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Team:Calgary/Project/Reporter
From 2011.igem.org
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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- | + | 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> | ||
| - | Our first challenge, was | + | 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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Revision as of 19:25, 28 September 2011
The Project
Reporter
What is a Reporter?
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.
Why Electrochemical?
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 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.
A Novel Application for the lacZ Gene
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.Building our System
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.For preliminary data, click here.
For characterization data, click here.
