Difference between revisions of "Part:BBa K902090:Experience"
 
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===Applications of BBa_K902090===
 
===Applications of BBa_K902090===
  
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The pre-existing registry part (BBa_I732901) was initially tested for functionality.  This was done by adding  a 1 mM solution of CPRG to the supernatant of cells grown overnight and induced with 200 mM IPTG.  As the LacZ enzyme, if produced, should be able to cleave CPRG which is yellow into CPR which is a bright red, a visual colorimetric assay was used where we looked for a color change.  Unfortunately, this color change did not occur, indicating that cleavage of CPRG was not occurring, likely occurring to a lack of LacZ production.  This part was sequenced, and it was determined to have a frameshift mutation.  A new part was constructed and the assay repeated.  The figure below (figure 1) shows the results of these two assays.
  
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This part was tested electrochemically.  CPRG was added to the solution, which is functional LacZ is produced, should be cleaved to CPR, which can be detected electrochemically using a potentiostat via an increase in current.  CPR is able to oxidize at a specific voltage resulting in a change in current.  The change in current can be correlated to the activity level of the promoter.  Our results can be seen in figure 1 below.
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[[Image:UofC_LacZ_color_change_data.png‎|thumb|600px|centre|Figure 1. Visual assay of LacZ cleavage of CPRG.  A 1 mM solution of CPRG was added to cells expressing an inducible LacZ circuit that were grown up ovenight in LB and then induced with 200 mM IPTG.  If LacZ was being produced, cleavage of CPRG into CPR should have occurred, resulting in a color change from yellow to a vivid red.  The image on the left shows this assay done on the BBa_i732901 part.  The image on the right shows a comparIson between the BBa_I232901 part on the left and the newly constructed BBa_K902090 part. ]]
  
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[[Image:UofC_LacZ_Data!.png|700px|centre|thumb|Figure 1. Current production from the lacZ and uidA systems under the IPTG inducible promoter. Both samples were run with the same conditions and held at the oxidation potentials of their respective analytes.]]
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<p>This data showed that the new circuit was able to restore its functionality.  Proper production of LacZ was occurring as demonstrated by the cleavage of CPRG indicated by the visible color change in the solution.</p>
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<p>This part was then characterized electrochemically.  When CPRG is present in a solution where the <i>lacZ</i> gene is being expressed, the functional enzyme can cleave the sugar bond to form galactose and CPR.  CPR is able to oxidize at a specific voltage (1.325V vs RHE), resulting in a change in current as electrons are released.  The change in current can be correlated to the activity level of the promoter from the amount of CPR produced.  Our results can be seen in figure 2 below.</p>
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[[Image:UofC_LacZ_Data!.png|700px|centre|thumb|Figure 2. Current production from the lacZ and uidA systems under the IPTG inducible promoter. Both samples were run with the same conditions and held at the oxidation potentials of their respective analytes in 25mL of 0.1M pH7 PBS.]]
  
 
In this figure we are measuring the change in current for this circuit, as well as for a circuit producing Beta-glucuronidase (UidA) under the same promoter.  This enzyme cleaves a different substrate, PNPG, into PNP which also oxidizes, but at a different voltage to CPR.  This allows for the two to be detected simultaneously.  The differenece that can be seen in their induction levels is due to the fact that the analyte produced through the action of UidA produces a product that oxidizes to release two electrons while the LacZ product only releases one electron when oxidized. As the current we measure is this release of electrons, a similar amount of the two enzymes would results in the doubling of current for uidA that we saw.  This graph thus indicates that our two circuits are working exactly as we expect them to!
 
In this figure we are measuring the change in current for this circuit, as well as for a circuit producing Beta-glucuronidase (UidA) under the same promoter.  This enzyme cleaves a different substrate, PNPG, into PNP which also oxidizes, but at a different voltage to CPR.  This allows for the two to be detected simultaneously.  The differenece that can be seen in their induction levels is due to the fact that the analyte produced through the action of UidA produces a product that oxidizes to release two electrons while the LacZ product only releases one electron when oxidized. As the current we measure is this release of electrons, a similar amount of the two enzymes would results in the doubling of current for uidA that we saw.  This graph thus indicates that our two circuits are working exactly as we expect them to!

Latest revision as of 03:17, 29 October 2012

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Applications of BBa_K902090

The pre-existing registry part (BBa_I732901) was initially tested for functionality. This was done by adding a 1 mM solution of CPRG to the supernatant of cells grown overnight and induced with 200 mM IPTG. As the LacZ enzyme, if produced, should be able to cleave CPRG which is yellow into CPR which is a bright red, a visual colorimetric assay was used where we looked for a color change. Unfortunately, this color change did not occur, indicating that cleavage of CPRG was not occurring, likely occurring to a lack of LacZ production. This part was sequenced, and it was determined to have a frameshift mutation. A new part was constructed and the assay repeated. The figure below (figure 1) shows the results of these two assays.

Figure 1. Visual assay of LacZ cleavage of CPRG. A 1 mM solution of CPRG was added to cells expressing an inducible LacZ circuit that were grown up ovenight in LB and then induced with 200 mM IPTG. If LacZ was being produced, cleavage of CPRG into CPR should have occurred, resulting in a color change from yellow to a vivid red. The image on the left shows this assay done on the BBa_i732901 part. The image on the right shows a comparIson between the BBa_I232901 part on the left and the newly constructed BBa_K902090 part.

This data showed that the new circuit was able to restore its functionality. Proper production of LacZ was occurring as demonstrated by the cleavage of CPRG indicated by the visible color change in the solution.


This part was then characterized electrochemically. When CPRG is present in a solution where the lacZ gene is being expressed, the functional enzyme can cleave the sugar bond to form galactose and CPR. CPR is able to oxidize at a specific voltage (1.325V vs RHE), resulting in a change in current as electrons are released. The change in current can be correlated to the activity level of the promoter from the amount of CPR produced. Our results can be seen in figure 2 below.

Figure 2. Current production from the lacZ and uidA systems under the IPTG inducible promoter. Both samples were run with the same conditions and held at the oxidation potentials of their respective analytes in 25mL of 0.1M pH7 PBS.

In this figure we are measuring the change in current for this circuit, as well as for a circuit producing Beta-glucuronidase (UidA) under the same promoter. This enzyme cleaves a different substrate, PNPG, into PNP which also oxidizes, but at a different voltage to CPR. This allows for the two to be detected simultaneously. The differenece that can be seen in their induction levels is due to the fact that the analyte produced through the action of UidA produces a product that oxidizes to release two electrons while the LacZ product only releases one electron when oxidized. As the current we measure is this release of electrons, a similar amount of the two enzymes would results in the doubling of current for uidA that we saw. This graph thus indicates that our two circuits are working exactly as we expect them to!

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