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From 2012.igem.org

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One major shortcoming however of using the bacterial arsenic-sensitive promoter (pArs) to control a biosensor device is that pArs has a very low threshold of activation. For example transformants of the Gronigen pArs part K190015 in the Endy lab expression vector J61002 are universally red without any added arsenic.

Reporters for bacterial biosensor devices created with E.coli pArs thus must measure an analog (or digital) output of signal to register the amount of arsenic present. We think that this will add unnecessary technical sophistication to what should be a simple device.

If then there was someway to raise the threshold of the bacterial arsenic promoter "genetically" this could form the basis of a simple binary device. This device would have then only two states: off =safe on=danger thus simplifying is use and maximizing its utility in the developing world.

We took this as the engineering goal for our project and entertained a number of approaches

A very leaky arsenic sensitive promoter possibly evolved to protect a cell from the danger of this metaloid and the basis of this is perhaps the affinity of the ArsR for the control region.

One approach that we entertained was to duplicate the promoter binding site for ArsR and to create a series of ArsR binding sites between the promoter and reporter. We discounted this idea because even in a free state, a series of control regions between the promoter and reporter might not be structurally conducive to RNA scanning to initiate transcription.

Another approach we entertained was to perhaps increase the expression of ArsR within the cell. We reasoned that this would have two non-mutually exclusive effects: 1. to increase the amount of bound ArsR through shifting the equilibrium to the right or, more likely, 2. to increase the binding of arsenic entering the cell (create an arsenic sink).

Thanks to the Edinburgh Team a composite part, J33201, had been created for us to test this idea!

The Edinburgh team assembled the following favorite device: pArs-ArsR-[open reading frame]

We therefore compared the sensitivity of J33201 to the Gronigen Team’s K190015 pArs site using the Endy Lab’s expression vector J61002. We loaded J33201 into a kanamycin resistant backbone pSB1K3 and exchanged it with K190015 by perfoming an upstream cut of both to leave the RFP in the J61002 backbone. [ratio of J33201 to K190015 plasmid DNA was 3:1) If the increased expression of ArsR had the effect of increasing the threshold of the arsenic response any resulting colonies of J33201 in J61002 we predicted would appear white. To our delight this is what we found.

[Photo of transformation results][Graph of the time course and sensitivity of J33201 and K190015 to increasing concentrations of sodium arsenite]

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