Part:BBa_K2066118:Experience

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Applications of BBa_K2066118

This synthetic enhancer part includes a 3x 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 houses the TetR and NRII expression needed to affect the rigidity of the spacer region and phosphorylation of activator NRI (bound to the enhancer), respectively.

For characterization of the multistate response, we used small chemical induction with aTc 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 two intermediate steps. 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 a plate reader to find the absolute fluorescent measurements over a gradient of induction. We are able to see four discrete steps allowing for control over a multistate output.

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.

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