Team:IvyTech-South Bend/Project

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== Project Details==
== Project Details==
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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  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.
 +
 
 +
Our observation is consistent with the idea that an increased expression of ArsR within the cell silences the leaky pArs and raises the threshold of the response.  Another possibility is that the RFP gene is expressed at a lower level by virtue of it being in a polycistronic location downstream from the ArsR.
 +
 
 +
To test this idea we first turned the BBa_J33201 part into an ArsR-generator by adding a doulbe terminar.  Our plan was that this would constitute the gain or tuneable element in a biosensor system. We then built BBa_K935002 to test the effect of the position of the ArsR generator on pArs controlled promoter activity.  We found that the responsiveness and the senstivity of J33201 in J61002 and our BBa_K935002 to be essentially the same. 
 +
 
 +
Not wanting apparent success to get in our way we hypothesized that the addition of a second ArsR generator under pArs control should further selectivley raise the threshold of the device. We therefore added assembled our "tuner element" BBa_K9350001 upstream once and twice to create BBa_K935004 and BBa-K935005, respectively. We subsequently observed a significant decrease in the sensitivity of pArs as measured by mRFP1 production.
 +
 
 +
We have subsequently employed our device to assess groundwater arsenate/arsenite presence in local waters and in samples of international waters as well.
 +
 
 +
“Behold the Arsenator!”
<img src=http://fc08.deviantart.net/fs10/i/2006/095/d/c/The_Water_by_leepawlowicz.jpg  
<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>
<b> Make sure your water is the best, use our arsenic test!!! </b>

Latest revision as of 02:53, 4 October 2012

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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.

Our observation is consistent with the idea that an increased expression of ArsR within the cell silences the leaky pArs and raises the threshold of the response. Another possibility is that the RFP gene is expressed at a lower level by virtue of it being in a polycistronic location downstream from the ArsR.

To test this idea we first turned the BBa_J33201 part into an ArsR-generator by adding a doulbe terminar. Our plan was that this would constitute the gain or tuneable element in a biosensor system. We then built BBa_K935002 to test the effect of the position of the ArsR generator on pArs controlled promoter activity. We found that the responsiveness and the senstivity of J33201 in J61002 and our BBa_K935002 to be essentially the same.

Not wanting apparent success to get in our way we hypothesized that the addition of a second ArsR generator under pArs control should further selectivley raise the threshold of the device. We therefore added assembled our "tuner element" BBa_K9350001 upstream once and twice to create BBa_K935004 and BBa-K935005, respectively. We subsequently observed a significant decrease in the sensitivity of pArs as measured by mRFP1 production.

We have subsequently employed our device to assess groundwater arsenate/arsenite presence in local waters and in samples of international waters as well.

“Behold the Arsenator!”

<img src=http://fc08.deviantart.net/fs10/i/2006/095/d/c/The_Water_by_leepawlowicz.jpg Make sure your water is the best, use our arsenic test!!!

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