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| − | <h2> Project Description </h2>
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| − | <p>The goal of our project is to create a biosensor for the detection of triclosan. Triclosan is an antimicrobial agent that works by preventing bacteria from synthesizing fatty acids. More specifically, triclosan competitively inhibits enoyl ACP reductase (FabI), the enzyme that catalyzes the last step of fatty acid synthesis.</p>
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| − | <img src="https://static.igem.org/mediawiki/2015/b/bb/Team_UC_Davis_biosynthesispathway.png" style="float:right;width:640px;height:280px;">
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| − | <p> FabI uses an electron from NADH to reduce crotonyl ACP. Because triclosan inhibits FabI from reducing crotonyl ACP, there will be higher levels of NADH in the presence of triclosan. By measuring levels of NADH, we will be able to infer levels of triclosan.
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| − | We will mix up enoyl reductase (enzyme), crotonyl-coA (substrate), NADH (cofactor), and triclosan (inhibitor) and drop a sample onto our screen-printed electrode, which will be connected to our potentiostat.
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| − | We will use the potentiostat to control the potential of the working electrode at a fixed value relative to the reference electrode. The applied potential serves as the driving force for the full reduction of the electroactive species (NADH).
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| − | It is important to note that the sample on the electrode is divided into two regions: the diffusion region and the bulk region and that the applied potential only affects the diffusion region. In this diffusion region, the current is governed by Fick’s Law of Diffusion, which states that the magnitude of current is proportional to the concentration gradient of NADH.
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| − | When we apply the potential, all of the NADH in the diffusion region will be oxidized. Since current is proportional to concentration gradient, there will be a huge spike in current. Over time, NADH from the bulk solution will diffuse back onto the electrode, making the concentration gradient less pronounced. This mellowing out of the concentration gradient translates to a steady stating out of current.
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| − | The steady state value of the current is proportional to the concentration of NADH in the bulk solution.
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| − | A solution with enzyme, substrate, and NADH that is allowed to incubate for some time will have less NADH than starting out with (due to the enzyme turning over NADH). If we add triclosan into the mix, the enzyme will still use up some NADH, but it will use up less because it is inhibited. Each of these solutions will have different levels of NADH and will thus generate different current values.
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| − | By comparing the current values, we will be able to determine how different levels of triclosan affect the magnitude of current. The idea is that for a sample of unknown triclosan, we will be able to correlate the current read out with the sample’s concentration of triclosan.
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| − | </html>
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