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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   <a href="https://2014.igem.org/Team:UC_Davis/Electrochemistry_Enzyme_Tests">
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     <span><h2>Enzyme Tests</h2></span>
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     <span><h2>Enzyme Dependent Activity</h2></span>
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<p>Overview</p>
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<p>
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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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<br><br>
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</p>
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</div>
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</div>
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<div class="mainTitleHeader">
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<p>Enzyme Activity</p>
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</div>
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<div class="mainContainer">
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<div class="mainContainerCenterTopPic">
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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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    <li><img src="https://static.igem.org/mediawiki/2014/f/f4/Pentanalstudyfigure1.png"/></li>
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    <li><img src="https://static.igem.org/mediawiki/2014/6/6c/Pentanalstudy2.png" /></li>
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</ul>
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</div><p><br><br>
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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).
 +
 
 +
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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<p>
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<br><br>
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After this conclusion, we have decided to test our enzyme specificity using 10µg/ml enzyme concentration.
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<br>
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</p>
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</div>
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</div>
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<div class="mainTitleHeader">
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<p>Enzyme Specificity</p>
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</div>
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<div class="mainContainer">
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<div class="mainContainerCenterTopPic">
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<p>
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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>
 +
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>
 +
Three engineered enzyme has been used to prove detection of enzyme specificity in our homemade device:<br>
 +
<ol>
 +
<li>enzyme 1 (referred as E.coli)
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<ul>
 +
<li>Proved to have higher response in shorter chain aldehyde than longer chain aldehyde </li>
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<li>Significantly lower response in unsaturated aldehyde(all chain length) </li>
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</ul></li>
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<li>enzyme 2 (referred as Rat)
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<ul><li>Active on all chain length aldehydes, regardless of saturity.</li></ul></li>
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<li>enzyme 3 (referred as mutant 3)
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<ul><li>Showed significantly higher activity on saturated aldehyde(in all chain length)</li></ul></li>
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</ul>
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</p>
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<p>
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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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<br>
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2) E. coli with C5, C10, EC10
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<br>
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3) NADH Standard Curve 2
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<br>
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4) Rat enzyme with C5, C10, EC10
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<br>
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5) NADH Standard Curve 3
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<br>
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6) E. coli Mutant 3 with C5, C10, EC10
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<br><br>
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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>
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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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<br><br><br>
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<p>
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<b>Enzyme Test Result</b><br><br></p>
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  <ul>
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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><img src="https://static.igem.org/mediawiki/2014/4/4f/Mut3specificity.png"/>
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    <li><img src="https://static.igem.org/mediawiki/2014/a/a1/Ecolienzymeactivity.png"/></li>
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    <li><img src="https://static.igem.org/mediawiki/2014/8/8b/Rat.png"/></li>
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    <li><img src="https://static.igem.org/mediawiki/2014/6/69/Mut3.png"/>
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  </ul> 
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</div><p>
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<br><br> </p>
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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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<p>
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<b>Conclusion</b>
 +
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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</p>
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</div>
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</div>
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<div class="mainTitleHeader">
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<p>Detailed protocols and data summaries</p>
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</div>
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<div class="mainContainer">
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<div class="mainContainerCenterTopPic">
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<p>
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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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</p>
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</div>
</div>
</div>
</body>
</body>
</html>
</html>

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"