igem.org/mediawiki/2013/c/cd/Goe-rt-riboRsystem.png" target="_blank"><img src="https://static.igem.org/mediawiki/2013/c/cd/Goe-rt-riboRsystem.png" width="750" /></a></html> | ||
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+ | ''Figure 2.2. In theory, E. coli cells transformed with the riboswitch reporter system will fluoresce blue when c-di-AMP is absent. When c-di-AMP is present, however, the ydaO riboswitch will prevent expression of CFP.'' | ||
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+ | <html> | ||
+ | <p><b>Reference:</b></p> | ||
+ | <p><a href="http://www.nature.com/nchembio/journal/v8/n12/abs/nchembio.1095.html" target="_blank">Peter Y Watson & Martha J Fedor (2012) “The ydaO motif is an ATP-sensing riboswitch in Bacillus subtilis“, Nature Chemical Biology No. 8, pp. 963-96</a></p> | ||
+ | </html> | ||
+ | |||
+ | |||
+ | ==Results== | ||
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Revision as of 09:47, 1 October 2013