Difference between revisions of "Part:BBa K2066117:Experience"

 
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===Applications of BBa_K2066117===
 
===Applications of BBa_K2066117===
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This synthetic enhancer part includes a 2x TetO binding cassette in the spacer region between the enhancer and sigma 54 promoter, and depending on the steady state of TetR, the rigidity of DNA changes and thus affects the thermodynamic capability of looping and activation of transcription.
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This part is sequence confirmed and synthetic enhancer sequences were derived from Amit et. al. 2011 and UNS sequences from Torella et. al. 2012.
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We were able to characterize the part by transforming it with Bba_K2066037, which expresses TetR and NRII to affect the rigidity of the spacer region and phosphorylation of activator NRI (bound to the enhancer), respectively.
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To characterize this part in BL21 and 10beta E.coli we used small chemical induction with aTc, which binds to and inactivates available TetR repressor. When a small amount of aTc is present, the steady state of TetR is still high enough to allow for complete repression of circuit. As you increase the amount of aTc in the circuit, the steady state of activated TetR decreases and the rigidity of the DNA, which previously prevented the looping and activation of transcription, decreases and gives rise to an intermediate step. At a high aTc concentrations, almost all of the TetR is bound and inactivated and you reach a saturated level of fluorescent expression.
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We used flow cytometry to find the absolute fluorescent measures over a gradient of aTc induction. However, we weren't able to observe discrete steps with this part. 
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For more information on the mechanism of the synthetic enhancer circuit, refer to our page: http://2016.igem.org/Team:William_and_Mary/Synthetic_Enhancer.
  
 
===User Reviews===
 
===User Reviews===

Revision as of 03:55, 29 October 2016


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_K2066117

This synthetic enhancer part includes a 2x TetO binding cassette in the spacer region between the enhancer and sigma 54 promoter, and depending on the steady state of TetR, the rigidity of DNA changes and thus affects the thermodynamic capability of looping and activation of transcription.

This part is sequence confirmed and synthetic enhancer sequences were derived from Amit et. al. 2011 and UNS sequences from Torella et. al. 2012.

We were able to characterize the part by transforming it with Bba_K2066037, which expresses TetR and NRII to affect the rigidity of the spacer region and phosphorylation of activator NRI (bound to the enhancer), respectively.

To characterize this part in BL21 and 10beta E.coli we used small chemical induction with aTc, which binds to and inactivates available TetR repressor. When a small amount of aTc is present, the steady state of TetR is still high enough to allow for complete repression of circuit. As you increase the amount of aTc in the circuit, the steady state of activated TetR decreases and the rigidity of the DNA, which previously prevented the looping and activation of transcription, decreases and gives rise to an intermediate step. At a high aTc concentrations, almost all of the TetR is bound and inactivated and you reach a saturated level of fluorescent expression.

We used flow cytometry to find the absolute fluorescent measures over a gradient of aTc induction. However, we weren't able to observe discrete steps with this part.

For more information on the mechanism of the synthetic enhancer circuit, refer to our page: http://2016.igem.org/Team:William_and_Mary/Synthetic_Enhancer.

User Reviews

UNIQ0c30e69b5fe311e0-partinfo-00000000-QINU UNIQ0c30e69b5fe311e0-partinfo-00000001-QINU