Team:William and Mary/Composite Part

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Composite Part

Bba_K2066114 is a composite part composed of a two step synthetic enhancer as well as a NRII2303 mutant integrated into the UNS region flanked onto the BioBrick backbone. This part, which was derived from Amit et al 2012 (“Building Enhancers from the Ground Up: a Synthetic Biology Approach”) and uses an enhancer mediated by NRI-P to loop around and kinetically interact with the poised σ54 polymerase (see synthetic enhancer page) coding for the proteins NRI and mCherry. By inserting a TetO cassette with three TetR repressor binding sites, one can modulate the flexibility of the spacer region of the DNA and ultimately affect the looping propensity and transcriptional activation.

Figure 1: Adapted from Amit et. al. 2011. The NRI-P activated enhancer can loop around to the poised promoter and activate transcription of NRI and fluorescent reporter. The presence of TetR repressor proteins makes the looping much harder and thermodynamically unfavorable as it makes the DNA less flexible. Using aTc inductions, we can modulate how much TetR is available to bind to the TetO sites and at different levels of aTc concentration, you get different binding states of the spacer region and thus discrete levels of output.

This part produces a transfer function that contains three discrete outputs, low, intermediate step and high, depending on the amount of apo-tetR. Since the synthetic enhancer requires both NRI and an NRII mutant, this circuit is complete and ready to be used in as part of the Circuit Control Toolbox. By putting NRII on the same plasmid as the synthetic enhancer suite, you minimize metabolic strain on the bacteria and decrease the interference of this genetic circuit with others present in the cell.

This part would be introduced as the final step in a given circuit and either modified to give the desired product (removal and replacement of mCherry with desired product), or used as is as a reporter. The existing circuit would then have its final output changed to TetR, so as to produce a 2 step transfer function.

Figure 2: This part was used in conjunction with Bba_I739001 to produce a multimodal output response curve and was compared to the model using the original plasmids. aTc induction did create a step-wise dose response regulatory graph for mCherry expression that looks similar to that of the Amit et. al. reproduction by WM iGEM 2016.


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