Team:UC Davis/Electrochemistry Enzyme Tests

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   <h2>Enzyme Tests</h2>
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   <h2>Enzyme Dependent Activity</h2>
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     <span><h2>Enzyme Tests</h2></span>
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     Once set up our electrochemical system to be comparable to our home-made detection device, testing our engineered enzymes in our system is the next step. Aldehyde dehydrogenase has been successfully engineered to have specificity in different chain length of aldehyde group or saturity of aldehyde groups. The data that proves these specificity has been collected through plate reader, which is the device measures the enzyme kinetics. However, unlike the plate reader, our system contains more complex and sensitive issue that closely related to electrochemistry. Therefore, planning out experiments that can measure current in our necessity level of detection, which will also be suitable for out system was the first part of enzyme testing; and the second part was to carry out the experimental data which can be comparable to plate reader data.<br><br>
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     Once set up our electrochemical system to be comparable to our home-made detection device, testing our engineered enzymes in our system is the next step. Aldehyde dehydrogenase has been successfully engineered to have specificity in different chain length of aldehyde group or saturity of aldehyde groups. The data that proves these specificity has been collected through plate reader, which is the device measures the enzyme kinetics. However, unlike the plate reader, our system contains more complex and sensitive issue that closely related to electrochemistry. Therefore, planning out experiments that can measure current in our necessity level of detection, which will also be suitable for out system was the first part of enzyme testing; and the second part was to carry out the experimental data which can be comparable to plate reader data.
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<b><br>Go to the bottom of the page for data!!!</b>
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<p>This portion of experiment is necessary to determine a maximum and minimum concentration of detecting enzyme activity. Since the necessary detection range for substrate concentration was limited to micromolar range(based on aldehyde concentration on rancid olive oil data), determining the amount of enzyme spiked into the substrate was a key. Our protocol was to mix different concentration of pentanal(as a substrate for this experiment) and enzyme to observe each response.
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<p>This portion of the experiment is necessary to determine a maximum and minimum concentration of detecting enzyme activity. Since the necessary detection range for substrate concentration was limited to micromolar range(based on aldehyde concentration on rancid olive oil data), determining the amount of enzyme spiked into the substrate was a key. Our protocol was to mix different concentrations of pentanal substrate with enzyme and to quantify the response with the potentiostat instrument. </p>
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There are many chemicals that are crucial for enzyme survival. This portion of experiment discusses the tests that uncovered 1. what chemicals were compatible with the electrode at various concentrations and 2. what chemicals would introduce the least noise to the signal. The creation of NADH standard curves was employed to demonstrate if the addition of a particular solute would obfuscate signal linearity, or our ability to distinguish signal at all.
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    <li><img src="https://static.igem.org/mediawiki/2014/f/f4/Pentanalstudyfigure1.png"/></li>
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Figure 1 displays the activities detected from 1000µM pentanal with 1µg/ml enzyme. This data indicates that our system can detect pentanal; figure 2 displays comparison of 500 and 1000µM pentanal with the negative control(0µM pentanal) with 1µg/ml enzyme, which implies that our system is able to detect smaller concentration than 1000µM aldehyde(pentanal).  
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With our original protocol, we expected to continue smaller pentanal concentration as 250µM. However, detection level was smaller than 100nA, which requires our resolution of device to be reset to 10nA - 100nA range. Unfortunately, after changing the resolution, noise level was so large that makes the collected data much less reliable, compared to the higher concentration detection. This experiment has suggested that <br><br>
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<ul><li>Necessity for higher enzyme concentration(>>>1µg/ml)</li>
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<li>Electrochemical approach of finding a solution to decrease noise level in the current system </li></ul></p>
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After this conclusion, we have decided to test our enzyme specificity using 10µg/ml enzyme concentration.
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<p>Buffer solution components include: <br></p><br>
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<p><u>Phosphate Buffer + salts(0.1M)</u><br>
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After reviewing multiple literature and research in the electrode that we decided to use(dropsens), we decided to use 0.1M KCl with phosphate buffer. A salt concentration of 0.1M in a buffer of pH 7.4 is a very common solution parameter in electrochemical experiments. This setups allows current to be passed through solution, and is thus crucial to our system. In addition, these ions are not oxidized at the electrode and thus do not contribute noise to the signal. (see figure(2)-referring to Dropsens NADH conductivity experimental data stated 0.1M salt concentration.) <br>
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<u>Reducing agents</u><br>
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<p>Enzyme Specificity</p>
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These reactive chemical species are crucial enzyme stability and ultimately survival, and therefore must be included in the buffer solution. We tested three different reducing agents to see which would be compatible. Their description and compatibility is discussed below.
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After our engineered proteins have successfully shows distinctive activities on different chain length and saturity of aldehydes in plate reader(device measuring enzyme kinetics), our next goal was to conduct an experiment that proves our electrochemical system can also detect these significant activities on each enzymes and display comparable detection to those data from plate reader. <br><br>
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Our experimental protocol always starts with measuring NADH Standard curve. This step is crucial to find out whether our electrode connection is reliable, and detection of damaged electrode. NADH standard run should be measured after and before each enzyme run to check our system. <br><br>
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Three engineered enzyme has been used to prove detection of enzyme specificity in our homemade device:<br>
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<li>enzyme 1 (referred as E.coli)
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<li><u>DTT</u></li><br>
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<li>Proved to have higher response in shorter chain aldehyde than longer chain aldehyde </li>
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Dithiothreitol (DTT) is a reducing agent that is used to stabilize enzymes that contain free sulfhydryl groups by reducing the amount of disulfide bonds formed. To determine the concentration of DTT that would be optimal for our system we generated 3 NADH standard curves. Each curve represents the how 3 different concentrations of DTT (0µM, 1 µM, and 10 µM) affect the linearity of the NADH standard curve. As seen in the graph below, we were able to generate linear NADH curves with all three concentrations of DTT. Therefore, for future experiments we decided to use the highest concentration, 10 µM DTT, for enzyme stabilization.
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<li>Significantly lower response in unsaturated aldehyde(all chain length) </li>
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<li><u>TCEP</u></li><br>
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<li>enzyme 2 (referred as Rat)
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TCEP (tris(2-carboxyethyl)phosphine is one of the reducing agents that we tested within our buffer system.
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<ul><li>Active on all chain length aldehydes, regardless of saturity.</li></ul></li>
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<br>"figure goes here"<br><br>
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<li>enzyme 3 (referred as mutant 3)  
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<li><u>Glycerol</u></li><br>
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<ul><li>Showed significantly higher activity on saturated aldehyde(in all chain length)</li></ul></li>
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Glycerol is a hardy perennial in any enzyme solution due to its important role in enzyme stability. Therefore, similar protocols as those above were conducted to examine its contribution to noise.
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<b> Extraction Buffer</b>
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In order to show reflect this result in our system, we have chose C5 as the representation of “short length aldehyde”; C10 as “long length aldehyde”; EC10 as “unsaturated aldehyde”<br><br><br>
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<b>Order of runs:</b><br>
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1) NADH Standard curve 1
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2) E. coli with C5, C10, EC10
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An objective of the wider project was the extraction of aldehydes from hydrophobic olive oil. This would allow the aldehydes to interact with enzymes and ultimately NADH to transmit a signal at the electrode. To make these aldehydes accessible, an extraction protocol was developed. This technique required the use of a number of solvents--in particular detergents, all of which needed to be tested for possible interfering effects with the electrode.<br>
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3) NADH Standard Curve 2
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Thus we had to examine which detergents, such as Tween and Isopropyl alcohol gave less noise and less interference with other chemicals in the final buffer solution.
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4) Rat enzyme with C5, C10, EC10
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<u>Tween</u>
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5) NADH Standard Curve 3
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One of the easiest and most common method for extracting aldehyde from olive oil is to add tween in the bugger. However, as shown in the figure, after tween has been added to our standard NADH control solution, we can clearly see the noise and interference in our electrode. <br>
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We wanted to test the effect of IPA in the buffer solution. We made a solution of 0M NADH with 1x PBKCl and varied the concentrations of IPA (10 µM, 50 µM, and 100 µM). All three solutions with the varying IPA concentrations gave the same signal. From this we can conclude that IPA within the buffer solution will not affect our ability to detect NADH.  <br>
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6) E. coli Mutant 3 with C5, C10, EC10
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As a system checkup run for NADH standard curve 1, 2, 3 came out to be very similar(as shown in below). This is an indication of no major problem/change in our system in between the enzyme comparison runs.<br><br>
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<p align="center"><img src="https://static.igem.org/mediawiki/2014/e/e3/NADH_comparison.png" width="900px"  style="border:3px solid black"></p>
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<b>Inhibition</b><br><br>
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<b>Enzyme Test Result</b><br><br></p>
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To more definitively correlate aldehyde concentration to output current, we needed to examine the possibility of substrate or product inhibition. Such effects could negate NADH generation via substrate conversion, and thus yield an incorrectly lower signal.<br><br>
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<u>Substrate inhibition</u><br><br>
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The compound capable of substrate inhibition was the aldehyde. We tested the aldehyde pentanal to ascertain possible potential inhibitory effects. We conducted 3 NADH standard curves with NADH concentrations of 0, 250, 500 and 1000µM. The curves generated were conducted as follows:
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    <li><img src="https://static.igem.org/mediawiki/2014/b/bb/Ecoliplatereader.10.png"/></li>
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    <li><img src="https://static.igem.org/mediawiki/2014/2/2c/Specificity2.png"/></li>
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<li>NADH curve without pentanal in solution</li>
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<li>NADH curve with 100µM pentanal in solution</li>
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<p align="center"><img src="https://static.igem.org/mediawiki/2014/b/b6/Threeenzymesalltogether.png" width="900px"  style="border:3px solid black"><br><br></p>
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<b>Conclusion</b>
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After completing each run we saw there was little to no effect on the detection of NADH. The graphs below show these results:
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Figures above shows our experimental data compared to the plate reader data, which has been collected at the same time, using same exact solutions, while we run our experiment on electrode. Data proves the predicted activity on all three engineered enzymes. This can be shown as clear indication of deconvolution of aldehyde concentrations(more than one types of aldehyde) in olive oil using the data from our device.
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<u>Product inhibition(NADH inhibition)</u>
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The effect of the product NADH on the forward reaction was assayed on a plate reader be preparing solutions with a constant concentration of NADH, pentanal, and enzyme, and variable concentrations of NAD+. If the enzyme velocity was approximately the same for all these combinations, then we could conclude that production inhibition would not feature.  
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As shown below, the notion of production inhibition was dispelled because the velocities were found to all be approximately the same.
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<p>Electrical Optimization</p>
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<p>Detailed protocols and data summaries</p>
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<u>Voltage</u><br>
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<a href="https://static.igem.org/mediawiki/2014/4/44/Coupling_enzyme_Protocols.zip">Download Protocols</a>
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<a href="https://static.igem.org/mediawiki/2014/5/5f/Coupling_enzyme_data_summaries.zip">Download Experiment Summaries</a>
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<li>hedging(current infusion-spontaneous current infusion) went away as we changes the current from 0.6 -> 0.7 V
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<li>used higher voltage to reduce noise
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<li>Tin foil cage to remove 60Hz noise -> didn't help
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Latest revision as of 03:58, 18 October 2014

UC Davis iGEM 2014

Electrode Choice

Electrode Choice

System Optimization

System Optimization

Enzyme Dependent Activity

Enzyme Dependent Activity

Overview

Once set up our electrochemical system to be comparable to our home-made detection device, testing our engineered enzymes in our system is the next step. Aldehyde dehydrogenase has been successfully engineered to have specificity in different chain length of aldehyde group or saturity of aldehyde groups. The data that proves these specificity has been collected through plate reader, which is the device measures the enzyme kinetics. However, unlike the plate reader, our system contains more complex and sensitive issue that closely related to electrochemistry. Therefore, planning out experiments that can measure current in our necessity level of detection, which will also be suitable for out system was the first part of enzyme testing; and the second part was to carry out the experimental data which can be comparable to plate reader data.
Go to the bottom of the page for data!!!

Enzyme Activity

This portion of the experiment is necessary to determine a maximum and minimum concentration of detecting enzyme activity. Since the necessary detection range for substrate concentration was limited to micromolar range(based on aldehyde concentration on rancid olive oil data), determining the amount of enzyme spiked into the substrate was a key. Our protocol was to mix different concentrations of pentanal substrate with enzyme and to quantify the response with the potentiostat instrument.



> <



Figure 1 displays the activities detected from 1000µM pentanal with 1µg/ml enzyme. This data indicates that our system can detect pentanal; figure 2 displays comparison of 500 and 1000µM pentanal with the negative control(0µM pentanal) with 1µg/ml enzyme, which implies that our system is able to detect smaller concentration than 1000µM aldehyde(pentanal). With our original protocol, we expected to continue smaller pentanal concentration as 250µM. However, detection level was smaller than 100nA, which requires our resolution of device to be reset to 10nA - 100nA range. Unfortunately, after changing the resolution, noise level was so large that makes the collected data much less reliable, compared to the higher concentration detection. This experiment has suggested that

  • Necessity for higher enzyme concentration(>>>1µg/ml)
  • Electrochemical approach of finding a solution to decrease noise level in the current system



After this conclusion, we have decided to test our enzyme specificity using 10µg/ml enzyme concentration.

Enzyme Specificity

After our engineered proteins have successfully shows distinctive activities on different chain length and saturity of aldehydes in plate reader(device measuring enzyme kinetics), our next goal was to conduct an experiment that proves our electrochemical system can also detect these significant activities on each enzymes and display comparable detection to those data from plate reader.

Our experimental protocol always starts with measuring NADH Standard curve. This step is crucial to find out whether our electrode connection is reliable, and detection of damaged electrode. NADH standard run should be measured after and before each enzyme run to check our system.

Three engineered enzyme has been used to prove detection of enzyme specificity in our homemade device:

  1. enzyme 1 (referred as E.coli)
    • Proved to have higher response in shorter chain aldehyde than longer chain aldehyde
    • Significantly lower response in unsaturated aldehyde(all chain length)
  2. enzyme 2 (referred as Rat)
    • Active on all chain length aldehydes, regardless of saturity.
  3. enzyme 3 (referred as mutant 3)
    • Showed significantly higher activity on saturated aldehyde(in all chain length)

    4. In order to show reflect this result in our system, we have chose C5 as the representation of “short length aldehyde”; C10 as “long length aldehyde”; EC10 as “unsaturated aldehyde”


    Order of runs:
    1) NADH Standard curve 1
    2) E. coli with C5, C10, EC10
    3) NADH Standard Curve 2
    4) Rat enzyme with C5, C10, EC10
    5) NADH Standard Curve 3
    6) E. coli Mutant 3 with C5, C10, EC10

    As a system checkup run for NADH standard curve 1, 2, 3 came out to be very similar(as shown in below). This is an indication of no major problem/change in our system in between the enzyme comparison runs.




    Enzyme Test Result

    > <


> <





Conclusion Figures above shows our experimental data compared to the plate reader data, which has been collected at the same time, using same exact solutions, while we run our experiment on electrode. Data proves the predicted activity on all three engineered enzymes. This can be shown as clear indication of deconvolution of aldehyde concentrations(more than one types of aldehyde) in olive oil using the data from our device.

Detailed protocols and data summaries

Download Protocols Download Experiment Summaries

Retrieved from "http://2014.igem.org/Team:UC_Davis/Electrochemistry_Enzyme_Tests"