Difference between revisions of "Part:BBa K1791000:Experience"
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The theophylline-responsive aptazyme used in our project was previously engineered to function in vivo in the yeast species Saccharomyces cerevisiae (Win and Smolke 2007). The device was designed to modulate the cleavage of the host RNA via addition of exogenous theophylline (theophylline is not normally present in yeast cells). Binding of theophylline to the aptazyme is predicted to induce a conformational change in the RNA that promotes a cleavage competent structure. Initially, we wanted to determine if the device was viable under in vitro conditions. To test this, we generated PCR products of the full length aptazyme and used this as a template for in vitro transcription by T7 RNA polymerase (Fig. 1). | The theophylline-responsive aptazyme used in our project was previously engineered to function in vivo in the yeast species Saccharomyces cerevisiae (Win and Smolke 2007). The device was designed to modulate the cleavage of the host RNA via addition of exogenous theophylline (theophylline is not normally present in yeast cells). Binding of theophylline to the aptazyme is predicted to induce a conformational change in the RNA that promotes a cleavage competent structure. Initially, we wanted to determine if the device was viable under in vitro conditions. To test this, we generated PCR products of the full length aptazyme and used this as a template for in vitro transcription by T7 RNA polymerase (Fig. 1). | ||
| − | [[Media: | + | [[Media:Uleth15_Uleth15_Theophylline_AptazymeIVT_Sept_1_2015.jpg]] |
We readily detected large amounts of aptazyme in vitro transcript (Fig 1), however we noted that a significant amount of the transcript was truncated and of a length consistent with cleaved aptazyme, even in the absence of theophylline. We hypothesized that the premature cleavage of the aptazyme may have resulted from the temperature conditions in which our in vitro transcription was performed; non-optimal temperatures may impair proper folding of the structure resulting in premature cleavage during in vitro transcription. We therefore tried performing the in vitro transcription at room temperature and 37°C (Fig. 2). | We readily detected large amounts of aptazyme in vitro transcript (Fig 1), however we noted that a significant amount of the transcript was truncated and of a length consistent with cleaved aptazyme, even in the absence of theophylline. We hypothesized that the premature cleavage of the aptazyme may have resulted from the temperature conditions in which our in vitro transcription was performed; non-optimal temperatures may impair proper folding of the structure resulting in premature cleavage during in vitro transcription. We therefore tried performing the in vitro transcription at room temperature and 37°C (Fig. 2). | ||
Revision as of 01:04, 22 September 2015
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Applications of BBa_K1791000
THEOPHYLLINE APTAZYME IN VITRO:
The theophylline-responsive aptazyme used in our project was previously engineered to function in vivo in the yeast species Saccharomyces cerevisiae (Win and Smolke 2007). The device was designed to modulate the cleavage of the host RNA via addition of exogenous theophylline (theophylline is not normally present in yeast cells). Binding of theophylline to the aptazyme is predicted to induce a conformational change in the RNA that promotes a cleavage competent structure. Initially, we wanted to determine if the device was viable under in vitro conditions. To test this, we generated PCR products of the full length aptazyme and used this as a template for in vitro transcription by T7 RNA polymerase (Fig. 1).
Media:Uleth15_Uleth15_Theophylline_AptazymeIVT_Sept_1_2015.jpg
We readily detected large amounts of aptazyme in vitro transcript (Fig 1), however we noted that a significant amount of the transcript was truncated and of a length consistent with cleaved aptazyme, even in the absence of theophylline. We hypothesized that the premature cleavage of the aptazyme may have resulted from the temperature conditions in which our in vitro transcription was performed; non-optimal temperatures may impair proper folding of the structure resulting in premature cleavage during in vitro transcription. We therefore tried performing the in vitro transcription at room temperature and 37°C (Fig. 2).
Media:Uleth15_Theophylline_Aptazyme_Sept_2_2015.jpg Fig 2.Theophylline Aptazyme in vitro transcription at 24°C and 37°C time course.
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