Team:IvyTech-South Bend/Project

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Project Details

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 on/off or safe/danger device.

If then there was however, there was someway to raise the threshold of the bacterial arsenic promoter this could form the basis of a simple binary device having 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

First: how the arsenic inducible promoter works.....

A very leaky arsenic sensitive promoter has 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 [is this not known?]

One approach that we entertained was to duplicate the control region to create a series of ArsR binding sites between the promoter and reporter.

We discounted this idea because, perhaps like the Lac operon, the control region includes the 3’ end of the RNA polymerase’ sigma subunit attachment site [known]. 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, to increase the binding of arsenic entering the cell.

Thanks to the Edinburgh Team a composite part, J33201, had been created for us to test this idea: [describe structure]

We 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

<img src= Make sure your water is the best, use our arsenic test!!!

@@ Line 18: / Line 18: @@
 == Project Details==
-We 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)
+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.
-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.
-Not wanting apparent success to get in our way we hypothesized that the addition of a second ArsR gene under pArs control should further raise the threshold of the device. We therefore added a terminiator to J33201 and coupled it upstream from J33201 and as a control K190015. A down stream cut of K190015 from J661002 expression plasmid yields pArs+RBS+RFP+Term.
+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 on/off or safe/danger device.
+If then there was however, there was someway to raise the threshold of the bacterial arsenic promoter this could form the basis of a simple binary device having 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
+First: how the arsenic inducible promoter works.....
+A very leaky arsenic sensitive promoter has 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 [is this not known?]
+One approach that we entertained was to duplicate the control region to create a series of ArsR binding sites between the promoter and reporter.
+We discounted this idea because, perhaps like the Lac operon, the control region includes the 3’ end of the RNA polymerase’ sigma subunit attachment site [known].  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, to increase the binding of arsenic entering the cell.
+Thanks to the Edinburgh Team a composite part, J33201, had been created for us to test this idea:  [describe structure]
+We 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
 <img src=http://fc08.deviantart.net/fs10/i/2006/095/d/c/The_Water_by_leepawlowicz.jpg
 <b> Make sure your water is the best, use our arsenic test!!! </b>