Team:Calgary/Project/Reporter

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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-B-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 detremining 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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<h2> Preliminary Testing</h2>
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<h2>Background and Rationale</h2>
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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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<p>For the purposes of testing out the LacZ reporter as an electrochecmical detector it was important to first assess how well the reporter performed at cleaving the chlorophenol red β-D-galactopyranoside  (CPRG) into Chlorophenol red (CPR). The LacZ gene that was chosen for this experiment as well as for the electrochemical testing was fused to an IPTG inducible promoter(LacI), or part I732901. Overall I732901 the purpose of the experiment was to ensure that the E. coli could be induced to produce β-galactosidase, which could then cleave chlorophenol red β-D-galactopyranoside  (CPRG) to produce Chlorophenol red (CPR). Since CPR has a very dark characteristic red color at pH 7 the reaction wasto be kept at that constant pH using a phosphate buffer. This experiment was also expected to provide useful data for electrochemical side of things (i.e. oxidizing the CPR and measuring either the current or voltage) by determining what sort of time frame would be needed for the E. coli to produce a significant amount of CPR by cleavage of CPRG providing a guideline for future electrochemical testing.</p>
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<h2>Methods</h2>
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<p>E. coli carrying the plasmid were subcultured cultured until log phase then exposed to PBS buffer containing 3mM CPRG and 1mM IPTG for fixed lengths of time before measuring the absorbance of the solution. A detailed procedure are recorded in <a href=”https://2011.igem.org/Team:Calgary/Notebook/Protocols/Process12”>protocols section.</a></p>
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<h2>Results</h2>
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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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</html>[[Image:UCalgary2011 CPR standard Curve.png|thumb|600px|center|<b>Figure 1.</b> A standard curve measuring the absorbance of known concentrations of CPR. Concentrations ranged from0.5 mM to 2.5 mM, and the CPR was dissolved in PBS buffer (pH 7).]]<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:UCalgary2011 CPR results in OD.png|thumb|600px|center|<b>Figure 2. </b> OD readings measuring the concentration of CPR which is formed by cleavage of CPRG. E. coli cells carrying I732901 were induced to cleave CPRG by the presence of IPTG the concentrations of which were 3mM and 1mM, respectively.]]<html>
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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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</html>[[Image:UCalgary2011 CPRG REsults in mM.png|thumb|600px|center|<b>Figure 3.</b> The concentration of CPR produced overtime by the E. coli in the presences of 3mM CPRG and 1mM IPTG at a constant pH of 7.]]<html>
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<h2>Discussion of Results</h2>
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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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<p>Figure 3 indicates that the concentration of CPR clearly increases over time in this mixture. Illustrating that the IPTG is successfully inducing the LacZ gene to produce β-galactosidase. This data will be useful to the electrochemical detector side of the project because they will now have data from which they can choose a time when they should begin oxidizing the CPR formed by the E. coli (by cleavage of CPRG). </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>
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<p>One drawback experienced in this experiment was that the Victor plate reader which was used to take OD readings could not give data past an OD of ~3 for CPR absorption, meaning that even samples with an extremely high concentration of CPR would at best have an absorbance reading similar to a ~3 mM sample. Thus, this method was not particularly accurate for highly concentrated solutions of CPR. However the results did reveal that under the specified conditions about 1mM of CPR could be produced in around 30 minutes revealing that the system responded quite rapidly when induced with IPTG.<p>
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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>.
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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