Difference between revisions of "Team:Stuttgart/Results"

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<h1>Results</h1>
 
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<p>Here you can describe the results of your project and your future plans. </p>
 
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<h5>What should this page contain?</h5>
 
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<ul>
 
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<li> Clearly and objectively describe the results of your work.</li>
 
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<li> Future plans for the project. </li>
 
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<li> Considerations for replicating the experiments. </li>
 
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<h5>You should also describe what your results mean: </h5>
 
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<li> Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for. </li>
 
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<li> Show data, but remember all measurement and characterization data must be on part pages in the Registry. </li>
 
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<li> Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project. </li>
 
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<h1 align=middle> Results </h1>
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<div class="container">
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    <div class="row section">
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<h3>Esterases and Lipases</h3>
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<br>
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<div class="col-xs-12 col-sm-2 col-md-2">
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<!--<https://static.igem.org/mediawiki/2017/4/45/EsteraseResults.png">-->
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<img src="https://static.igem.org/mediawiki/2017/7/7f/EsteraseundLipase.png" class="img-responsive"/>
 
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<h4>Enzyme activity assay: Esterases</h4>
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            <p>In the following figures enzyme activities of the supernatant of the esterases EstCS2 and LIpB are shown. All results were obtained from biological triplicates. The detailed method for the activity determination is shown here: LINK
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                At the N-terminal end of the esterase EstCS2 a signal peptide (PelB) is added. Therefore the gene induction leads to enzyme expression and the expression of the signalpeptide and the secretion of the enzyme is enabled.  
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<h5> Project Achievements </h5>
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<p>You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.</p>
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<li>A list of linked bullet points of the successful results during your project</li>
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<li>A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.</li>
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<!--<https://static.igem.org/mediawiki/2017/4/45/EsteraseResults.png">-->
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<h5>Inspiration</h5>
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<h8>Figure 3: Illustration of the esterase activity [U] of EstCS2 in the supernatant dependend on time and substrate concentration (2.5, 5, 10, 15, 20 mM). The standard abbreviation was calculated from biological triplicates.</h8>
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<p>See how other teams presented their results.</p>
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<li><a href="https://2014.igem.org/Team:TU_Darmstadt/Results/Pathway">2014 TU Darmstadt </a></li>
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<li><a href="https://2014.igem.org/Team:Imperial/Results">2014 Imperial </a></li>
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<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Results">2014 Paris Bettencourt </a></li>
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<p>The enzyme activity of EstCS2 raises with the induction level for gene expression and with the substrate concentration. A maximum of 246,5 U is gained with 20 mM p-Nitrophenyl butyrate and 1 mM arabinose. The enzyme activities between the induction levels of 2 mM arabinose and 3 mM arabinose isn’t different. The enzyme activity is highest with an induction level of 1 mM arabinose instead of an induction level of 2 or 3 mM arabinose. This could be explained with less movement flexibility of the esterases if high gene expression rates are induced. We would further recommend to induce the gene expression with less arabinose concentrations and investigate the enzyme activity with 0.1 mM, 0.5 mM and 1 mM arabinose. </p>
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<!--<https://static.igem.org/mediawiki/2017/a/a0/EsteraseResults2.png">-->
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<img src="https://static.igem.org/mediawiki/2017/a/a0/EsteraseResults2.png" class="img-responsive"/>
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<h8>Figure 4: Illustration of the esterase activity [U] of E. coli wilde types in the supernatant dependend on time and substrate concentration (2.5, 5, 10, 15, 20 mM). The standard abbreviation was calculated from biological triplicates.
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</h8>
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        <div class="col-xs-12 col-sm-4 col-md-4">
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            <p>In comparison we investigated the enzyme activity of two E. coli wilde type strains (E. coli DH5α and MG1655). We aimed to aquire knowledge about the esterase activity of the wild typs to identify the effect of genetically engineered organisms. The figures show less enzyme activity of the wild types in comparison to the genetically engineered E. coli that contain the plasmid with the esterase genes. This means that the investigated EstCS2 is an appropriate enzyme to degradade fat layers on hair.
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<div class="col-xs-12 col-sm-8 col-md-8">
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  <!--<https://2017.igem.org/File:EsteraseResults3.png">-->
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  <img src="https://static.igem.org/mediawiki/2017/3/3a/EsteraseResults3.png" class="img-responsive"/>
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  <h8>Figure 5:Illustration of the esterase activity [U] of LipB in the supernatant dependend on time and substrate concentration (2.5, 5, 10, 15, 20 mM). The standard abbreviation was calculated from biological triplicates.
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  </h8>
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              <p>Additionally we investigated the enzyme activity of LipB. To compare the enzyme activities of LipB and EstCS2, we used the same induction levels and substrate concentrations for the assays. The figures show that the enzyme activity of the supernatant isn’t higher than the enzyme activity of the supernatant of the wild type cells. These results can be declared with the absence of a signal peptide at the N-terminal side of the esterase gene. Thus, no enzyme secretion is performed and less enzyme activity can be detected in the supernatant. This leads to the conclusion that a signal peptide has to be added at the N-terminal side of the esterase gene to obtain enzyme secretion and extracellular enzyme activity.
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Latest revision as of 10:30, 30 October 2017

Results

Esterases and Lipases


Enzyme activity assay: Esterases

In the following figures enzyme activities of the supernatant of the esterases EstCS2 and LIpB are shown. All results were obtained from biological triplicates. The detailed method for the activity determination is shown here: LINK At the N-terminal end of the esterase EstCS2 a signal peptide (PelB) is added. Therefore the gene induction leads to enzyme expression and the expression of the signalpeptide and the secretion of the enzyme is enabled.

Figure 3: Illustration of the esterase activity [U] of EstCS2 in the supernatant dependend on time and substrate concentration (2.5, 5, 10, 15, 20 mM). The standard abbreviation was calculated from biological triplicates.

The enzyme activity of EstCS2 raises with the induction level for gene expression and with the substrate concentration. A maximum of 246,5 U is gained with 20 mM p-Nitrophenyl butyrate and 1 mM arabinose. The enzyme activities between the induction levels of 2 mM arabinose and 3 mM arabinose isn’t different. The enzyme activity is highest with an induction level of 1 mM arabinose instead of an induction level of 2 or 3 mM arabinose. This could be explained with less movement flexibility of the esterases if high gene expression rates are induced. We would further recommend to induce the gene expression with less arabinose concentrations and investigate the enzyme activity with 0.1 mM, 0.5 mM and 1 mM arabinose.

Figure 4: Illustration of the esterase activity [U] of E. coli wilde types in the supernatant dependend on time and substrate concentration (2.5, 5, 10, 15, 20 mM). The standard abbreviation was calculated from biological triplicates.

In comparison we investigated the enzyme activity of two E. coli wilde type strains (E. coli DH5α and MG1655). We aimed to aquire knowledge about the esterase activity of the wild typs to identify the effect of genetically engineered organisms. The figures show less enzyme activity of the wild types in comparison to the genetically engineered E. coli that contain the plasmid with the esterase genes. This means that the investigated EstCS2 is an appropriate enzyme to degradade fat layers on hair.

Figure 5:Illustration of the esterase activity [U] of LipB in the supernatant dependend on time and substrate concentration (2.5, 5, 10, 15, 20 mM). The standard abbreviation was calculated from biological triplicates.

Additionally we investigated the enzyme activity of LipB. To compare the enzyme activities of LipB and EstCS2, we used the same induction levels and substrate concentrations for the assays. The figures show that the enzyme activity of the supernatant isn’t higher than the enzyme activity of the supernatant of the wild type cells. These results can be declared with the absence of a signal peptide at the N-terminal side of the esterase gene. Thus, no enzyme secretion is performed and less enzyme activity can be detected in the supernatant. This leads to the conclusion that a signal peptide has to be added at the N-terminal side of the esterase gene to obtain enzyme secretion and extracellular enzyme activity.