Difference between revisions of "Team:BGU ISRAEL/ImprovedParts"

 
(7 intermediate revisions by 2 users not shown)
Line 3: Line 3:
 
<html>
 
<html>
  
−
<div class="container-fluid">
+
<nav class="navbar navbar-inverse navbar-fixed-top" role="navigation">
 +
            <div class="container">
 +
                <div class="navbar-header">
 +
                    <button type="button" class="navbar-toggle collapsed" data-toggle="collapse" data-target="#navbar" aria-expanded="false" aria-controls="navbar">
 +
                        <span class="sr-only">Toggle navigation</span>
 +
                        <span class="icon-bar"></span>
 +
                        <span class="icon-bar"></span>
 +
                        <span class="icon-bar"></span>
 +
                    </button>
 +
                    <a class="navbar-brand" href="https://2016.igem.org/Team:BGU_ISRAEL"><img class="OurLogo" alt="bgu_logo_plasticure" src="https://static.igem.org/mediawiki/2016/b/b2/White_plasticure_for_bar_site.png">
 +
                    </a>
 +
                </div>
 +
                <div id="navbar" class="navbar-collapse collapse">
 +
                    <ul class="mainmenu nav navbar-nav">
 +
                        <li class="dropdown" id="HomeB"><a class="dropdown-toggle" href="https://2016.igem.org/Team:BGU_ISRAEL">Home</a>
 +
                        <li class="dropdown" id="projectB"><a class="dropdown-toggle" href="https://2016.igem.org/Team:BGU_ISRAEL/Project">Project</a>
 +
                            <ul class="dropdown-menu submenubgu">
 +
                                <li><a href="https://2016.igem.org/Team:BGU_ISRAEL/Description">Description</a></li>
 +
                                <li><a href="https://2016.igem.org/Team:BGU_ISRAEL/Design">Design</a></li>
 +
                                <li><a href="https://2016.igem.org/Team:BGU_ISRAEL/Experiments">Experiments</a></li>
 +
                                <li><a href="https://2016.igem.org/Team:BGU_ISRAEL/Results">Results</a></li>
 +
                                <li><a href="https://2016.igem.org/Team:BGU_ISRAEL/Protocols">Protocols</a></li>
 +
                                <li><a href="https://2016.igem.org/Team:BGU_ISRAEL/Notebook">Notebook</a></li>
 +
                                <li><a href="https://2016.igem.org/Team:BGU_ISRAEL/Achievements">Achievements</a></li>
 +
                            </ul>
 +
                        </li>
 +
                        <li class="dropdown" id="teamB"><a href="https://2016.igem.org/Team:BGU_ISRAEL/Team">Team</a>
 +
                            <ul class="dropdown-menu submenubgu">
 +
                                <li><a href="https://2016.igem.org/Team:BGU_ISRAEL/TeamMembers">Members</a></li>
 +
                                <li><a href="https://2016.igem.org/Team:BGU_ISRAEL/MeetUniversity">Meet Our University</a></li>
 +
                                <li><a href="https://2016.igem.org/Team:BGU_ISRAEL/MeetTown">Meet Our Town</a></li>
 +
                                <li><a href="https://2016.igem.org/Team:BGU_ISRAEL/Collaborations">Collaborations</a></li>
 +
                            </ul>
 +
                        </li>
 +
 
 +
                        <li class="dropdown" id="partsB" style="background-color: #77DD77;"><a class="dropdown-toggle" href="https://2016.igem.org/Team:BGU_ISRAEL/Parts">Parts</a>
 +
                            <ul class="dropdown-menu submenubgu">
 +
                                <li><a href="https://2016.igem.org/Team:BGU_ISRAEL/Parts#Overview">Overview</a></li>
 +
                                <li><a href="https://2016.igem.org/Team:BGU_ISRAEL/Basic_Part">Basic Parts</a></li>
 +
                                <li><a href="https://2016.igem.org/Team:BGU_ISRAEL/ImprovedParts">Improved Parts</a></li>
 +
                            </ul>
 +
                        </li>
 +
                        <li class="dropdown" id="humanB"><a class="dropdown-toggle" href="https://2016.igem.org/Team:BGU_ISRAEL/Integrated_Practices">Human Practices</a>
 +
                            <ul class="dropdown-menu submenubgu">
 +
                                <li><a href="https://2016.igem.org/Team:BGU_ISRAEL/Integrated_Practices">Integrated Practices</a></li>
 +
                                <li><a href="https://2016.igem.org/Team:BGU_ISRAEL/PlasticArt">Plastic Art</a></li>
 +
                                <li><a href="https://2016.igem.org/Team:BGU_ISRAEL/EthicsAndSafety">Ethics & Safety</a></li>
 +
                            </ul>
 +
                        </li>
 +
                        <li class="dropdown" id="attributionsB"><a class="dropdown-toggle" href="https://2016.igem.org/Team:BGU_ISRAEL/Attributions">Attributions</a></li>
 +
                        <li class="dropdown" id="awardsB"><a class="dropdown-toggle" href="https://2016.igem.org/Team:BGU_ISRAEL/Awards">Awards</a>
 +
                            <ul class="dropdown-menu submenubgu">
 +
                                <li><a href="https://2016.igem.org/Team:BGU_ISRAEL/Entrepreneurship">Entrepreneurship</a></li>
 +
                                <li><a href="https://2016.igem.org/Team:BGU_ISRAEL/Measurements">Measurements</a></li>
 +
                                <li><a href="https://2016.igem.org/Team:BGU_ISRAEL/Proof">Proof Of Concept</a></li>
 +
                                <li><a href="https://2016.igem.org/Team:BGU_ISRAEL/Demonstrate">Demonstrate</a></li>
 +
                                <li><a href="https://2016.igem.org/Team:BGU_ISRAEL/HP/Silver">HP - Silver</a></li>
 +
                                <li><a href="https://2016.igem.org/Team:BGU_ISRAEL/HP/Gold">HP - Gold</a></li>
 +
                                <li><a href="https://2016.igem.org/Team:BGU_ISRAEL/Engagement">Engagements</a></li>
 +
                            </ul>
 +
                        </li>
 +
                    </ul>
 +
                </div><!--/.navbar-collapse -->
 +
            </div>
 +
        </nav>
 +
 
 +
 
 +
        <div class="container-fluid">
 +
            <div class="row">
 +
                <div class="PartColor">
 +
                    <p>Improved Parts</p>
 +
                </div>
 +
            </div>
 +
        </div>
 +
        <div class="container-fluid">
 
             <div class='row  whiteback'>
 
             <div class='row  whiteback'>
−
                 <div class="col-lg-1"></div>
+
                 <div class="col-lg-2"></div>
−
                 <div class="col-lg-10">
+
                 <div class="col-lg-8">
 
                     <div class='firstrowPart'></div>
 
                     <div class='firstrowPart'></div>
 
                     <div class='partText'>
 
                     <div class='partText'>
Line 13: Line 87:
 
                         </p>
 
                         </p>
 
                         <p>
 
                         <p>
−
                             We have chosen to test our improved variants' activity using a pNP-Butyrate degradation assay at different substrate and enzyme concentrations.
+
                             We have chosen to test our improved variants' activity using a pNP-Butyrate degradation assay with different substrate and enzyme concentrations.
 
                         </p>
 
                         </p>
 
                         <p>
 
                         <p>
−
                             We believe that our proteins are significant improvements of existing part <a href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K936000'>BBa_K936000</a> due to the following reasons:
+
                             Our experiments support that our designed proteins are significantly improved compared to the existing part <a href='http://parts.igem.org/wiki/index.php?title=Part:BBa_K936000'>BBa_K936000</a> due to the following reasons:
 
                         </p>
 
                         </p>
−
                         <ol type='A'>
+
                         <p>
−
                             <li>Our experiments show a significant improvement of pNP-B degradation activity in 2 of our variants: <br>
+
                             <b>First</b>, our experiments showed a significant improvement of pNP-B degradation activity in two of our LC-Cutinase variants:
−
                                <ol>
+
                        </p>
−
                                    <li>The codon optimized version (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2091004">BBa_K2091004</a>) </li>
+
                        <ol>
−
                                    <li>The F4 variant (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2091005">BBa_K2091005</a>)</li>
+
                            <li>The codon optimized version (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2091004">BBa_K2091004</a>) </li>
−
                                </ol>
+
                            <li>The F4 variant (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2091005">BBa_K2091005</a>)</li>
−
                                <p>
+
                        </ol>
−
                                    Our conclusion is based on kinetic test results which showed faster rise in O.D levels compared to the W.T. protein in 3 different substrate concentration (Fig. 1,2,3):
+
                        <p>
−
                                </p>
+
                            Our conclusion is based on kinetic test results which showed faster rise in O.D levels compared to the W.T. protein in three different substrate concentration (Fig. 1,2,3):
−
                                <div class='row'>
+
                        </p>
−
                                    <div class='col-lg-6'>
+
                        <div class='row'>
−
                                        <figure>
+
                            <div class='col-lg-6'>
−
                                            <img src='https://static.igem.org/mediawiki/2016/5/5f/Pnp_50uMBGU.jpg' class='ImprovedPics'>
+
                                <figure class="improvedPicsWrapper">
−
                                            <figcaption>
+
                                    <img src='https://static.igem.org/mediawiki/2016/5/5f/Pnp_50uMBGU.jpg' class='ImprovedPics'>
−
                                                <p>
+
                                    <figcaption>
−
                                                    <b>Figure 1:</b> pNP-Butyrate degradation activity of all LC-Cutinase variants and W.T. at a substrate concentration of 50μM.  
+
                                        <p>
−
                                                    For control we used E. coli strain BL-21 without any vector ("BL-21") with pACYC plasmid backbone only ("pACYC").
+
                                            <b>Figure 1:</b> pNP-Butyrate degradation activity of all LC-Cutinase variants and W.T. at a substrate concentration of 50μM.  
−
                                                </p>
+
                                            For negative control we used <i>E. coli</i> strain BL-21 without any vector ("BL-21") and also <i>E. coli</i> strain BL-21 with only the pACYC plasmid backbone ("pACYC").
−
                                            </figcaption>
+
                                        </p>
−
                                        </figure>
+
                                    </figcaption>
−
                                    </div>
+
                                </figure>
−
                                    <div class='col-lg-6'>
+
                            </div>
−
                                        <figure>
+
                            <div class='col-lg-6'>
−
                                            <img src='https://static.igem.org/mediawiki/2016/6/68/Pnp125uMBGU.jpg' class='ImprovedPics'>
+
                                <figure class="improvedPicsWrapper">
−
                                            <figcaption>
+
                                    <img src='https://static.igem.org/mediawiki/2016/6/68/Pnp125uMBGU.jpg' class='ImprovedPics'>
−
                                                <p>
+
                                    <figcaption>
−
                                                    <b>Figure 2:</b> pNP-Butyrate degradation activity of all LC-Cutinase variants and W.T. at a substrate concentration of 125μM.  
+
                                        <p>
−
                                                    Controls are the same as with 50μM concentration.
+
                                            <b>Figure 2:</b> pNP-Butyrate degradation activity of all LC-Cutinase variants and W.T. at a substrate concentration of 125μM.  
−
                                                </p>
+
                                            Controls are the same as with 50μM concentration.
−
                                            </figcaption>
+
                                        </p>
−
                                        </figure>
+
                                    </figcaption>
−
                                    </div>
+
                                </figure>
−
                                </div>
+
                            </div>
−
                                <div class='row'>
+
                        </div>
−
                                    <div class='col-lg-3'></div>
+
                        <div class='row'>
−
                                    <div class='col-lg-6'>
+
                            <div class='col-lg-3'></div>
−
                                        <figure>
+
                            <div class='col-lg-6'>
−
                                            <img src='https://static.igem.org/mediawiki/2016/c/ca/Pnp250uMBGU.jpg' class='ImprovedPics'>
+
                                <figure class="improvedPicsWrapper">
−
                                            <figcaption>
+
                                    <img src='https://static.igem.org/mediawiki/2016/c/ca/Pnp250uMBGU.jpg' class='ImprovedPics'>
−
                                                <p>
+
                                    <figcaption>
−
                                                    <b>Figure 3:</b> pNP-Butyrate degradation activity of all LC-Cutinase variants and W.T. at a substrate concentration of 250μM.  
+
                                        <p>
−
                                                    Controls are the same as with 50μM concentration.
+
                                            <b>Figure 3:</b> pNP-Butyrate degradation activity of all LC-Cutinase variants and W.T. at a substrate concentration of 250μM.  
−
                                                </p>
+
                                            Controls are the same as with 50μM concentration.
−
                                            </figcaption>
+
                                        </p>
−
                                        </figure>
+
                                    </figcaption>
−
                                    </div>
+
                                </figure>
−
                                </div>
+
                            </div>
−
                                <p>
+
                        </div>
−
                                    These tests were conducted using a bacterial supernatant, without measurement of enzyme concentrations, which might suggest that the improvement in activity is accomplished by an increase of enzyme expression.
+
                        <p>
−
                                    Although it suggests an improvement in expression and not efficiency, this hypothesis is still considered an improvement over the W.T. as it increases the degradation rate of the substrate.  
+
                            These tests were conducted using a bacterial supernatant, without measurement of enzyme concentrations, which might suggest that the improvement in activity is accomplished by an increase of enzyme expression/concentration.
−
                                </p>
+
                            Although it suggests an improvement in expression levels and not efficiency, this hypothesis is still considered an improvement over the W.T. as it increases the degradation rate of the substrate.  
−
                                <p>
+
                        </p>
−
                                    In order to minimize the effect of expression levels on activity, we isolated and cleaned the enzymes and performed the experiment again, with equal enzyme concentrations.
+
                        <p>
−
                                </p>
+
                            In order to minimize the effect of expression levels on activity, we isolated and cleaned the enzymes and performed the experiment again, with equal enzyme concentrations.
−
                                <div class='row'>
+
                        </p>
−
                                    <div class='col-lg-3'></div>
+
                        <div class='row'>
−
                                    <div class='col-lg-6'>
+
                            <div class='col-lg-3'></div>
−
                                        <figure>
+
                            <div class='col-lg-6'>
−
                                            <img src='https://static.igem.org/mediawiki/2016/6/67/Normal_cutiBGU.png' class='ImprovedPics'>
+
                                <figure class="improvedPicsWrapper">
−
                                            <figcaption>
+
                                    <img src='https://static.igem.org/mediawiki/2016/6/67/Normal_cutiBGU.png' class='ImprovedPics'>
−
                                                <p>
+
                                    <figcaption>
−
                                                    <b>Figure 4:</b> pNP-Butyrate degradation activity of 2 LC-Cutinase variants - CO, F4 and the W.T., at a substrate concentration of 125μM.  
+
                                        <p>
−
                                                    Enzyme concentration is 0.11mg/ml.
+
                                            <b>Figure 4:</b> pNP-Butyrate degradation activity of two LC-Cutinase variants - CO, F4 and the W.T., at a substrate concentration of 125μM.  
−
                                                </p>
+
                                            Enzyme concentration is 0.11mg/ml.
−
                                            </figcaption>
+
                                        </p>
−
                                        </figure>
+
                                    </figcaption>
−
                                    </div>
+
                                </figure>
−
                                </div>
+
                            </div>
−
                                <p>
+
                        </div>
−
                                    As seen from the results above, even in equal enzyme concentrations our LC-Cutinase variants display a significant improvement in pNP-B degradation in comparison to the W.T. LC-Cutinase.
+
                        <p>
−
                                </p>
+
                            As seen from the results above, even in equal enzyme concentrations our LC-Cutinase variants display a significant improvement in pNP-B degradation in comparison to the W.T. LC-Cutinase.
 +
                        </p>
 +
                        <p>
 +
                            <b>Secondly</b>, we have improved the characterization of the LC-Cutinase protein with regards to:
 +
                        </p>
 +
                        <ol>
 +
                            <li>
 +
                                pNP-Butyrate degradation - Previous data does not include kinetic tests, only final O.D values. We have also measured activity with different substrate concentrations at known enzyme concentrations, also, not performed previously.
 
                             </li>
 
                             </li>
 
                             <li>
 
                             <li>
−
                                We have improved the characterization of the LC-Cutinase protein with regards to:
 
−
                                <ol>
 
−
                                    <li>
 
−
                                        pNP-Butyrate degradation - Previous data does not include kinetic tests, only final O.D values. We have also measured activity with different substrate concentrations at known enzyme concentrations, also, not performed previously.
 
−
                                    </li>
 
−
                                    <li>
 
  
−
                                        PET degradation - We have shown evidence of LC-Cutinase PET degradation activity using two different experiments - Agar plates containing shredded PET and Electron microscopy of PET samples incubated with LC-Cutinase. All previous data in the registry does not include records of PET degradation activity, only pNP-B. <br>
+
                                PET degradation - We have shown evidence of LC-Cutinase PET degradation activity using two different experiments - Agar plates containing shredded PET and Electron microscopy of PET samples incubated with LC-Cutinase. All previous data in the registry does not include records of PET degradation activity, only pNP-B. <br>
−
                                    </li>
+
−
                                </ol>
+
−
                                <div class='row'>
+
−
                                    <div class='col-lg-6'>
+
−
                                        <figure>
+
−
                                            <img src='https://static.igem.org/mediawiki/2016/3/30/WT_plate_markedBGU.png' class='ImprovedPics'>
+
−
                                            <figcaption>
+
−
                                                <b>Figure 5:</b> <i>E. coli</i> expressing the W.T LC-Cutinase grown on a M9 soft agar plate with shredded PET pellets as a sole carbon source.
+
−
                                                Colonies are marked in red.
+
−
                                            </figcaption>
+
−
                                        </figure>
+
−
                                    </div>
+
−
                                    <div class='col-lg-6'>
+
−
                                        <figure>
+
−
                                            <img src='https://static.igem.org/mediawiki/2016/0/01/PET_LB_CO_II_11BGU.png' class='ImprovedPics'>
+
−
                                            <figcaption>
+
−
                                                <b>Figure 6:</b> A scanning electron microscope (SEM) image of a PET pellet after 2 days of incubation in a liquid LB broth with <i>E. coli</i> expressing the codon-optimized LC-Cutinase protein. X5000 magnification.
+
−
                                            </figcaption>
+
−
                                        </figure>
+
−
                                    </div>
+
−
                                </div>
+
−
                                <br>
+
 
                             </li>
 
                             </li>
 
                         </ol>
 
                         </ol>
 +
                        <div class='row'>
 +
                            <div class='col-lg-6'>
 +
                                <figure class="improvedPicsWrapper">
 +
                                    <img src='https://static.igem.org/mediawiki/2016/3/30/WT_plate_markedBGU.png' class='ImprovedPics'>
 +
                                    <figcaption>
 +
                                        <b>Figure 5:</b> <i>E. coli</i> expressing the W.T LC-Cutinase grown on a M9 soft agar plate with shredded PET pellets as a sole carbon source.
 +
                                        Colonies are marked in red.
 +
                                    </figcaption>
 +
                                </figure>
 +
                            </div>
 +
                            <div class='col-lg-6'>
 +
                                <figure class="improvedPicsWrapper">
 +
                                    <img src='https://static.igem.org/mediawiki/2016/0/01/PET_LB_CO_II_11BGU.png' class='ImprovedPics'>
 +
                                    <figcaption>
 +
                                        <b>Figure 6:</b> A scanning electron microscope (SEM) image of a PET pellet after 2 days of incubation in a liquid LB broth with <i>E. coli</i> expressing the codon-optimized LC-Cutinase protein. X5000 magnification.
 +
                                    </figcaption>
 +
                                </figure>
 +
                            </div>
 +
                        </div>
 +
                        <br>
 
                         <p>
 
                         <p>
 
                             For more results see our parts' pages - <a href='http://parts.igem.org/Part:BBa_K2091004'>BBa_K2091004</a>, <a href='http://parts.igem.org/Part:BBa_K2091005'>BBa_K2091005</a>.
 
                             For more results see our parts' pages - <a href='http://parts.igem.org/Part:BBa_K2091004'>BBa_K2091004</a>, <a href='http://parts.igem.org/Part:BBa_K2091005'>BBa_K2091005</a>.

Latest revision as of 14:56, 19 October 2016

PlastiCure

Improved Parts

Our team has chosen to improve the LC-Cutinase eznyme using a rational mutagenesis approach.

We have chosen to test our improved variants' activity using a pNP-Butyrate degradation assay with different substrate and enzyme concentrations.

Our experiments support that our designed proteins are significantly improved compared to the existing part BBa_K936000 due to the following reasons:

First, our experiments showed a significant improvement of pNP-B degradation activity in two of our LC-Cutinase variants:

  1. The codon optimized version (BBa_K2091004)
  2. The F4 variant (BBa_K2091005)

Our conclusion is based on kinetic test results which showed faster rise in O.D levels compared to the W.T. protein in three different substrate concentration (Fig. 1,2,3):

Figure 1: pNP-Butyrate degradation activity of all LC-Cutinase variants and W.T. at a substrate concentration of 50μM. For negative control we used E. coli strain BL-21 without any vector ("BL-21") and also E. coli strain BL-21 with only the pACYC plasmid backbone ("pACYC").

Figure 2: pNP-Butyrate degradation activity of all LC-Cutinase variants and W.T. at a substrate concentration of 125μM. Controls are the same as with 50μM concentration.

Figure 3: pNP-Butyrate degradation activity of all LC-Cutinase variants and W.T. at a substrate concentration of 250μM. Controls are the same as with 50μM concentration.

These tests were conducted using a bacterial supernatant, without measurement of enzyme concentrations, which might suggest that the improvement in activity is accomplished by an increase of enzyme expression/concentration. Although it suggests an improvement in expression levels and not efficiency, this hypothesis is still considered an improvement over the W.T. as it increases the degradation rate of the substrate.

In order to minimize the effect of expression levels on activity, we isolated and cleaned the enzymes and performed the experiment again, with equal enzyme concentrations.

Figure 4: pNP-Butyrate degradation activity of two LC-Cutinase variants - CO, F4 and the W.T., at a substrate concentration of 125μM. Enzyme concentration is 0.11mg/ml.

As seen from the results above, even in equal enzyme concentrations our LC-Cutinase variants display a significant improvement in pNP-B degradation in comparison to the W.T. LC-Cutinase.

Secondly, we have improved the characterization of the LC-Cutinase protein with regards to:

  1. pNP-Butyrate degradation - Previous data does not include kinetic tests, only final O.D values. We have also measured activity with different substrate concentrations at known enzyme concentrations, also, not performed previously.
  2. PET degradation - We have shown evidence of LC-Cutinase PET degradation activity using two different experiments - Agar plates containing shredded PET and Electron microscopy of PET samples incubated with LC-Cutinase. All previous data in the registry does not include records of PET degradation activity, only pNP-B.
Figure 5: E. coli expressing the W.T LC-Cutinase grown on a M9 soft agar plate with shredded PET pellets as a sole carbon source. Colonies are marked in red.
Figure 6: A scanning electron microscope (SEM) image of a PET pellet after 2 days of incubation in a liquid LB broth with E. coli expressing the codon-optimized LC-Cutinase protein. X5000 magnification.

For more results see our parts' pages - BBa_K2091004, BBa_K2091005.

Address:

Ben-Gurion University of the Negev
Ben Gurion 1, Beer Sheva 8410501, Israel

Mail: igembgu2016@gmail.com

Connect With Us!