Team:MIT mammalian Switch
From 2010.igem.org
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<br> The three components of this system has been fully constructed and are ready for transfection into mammalian cells. Unfortunately at the present, due to time constraints, we are unable to provide conclusive data supporting the functionality of the toggle. The figure below show a preliminary experiment during which PonS was added post transient calcium phosphate transfection of a HEK293FT cell line containing the Hef1a_VgEcR_RxR construct with the other two components of the toggle. CMV_EGFP was transfected in other wells to control for transfection efficiency. | <br> The three components of this system has been fully constructed and are ready for transfection into mammalian cells. Unfortunately at the present, due to time constraints, we are unable to provide conclusive data supporting the functionality of the toggle. The figure below show a preliminary experiment during which PonS was added post transient calcium phosphate transfection of a HEK293FT cell line containing the Hef1a_VgEcR_RxR construct with the other two components of the toggle. CMV_EGFP was transfected in other wells to control for transfection efficiency. | ||
</tr> | </tr> | ||
- | <tr><td><img src=https://static.igem.org/mediawiki/2010/0/07/Simplified_Toggle_Micrographs.png> | + | <tr><td><img src=https://static.igem.org/mediawiki/2010/0/07/Simplified_Toggle_Micrographs.png width=100%> |
<p>There is significant increase in fluorescence in the cells transfected with the toggle within 7 hours after the addition of ponS. We did not have enough DNA to perform a parallel transfection without PonS addition; however, we argue that the increase in fluorescence we saw 7 hours after PonS addition is much greater than in the previous 41 hours post co-transfection. Thus it can be concluded that the co-tranfescted construct shows EYFP fluorescence with, but not without, PonS induction. | <p>There is significant increase in fluorescence in the cells transfected with the toggle within 7 hours after the addition of ponS. We did not have enough DNA to perform a parallel transfection without PonS addition; however, we argue that the increase in fluorescence we saw 7 hours after PonS addition is much greater than in the previous 41 hours post co-transfection. Thus it can be concluded that the co-tranfescted construct shows EYFP fluorescence with, but not without, PonS induction. | ||
+ | <li> No DNA is shown as a control for auto-fluorescence. | ||
+ | <li> CMV_EGFP transfection showed >30% transfection efficiency (not shown). | ||
</td> | </td> | ||
Revision as of 10:40, 27 October 2010
Synthetic Switch: Implementation of Bistable Toggles in Mammalian Cells |
It is useful for a synthetic system capable of sensing mechanical stimuli to be able to convert a transient signal into a constitutive response. During the summer we designed both a complete and a simplified bistable toggle with a low and high output state using positive feedback loops.
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The three components of this system has been fully constructed and are ready for transfection into mammalian cells. Unfortunately at the present, due to time constraints, we are unable to provide conclusive data supporting the functionality of the toggle. The figure below show a preliminary experiment during which PonS was added post transient calcium phosphate transfection of a HEK293FT cell line containing the Hef1a_VgEcR_RxR construct with the other two components of the toggle. CMV_EGFP was transfected in other wells to control for transfection efficiency. |
![]() There is significant increase in fluorescence in the cells transfected with the toggle within 7 hours after the addition of ponS. We did not have enough DNA to perform a parallel transfection without PonS addition; however, we argue that the increase in fluorescence we saw 7 hours after PonS addition is much greater than in the previous 41 hours post co-transfection. Thus it can be concluded that the co-tranfescted construct shows EYFP fluorescence with, but not without, PonS induction. |