Team:Calgary/Project/Reporter

From 2011.igem.org

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       <span id="theory"><h2>What is a Reporter?</h2></span>
       <span id="theory"><h2>What is a Reporter?</h2></span>
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       <p> It isn't enough to just be able to detect something - after the presence of naphthenic acids are confirmed by our promoter, we require some way for our bacteria to report back results that can be interpreted by an observer. Over the course of the project, Team Calgary considered three different reporter systems - colorimetric, fluorescent, and electrochemical.</p>
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       <p> Being able to detect a particle in laboratory conditions can vary drastically from conditions <i>in situ.</i> After the presence of naphthenic acids is confirmed, we will require a reporter system for our bacteria to report their concentration accurately and with high resolution. Over the course of the project, Team Calgary considered three different reporter systems - colorimetric, fluorescent, and electrochemical. After weighing the advantages and disadvantages of each approach, we decided to use the electrochemical approach.  </p>
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      <span id="practice"><h2>Why Electrochemical?</h2></span>
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<span id="practice"><h2>Why Electrochemical?</h2></span>
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      <p>Oilsands tailings ponds samples are difficult to work with for several different reasons. First of all, tailings ponds samples are murky, and can have greatly varying compositions from pond to pond. It would be difficult to obtain accurate colorimetric data from these samples, and it would be similarly difficult to readily observe fluorescence. Because an electrochemical response does not rely on being able to see anything, Team Calgary determined the electrochemical reporter to be ideal for this purpose.</p>
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</html>[[image:UofC_tpw.jpg|thumb|200px|right|<b>Figure 1</b> Tailings pond water is often murky and turbid, with an unknown composition of pollutants within.]]<html>
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<p>Electrochemical reporters offer several advantages over visually-determined reporters. The signal is robust - environmental factors such as the presence of pollutants or sample turbidity do not affect the signal. Every molecule has a different oxidation potential - because we know the exact oxidation potential for our analyte, we can easily tell the difference between our desired signal and background noise. Another advantage of an electrochemical response is that the data is immediately available in digital form. This allows for fast graphical interpretation of the data as well as maximum information collection from the data. </p>
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<p>Oilsands tailings ponds samples are difficult to work with for several different reasons. First of all, tailings ponds samples are murky, and can have greatly varying compositions from pond to pond. It would be difficult to obtain accurate colorimetric data from these samples, and it would be similarly difficult to readily observe fluorescence. Because an electrochemical response does not rely on being able to see anything, Team Calgary determined the electrochemical reporter to be ideal for this purpose.</p>
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       <h2>A Novel Application for the lacZ Gene</h2></span>
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<p>Electrochemical reporters offer several advantages over colorimetric and fluorometric reporters: the signal is robust enough to not be altered by the presence of pollutants or sample turbidity; the oxidation potential of our analyte is distinct and can be readily distinguished between our desired signal and background noise; and the data is immediately available for analysis. </p>
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<h2>A Novel Application for the <i>lacZ</i> Gene</h2></span>
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</html>[[image:UofC2011_BGAL.png|thumb|400px|left|<b>Figure 2</b> Schematic diagram for the conversion of CPGR to CPR and Galactose.  CPR can be oxidized at a specific voltage to produce an electrochemical output.]]<html>
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<p>We decided to use the<i> lacZ </i>gene in our electrochemical reporter.  Although traditionally, the <i>lacZ</i> gene (BBa_I732005) is used as a reporter for colorimetric assays (cleaving X-gal to create a blue pigment), the gene product ß-galactosidase, is capable of cleaving a variety of substrates.  It has been shown by Biran <i>et al.</i> (1999) that ß-galactosidase can cleave p-aminophenyl-ß-D-galactopyranoside (PAPG), producing p-aminophenol (PAP).  PAP can be oxidized at an electrode enabling detection of an electrochemical signal.  It’s been shown that chlorophenolred-ß-D-galactopyranoside (CPRG) can also be cleaved by beta-galactosidase, producing chlorophenol red (CPR) and galactose.  This substrate produces both a distinct color change (yellow to purple), and an electrochemical signal.</p>
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We decided to use the lacZ gene in our electrochemical reporter.  Traditionally, the lacZ gene is used as a reporter for blue-white screening.  The gene product, beta-galactosidase, is an enzyme which is capable of cleaving various substrates.  When x-gal is added for exmaple, beta-galactosidase cleaves it, producing a visible blue color.  Recently however, Biran et al showed that beta-galactosidase can also cleave p-aminophenyl-ß-D-galactopyranoside (PAPG), producing p-aminophenol (PAP).  PAP can be oxidized at an electrode and the signal converted into a current signal.  It’s been shown that chlorophenolred-ß-D-galactopyranoside (CPRG) can also be cleaved by beta-galactosidase, producing chlorophenol red (CPR) and galactosidase.  This produces a distinct color change (yellow to purple), and can also be oxidized at an electrode and converted into a current signal.
 
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<h2>Building our System</h2></span>
<h2>Building our System</h2></span>
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Our first challenge, was determining how we would read a signal from our system.  We spent a lot of time thinking about what hardware could be used and found out that a potentiostat was the best option. A potentiostat is the electronic hardware needed to control the three electrode system used to measure the data in our experiments. It keeps the working electrode at a constant potential with respect to the reference electrode by adjusting and measuring the current between the working electrode and counter electrode. We obtained our data using a professional lab grade potentiostat, but we also spent some time looking into how they are designed and managed to built our own prototype. While it is built on a breadboard and is bigger than the potentiostat we used for the measurements, it is a valid proof of concept that potentiostats can be built using electrical circuit components no more complicated than an operational amplifier.
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<p>One of the main reasons we selected a primarily electrochemical response for our system was that the response can be detected and processed digitally, without the need for subjective qualitative observations. The CPR analyte has an oxidation potential that is distinct from the tailings pond environment making it distinguishable from the background.</p>
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<p>The voltammetric technique we used to detect chlorophenol red was cyclic voltammetry - the input voltage is shown below.</p>
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</html>[[image:Calgary2011_appliedpotential.png|thumb|400px|right|<b>Figure 3</b> Applied potential across the cell during cyclic voltammetry.]]<html>
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<p>As voltage slowly increases and decreases, each species present in solution will undergo a redox reaction at its respective oxidation potential. Because we know the oxidation potential of our analyte, chlorophenol red (CPR), we can ignore any background noise or pollution using this method.</p>
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<p>A three-electrode setup is required to perform cyclic voltammetry. The electrical current passes between the working and counter electrodes while the potential difference between the reference electrode and counter electrode is controlled. This setup allows us to take voltage and current readings that do not interfere with each other.</p>
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</html>[[image:Calgary2011_potentiostat.png|thumb|400px|center|<b>Figure 4</b> A schematic for a potentiostat suitable for performing cyclic voltammetry. Source: Gopinath and Russell, 2005.]]<html>
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<p>In order to perform cyclic voltammetry, specialized equipment is usually required. Such equipment is rare in biology labs, so we have created potentiostat prototypes - a device that outputs a known voltage and measures a corresponding current between two electrodes. These prototypes are cheap to build and have been shown to function as expected. However, for our measurements we used a commercial potentiostat and it's accompanying software to reduce the amount of time needed for data analysis.</p>
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</html>[[image:Calgary2011_ourpotentiostat.jpg|thumb|600px|center|<b>Figure 5</b> Two homemade potentiostats capable of detecting chlorophenol red oxidation. The one on the left uses a breadboard as it's backbone, while the one on the right is soldered onto a prototyping board.]]<html>
For preliminary data, <a href="https://2011.igem.org/Team:Calgary/Project/Preliminary_Data">click here</a>.
For preliminary data, <a href="https://2011.igem.org/Team:Calgary/Project/Preliminary_Data">click here</a>.
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For characterization data, <a href="https://2011.igem.org/Team:Calgary/Project/Promoter/Final_Data">click here</a>.
For characterization data, <a href="https://2011.igem.org/Team:Calgary/Project/Promoter/Final_Data">click here</a>.
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For electrochemical optimization, <a href="https://2011.igem.org/Team:Calgary/Project/Reporter/Optimization">click here</a><br>
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Latest revision as of 03:32, 29 October 2011


Reporter

What is a Reporter?

Being able to detect a particle in laboratory conditions can vary drastically from conditions in situ. After the presence of naphthenic acids is confirmed, we will require a reporter system for our bacteria to report their concentration accurately and with high resolution. Over the course of the project, Team Calgary considered three different reporter systems - colorimetric, fluorescent, and electrochemical. After weighing the advantages and disadvantages of each approach, we decided to use the electrochemical approach.

Why Electrochemical?

Figure 1 Tailings pond water is often murky and turbid, with an unknown composition of pollutants within.

Oilsands tailings ponds samples are difficult to work with for several different reasons. First of all, tailings ponds samples are murky, and can have greatly varying compositions from pond to pond. It would be difficult to obtain accurate colorimetric data from these samples, and it would be similarly difficult to readily observe fluorescence. Because an electrochemical response does not rely on being able to see anything, Team Calgary determined the electrochemical reporter to be ideal for this purpose.

Electrochemical reporters offer several advantages over colorimetric and fluorometric reporters: the signal is robust enough to not be altered by the presence of pollutants or sample turbidity; the oxidation potential of our analyte is distinct and can be readily distinguished between our desired signal and background noise; and the data is immediately available for analysis.

A Novel Application for the lacZ Gene

Figure 2 Schematic diagram for the conversion of CPGR to CPR and Galactose. CPR can be oxidized at a specific voltage to produce an electrochemical output.

We decided to use the lacZ gene in our electrochemical reporter. Although traditionally, the lacZ gene (BBa_I732005) is used as a reporter for colorimetric assays (cleaving X-gal to create a blue pigment), the gene product ß-galactosidase, is capable of cleaving a variety of substrates. It has been shown by Biran et al. (1999) that ß-galactosidase can cleave p-aminophenyl-ß-D-galactopyranoside (PAPG), producing p-aminophenol (PAP). PAP can be oxidized at an electrode enabling detection of an electrochemical signal. It’s been shown that chlorophenolred-ß-D-galactopyranoside (CPRG) can also be cleaved by beta-galactosidase, producing chlorophenol red (CPR) and galactose. This substrate produces both a distinct color change (yellow to purple), and an electrochemical signal.


Building our System

One of the main reasons we selected a primarily electrochemical response for our system was that the response can be detected and processed digitally, without the need for subjective qualitative observations. The CPR analyte has an oxidation potential that is distinct from the tailings pond environment making it distinguishable from the background.

The voltammetric technique we used to detect chlorophenol red was cyclic voltammetry - the input voltage is shown below.

Figure 3 Applied potential across the cell during cyclic voltammetry.

As voltage slowly increases and decreases, each species present in solution will undergo a redox reaction at its respective oxidation potential. Because we know the oxidation potential of our analyte, chlorophenol red (CPR), we can ignore any background noise or pollution using this method.

A three-electrode setup is required to perform cyclic voltammetry. The electrical current passes between the working and counter electrodes while the potential difference between the reference electrode and counter electrode is controlled. This setup allows us to take voltage and current readings that do not interfere with each other.

Figure 4 A schematic for a potentiostat suitable for performing cyclic voltammetry. Source: Gopinath and Russell, 2005.

In order to perform cyclic voltammetry, specialized equipment is usually required. Such equipment is rare in biology labs, so we have created potentiostat prototypes - a device that outputs a known voltage and measures a corresponding current between two electrodes. These prototypes are cheap to build and have been shown to function as expected. However, for our measurements we used a commercial potentiostat and it's accompanying software to reduce the amount of time needed for data analysis.

Figure 5 Two homemade potentiostats capable of detecting chlorophenol red oxidation. The one on the left uses a breadboard as it's backbone, while the one on the right is soldered onto a prototyping board.
For preliminary data, click here.
For characterization data, click here.
For electrochemical optimization, click here