Team:UC Davis/Project/Directed Evolution

From 2012.igem.org

(Difference between revisions)
 
(52 intermediate revisions not shown)
Line 227: Line 227:
     padding: 0;
     padding: 0;
     padding-bottom: 10px;
     padding-bottom: 10px;
-
#    background:url(http://img.photobucket.com/albums/v26/bluemelon/bg-2-1.jpg);
+
#    background:url(https://static.igem.org/mediawiki/2012/c/cc/UCD_Bg_1.jpg);
#    background-repeat: no-repeat;
#    background-repeat: no-repeat;
#    background-attachment: fixed;
#    background-attachment: fixed;
Line 248: Line 248:
body {
body {
background-color: rgba(255,255,255,1);
background-color: rgba(255,255,255,1);
-
background-image: url('http://img.photobucket.com/albums/v26/bluemelon/bg-2-1.jpg');
+
background-image: url('https://static.igem.org/mediawiki/2012/c/cc/UCD_Bg_1.jpg');
background-size: 100%;
background-size: 100%;
background-repeat: no-repeat;
background-repeat: no-repeat;
Line 1,036: Line 1,036:
   <div id="newnavi">
   <div id="newnavi">
     <ul class="newmenu">
     <ul class="newmenu">
-
        <li ><a href="https://2012.igem.org/" title="Back to iGEM">iGEM</a></li>
+
    <li ><a target="new" href="https://2012.igem.org/" title="Back to iGEM">iGEM</a>
 +
          <ul>
 +
          <li><a target="new" href="https://2012.igem.org/">Main iGEM</a></li>
 +
          <li><a href="https://2012.igem.org/Team:UC_Davis/Criteria">Criteria</a></li>
 +
          <li><a href="https://2012.igem.org/Team:UC_Davis/Human_Practices">Human Practices</a></li>
 +
          </ul>
 +
        </li>
         <li ><a href="https://2012.igem.org/Team:UC_Davis/Attributions" title="Attributions">Attributions</a></li>
         <li ><a href="https://2012.igem.org/Team:UC_Davis/Attributions" title="Attributions">Attributions</a></li>
-
         <li ><a href="https://2012.igem.org/Team:UC_Davis/Data" title="Data">Data</a>
+
         <li ><a title="https://2012.igem.org/Team:UC_Davis/Data" title="Data">Data</a>
           <ul>
           <ul>
-
             <li ><a href="./Data.htm ">Data 1</a></li>
+
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Data/Cutinase_Activity" title="Data">Cutinase Activity</a></li>
-
             <li ><a href="./Data.htm ">Data 2</a></li>
+
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Data/Ethylene_Glycol"
-
            <li ><a href="./Data.htm ">Data 3</a></li>
+
title="Data">Ethylene Glycol</a></li>
-
        </ul>
+
<li ><a href="https://2012.igem.org/Team:UC_Davis/Data/Modeling"
 +
title="Data">Modeling</a></li>
 +
 
 +
            <li ><a href="https://2012.igem.org/Team:UC_Davis/Parts">Parts</a></li>
 +
          </ul>
         </li>
         </li>
-
         <li ><a title="https://2012.igem.org/Notebook" title="Notebook">Notebook</a>
+
         <li ><a href="https://2012.igem.org/Team:UC_Davis/Notebook" title="Notebook">Notebook</a>
           <ul>
           <ul>
-
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Notebook ">Overview</a></li>
+
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Notebook">Notebook</a></li>
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Notebook/Protocols ">Protocols</a></li>
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Notebook/Protocols ">Protocols</a></li>
-
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Notebook ">Notebook</a></li>
+
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Notebook/Gallery">Gallery</a></li>
           </ul>
           </ul>
         </li>
         </li>
Line 1,057: Line 1,067:
         <li ><a href="https://2012.igem.org/Team:UC_Davis/Safety" title="Safety">Safety</a></li>
         <li ><a href="https://2012.igem.org/Team:UC_Davis/Safety" title="Safety">Safety</a></li>
-
         <li class="selected"><a title="https://2012.igem.org/Team:UC_Davis/Project" title="Project">Project</a>
+
         <li class="selected"><a href="https://2012.igem.org/Team:UC_Davis/Project" title="Project">Project</a>
           <ul>
           <ul>
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Project">Project Overview</a></li>
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Project">Project Overview</a></li>
-
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Project/Catalyst">Modular Engineering</a></li>
+
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Project/Catalyst">Module Engineering</a></li>
-
             <li ><a title="https://2012.igem.org/Team:UC_Davis/Project/Strain">Chassis Engineering</a>
+
            <li ><a href="https://2012.igem.org/Team:UC_Davis/Project/Protein_Engineering">Protein Engineering</a></li>
 +
             <li ><a title="https://2012.igem.org/Team:UC_Davis/Project/Strain">Chassis Engineering </a>
  <ul>
  <ul>
-
                 <li><a href="https://2012.igem.org/Team:UC_Davis/Project/Strain">Chassis Engineering</a></li>
+
                 <li><a href="https://2012.igem.org/Team:UC_Davis/Project/Strain">Background</a></li>
        <li><a href="https://2012.igem.org/Team:UC_Davis/Project/Directed_Evolution">Directed Evolution</a></li>
        <li><a href="https://2012.igem.org/Team:UC_Davis/Project/Directed_Evolution">Directed Evolution</a></li>
                 <li><a href="https://2012.igem.org/Team:UC_Davis/Project/Our_Strain">Rational Engineering </a></li>
                 <li><a href="https://2012.igem.org/Team:UC_Davis/Project/Our_Strain">Rational Engineering </a></li>
</ul>
</ul>
</li>
</li>
-
            <li ><a href="https://2012.igem.org/Team:UC_Davis/Project/Protein_Engineering">Protein Engineering</a></li>
 
-
            <li ><a href="https://2012.igem.org/Team:UC_Davis/Parts">Parts</a></li>
 
           </ul>
           </ul>
         </li>
         </li>
Line 1,079: Line 1,088:
<div id="slides">
<div id="slides">
-
     <img  src="http://img.photobucket.com/albums/v26/bluemelon/slide-1-2.jpg" width="850" height="280" alt="" class="current" />
+
     <img  src="https://static.igem.org/mediawiki/2012/c/cf/UCD_slide1.jpg" width="850" height="280" alt="" class="current" />
-
     <img  src="http://img.photobucket.com/albums/v26/bluemelon/slide-2-2.jpg" width="850" height="280" alt="" />
+
     <img  src="https://static.igem.org/mediawiki/2012/e/e3/UCD_Slide_2.jpg" width="850" height="280" alt="" />
-
     <img  src="http://img.photobucket.com/albums/v26/bluemelon/slide-3-2.jpg" width="850" height="280" alt="" />
+
     <img  src="https://static.igem.org/mediawiki/2012/d/d9/UCD_Slide_3.jpg" width="850" height="280" alt="" />
-
     <img  src="http://img.photobucket.com/albums/v26/bluemelon/slide-4-2.jpg" width="850" height="280" alt="" />
+
     <img  src="https://static.igem.org/mediawiki/2012/a/a4/UCD_Slide_4.jpg" width="850" height="280" alt="" />
</div>
</div>
     <ul class="progress">
     <ul class="progress">
Line 1,115: Line 1,124:
</div>
</div>
<div id="myleftrightbox"  class="fourboxes-3">
<div id="myleftrightbox"  class="fourboxes-3">
-
<a href="https://2012.igem.org/Team:UC_Davis/Project/Protein_Engineering"><img src="http://img.photobucket.com/albums/v26/bluemelon/protein_small_banner-2.jpg"></a>
+
<a href="https://2012.igem.org/Team:UC_Davis/Project/Protein_Engineering"><img src="https://static.igem.org/mediawiki/2012/f/ff/UCD_protein_small_banner.jpg"></a>
</div>
</div>
<div id="myleftrightbox"  class="spacebox">
<div id="myleftrightbox"  class="spacebox">
Line 1,133: Line 1,142:
<div id="sponsorbox" class="sponsorfloat1">
<div id="sponsorbox" class="sponsorfloat1">
-
<a href="https://2012.igem.org/Team:UC_Davis/Project/Directed_Evolution"><img src="http://img.photobucket.com/albums/v26/bluemelon/directed_small_banner-1.jpg"></a>
+
<a href="https://2012.igem.org/Team:UC_Davis/Project/Directed_Evolution"><img src="https://static.igem.org/mediawiki/2012/d/df/UCD_Directed_small_banner.jpg"></a>
</div>
</div>
<div id="sponsorbox" class="sponsorfloat2">
<div id="sponsorbox" class="sponsorfloat2">
-
<a href="https://2012.igem.org/Team:UC_Davis/Project/Our_Strain"><img src="http://img.photobucket.com/albums/v26/bluemelon/rational_small_banner-1.jpg"></a>
+
<a href="https://2012.igem.org/Team:UC_Davis/Project/Our_Strain"><img src="https://static.igem.org/mediawiki/2012/4/47/UCD_Rational_small_banner.jpg"></a>
</div>
</div>
Line 1,220: Line 1,229:
<center>
<center>
-
<a href="http://provost.ucdavis.edu/" target="_blank"><img src="https://static.igem.org/mediawiki/2012/9/99/UCD_Research_sponsor.jpg" width="200"></a>
+
<a href="http://www.research.ucdavis.edu/" target="_blank"><img src="https://static.igem.org/mediawiki/2012/9/99/UCD_Research_sponsor.jpg" width="200"></a>
</center>
</center>
<center>
<center>
-
<a href="http://provost.ucdavis.edu/" target="_blank"><img src="https://static.igem.org/mediawiki/2012/b/b4/UCD_Communications_sponsor.jpg" width="200"></a>
+
<a href="http://ucomm.ucdavis.edu/" target="_blank"><img src="https://static.igem.org/mediawiki/2012/b/b4/UCD_Communications_sponsor.jpg" width="200"></a>
</center>
</center>
Line 1,241: Line 1,250:
<h1>Directed Evolution</h1>
<h1>Directed Evolution</h1>
<article>
<article>
-
Evolution occurs naturally by selection pressure, but in an overall slow pace. To speed up the process for certain desired traits, it is possible to re-passage cells to grow on a certain type of media, or expose certain mutagens to the cells to select for surviving mutants. Our team has carried out both of these selection pressures in hopes to isolate an ethylene glycol utilizing bacterium.
+
Evolution occurs naturally by selection pressure, but in an overall slow pace. To speed up the process for certain desired traits, it is possible to re-passage cells to grow on a certain type of media, or expose certain mutagens to increase the variation in the cells to select for surviving mutants. Our team has carried out both of these selection pressures in hopes to isolate an ethylene glycol utilizing bacterium.
</article></div>
</article></div>
Line 1,247: Line 1,256:
<h1>History</h1>
<h1>History</h1>
<article>
<article>
-
Mutants of E. coli able to grow on propylene glycol were selected for ethylene glycol enzymatic breakdown after ethyl methane sulfonate mutagenesis. The culture conditions strongly selected for ethylene glycol utilizing mutants, by having ethylene glycol as the sole carbon source in liquid culture. Through directed evolution, colonies were continuously streaked on ethylene glycol plates for three more generations, in order to isolate ethylene glycol degrading mutants. Through spectrophotometric assays, increased activities of glycolaldehyde reductase and glycolaldehyde dehydrogenase were observed in the ethylene glycol isolates.  
+
Mutants of <i>E. coli</I> able to grow on propylene glycol were selected for ethylene glycol enzymatic breakdown after ethyl methanesulfonate (EMS) mutagenesis by the University of Barcelona [1]. The culture conditions strongly selected for ethylene glycol utilizing mutants, by having ethylene glycol as the sole carbon source in liquid culture. Through directed evolution, colonies were continuously streaked on ethylene glycol plates for three more generations, in order to isolate ethylene glycol degrading mutants. Through spectrophotometric assays, increased activities of glycolaldehyde reductase and glycolaldehyde dehydrogenase were observed in the ethylene glycol isolates.  
 +
<br><br>
 +
<center>
 +
<img src="https://static.igem.org/mediawiki/2012/e/ee/UCD_DE_Tecan_3.jpg" align="left">
 +
<img src="https://static.igem.org/mediawiki/2012/b/bb/UCD_DE_Tecan_2.jpg" align="right">
 +
</center>
 +
<br>
 +
 
 +
</center>
</article></div>
</article></div>
Line 1,253: Line 1,270:
<h1>Genome Sequencing</h1>
<h1>Genome Sequencing</h1>
<article>
<article>
-
In progress ...
+
We are sequencing the genome of Strain E-15 EG3 so that we can see what changes are present between the MG1655 and Strain E-15 EG3. Once sequenced, we will be able to compare the sequences between each strain which would show single nucleotide polymorphisms (SNPs), as well as any deletions or insertions. From this, we will be able to apply the knowledge to a hybrid approach with the rational engineering.
 +
Using the transposase, Tn5, we have constructed an Illumina compatible library that we plan to submit to our Sequencing Core Facilities. Unfortunately, the sequencing run on the MiSeq takes around one week and we were not able to get sequence data before the competition.
</article></div>
</article></div>
Line 1,259: Line 1,277:
<h1>Tecan Experiments</h1>
<h1>Tecan Experiments</h1>
<article>
<article>
-
The Tecan experiments with MG1655 and DH5a show us that the ethylene glycol does not hinder the growth and development of the strains, as long as it is mixed with LB media. The growth curves all had the same shape, independent of the amount of ethylene glycol in solution. We chose a broad, nearly exponential range of ethylene glycol concentrations to allow a broad range to test the toxicity. We attempted to find the lower limit of toxicity due to a saturation of ethylene glycol. However, we had not reached it. In our engineered strain, we will not expect to see a concentration of ethylene glycol above 150mM, so we can expect our strain to be able to live in an environment with a concentration as high as that.
+
<p>Re-streaks</p>
-
<br><br>
+
After receiving Strain E-15 EG3, the stab culture was re-streaked on ethylene glycol (EG) agar for three generations to ensure that the strain retained the two functional enzymes key for ethylene glycol utilization after thirty years of storage. Colonies became defined on the EG plates every three to four days, and were immediately re-streaked onto a new EG plate. With this selection pressure to choose the fastest developing colony, our hope is to isolate an evolved version of Strain E-15 EG3, that is capable of replicating on EG at a faster pace.
-
After seeing that ethylene glycol does not pose a threat to MG1655 and DH5a, we subjected the Barcelona strain to the same broad range of ethylene glycol. We sought out to find the most efficient concentration of ethylene glycol for this strain, as a guideline for the efficient concentration of EG for our engineered strain. While analyzing the data, we realized that we have to define efficiency more clearly. Efficiency can mean faster growth on low amounts of ethylene glycol or it could mean a higher optical density after a certain amount of time, where it reaches the stationary phase. We saw that once the ethylene glycol concentration reaches a certain threshold (49.34 mM), the growth curves are all the same in terms of time when the stationary phase has been reached. We saw that some of the E. coli were efficient at low concentrations, making us focus on the fast growth efficiency at lower concentrations of EG because the LC-cutinase will not degrade quickly enough to produce 49.34 mM in a cell’s solution. Now, we have an ongoing experiment where we re-passage cells between ethylene glycol media at 30 mM. We discuss this in more detail in our directed evolution section.  
+
<br>We are comparing the most recent re-streak (re-streak #12) with the earliest re-streak (re-streak #3) to test if constantly selecting Strain E-15 EG3 improves the overall growth rate of the strain. <br><br>
 +
<p>Liquid Cultures</p>
 +
We made liquid cultures of the re-streaked Strain E-15 EG3 and re-passaged the strain in new ethylene glycol media for twenty-five generation cycles each time the tubes reached an OD 600 of 0.2. By repeating this process, we were constantly selecting the fastest growing cells on ethylene glycol. We hoped to create a population whose growth rate was even greater than the original Strain E-15 EG3. (Click <a href="https://2012.igem.org/Team:UC_Davis/Data/Ethylene_Glycol#Directed">here</a> for data relating to the repassaging.) Since the liquid culture contains a mixed population of EG utilizing cells, it is important to first plate the cells and select for the fastest developing colonies. After observing the presence of colonies, the first fifty-five colonies were selected, tested for rate of growth in EG media, and compared to the standard, Strain E-15 EG3 from re-streak #3. <br>
 +
<br><center><img src="https://static.igem.org/mediawiki/2012/3/3c/ODcuvettereading.JPG" align="left"><img src="https://static.igem.org/mediawiki/2012/8/85/Clearflaskwithtip.JPG" align="right"></center><br><br><br><br><br><br><br><br><br><br><br><br>
 +
<p>EMS mutagenesis</p>
 +
Ethyl methanesulfonate (EMS) is a liquid mutagen that favors a G/C to A/T transition. We are employing the EMS to both the MG1655 and E-15 EG3 liquid cultures in different concentrations (28 μL, 56 μL, 84 μL to 4 mL of cells) for different amounts of time (0 minute, 15 minutes, 30 minutes, and 45 minutes) [3]. However, we are not focusing on a specific gene with a high G/C content, so the EMS just serves the purpose of adding variation to the population. We wanted to compare the effects of EMS and whether mutagenesis introduced any variation in the utilization of ethylene glycol at different rates. To set up the comparison across plates for relative growth rates, a set of Strain E-15 EG3 standards were included in each plate. We expect that the EMS caused some variation in each of the populations. However, they are probably minute variations, and are likely to be similar in efficiency. <br><br><img src="https://static.igem.org/mediawiki/2012/6/67/EMSatArcadia1.JPG" align="left"><img src="https://static.igem.org/mediawiki/2012/4/4f/EMSatArcadia2.JPG" align="right"><br><br><br><br><br><br><br><br><br><br><br><br>
 +
The cultures that were exposed to EMS for 45 minutes still showed colonies, meaning that the EMS was not strong enough to kill off the cells. Therefore, these colonies should theoretically show the most variation. The first fifty-five colonies that developed in both MG1655 and E-15 EG13 were compared at the 0 minute time point and the 45 minute time point using the Tecan (here's our <a href="https://2012.igem.org/Team:UC_Davis/Data/Ethylene_Glycol#EMS">data</a>). <br>
 +
Given more time, these colonies would be sequenced and compared to the E-15 EG13 to see how any base changes introduced by EMS will affect the efficiency of the enzymes.
 +
 
 +
 
 +
 
 +
</article></div>
</article></div>
Line 1,268: Line 1,297:
<article>
<article>
1. Boronat, Albert, Caballero, Estrella, and Juan Aguilar. “Experimental Evolution of a Metabolic Pathway for Ethylene Glycol Utilization by Escherichia coli.” Journal of Bacteriology, Vol. 153 No. 1, pp. 134-139, January 1983.  <br>
1. Boronat, Albert, Caballero, Estrella, and Juan Aguilar. “Experimental Evolution of a Metabolic Pathway for Ethylene Glycol Utilization by Escherichia coli.” Journal of Bacteriology, Vol. 153 No. 1, pp. 134-139, January 1983.  <br>
-
2. Andrianantoandro, Ernesto, Subhayu Basu, David K. Karig, and Ron Weiss. "Synthetic biology: new engineering rules for an emerging discipline." Nature - Molecular Systems Biology. (2006): n. page. Web. 29 Aug. 2012. <http://www.nature.com/msb/journal/v2/n1/full/msb4100073.html>.
+
2. Andrianantoandro, Ernesto, Subhayu Basu, David K. Karig, and Ron Weiss. "Synthetic biology: new engineering rules for an emerging discipline." Nature - Molecular Systems Biology. (2006): n. page. Web. 29 Aug. 2012. <http://www.nature.com/msb/journal/v2/n1/full/msb4100073.html>. <br>
 +
3. Cupples, Claire, and Jeffrey Miller. "A set of lacZ mutations in Escherichia coli that allow rapid detection of each of the six base substitutions." Proceedings of the National Academy of Sciences. 86. (1989): 5345-5349. Print.
</article>
</article>
Line 1,278: Line 1,308:
<!-- site map starts here -->
<!-- site map starts here -->
<div id="myleftbox"  class="smallboxsite">
<div id="myleftbox"  class="smallboxsite">
-
<ul style="font-size:10px;list-style-image:none;list-style-type:none;float:left;display:inline;color:#000000;"  >
+
<ul style="font-size:10px;list-style-image:none;list-style-type:none;float:left;display:inline;color:#000000;"
 +
  >
 +
 
 +
<li style="float:left;margin:0 10px;"><a
 +
href="https://2012.igem.org/Team:UC_Davis"><p>Home</p><ul
 +
style="text-indent:-15px;
 +
list-style-image:none;list-style-type:none;color:#000000;"><li><a
 +
style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis">Welcome</a> </li><li><a
 +
style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis">Tweets</a></li><li><a
 +
style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis">Sponsors</a> </li><li><a
 +
style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Criteria">Criteria</a> </li>
 +
</ul> </a> </li>
-
<li style="float:left;margin:0 10px solid red"><a href="https://2012.igem.org/Team:UC_Davis"><p>Home</p><ul style="text-indent:-15px;
+
<li style="float:left;margin:0 10px;"><a
-
list-style-image:none;list-style-type:none;color:#000000;"><li>Welcome </li><li>Tweets </li><li>Criteria </li><li>Sponsors </li> </ul> </a> </li>
+
href="https://2012.igem.org/Team:UC_Davis/Team"><p>Team</p><ul
 +
style="text-indent:-15px;
 +
list-style-image:none;list-style-type:none;color:#000000;"><li><a
 +
style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Team">Who we are</a>
 +
</li><li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Team">Students</a></li><li><a
 +
style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Team">Advisors</a> </li>
 +
</ul> </a> </li>
-
<li style="float:left ;margin:0 10px;"><a href="https://2012.igem.org/Team:UC_Davis/Team"><p>Team</p> <ul style="text-indent:-15px;list-style-image:none;
+
<li style="float:left ;margin:0 10px;"><a
-
list-style-type:none;color:#000000 "><li>Who we are </li><li>Students </li><li>Advisors</li></ul></a> </li>
+
href="https://2012.igem.org/Team:UC_Davis/Project "><p>Project</p></a>
 +
<ul style="text-indent:-15px;list-style-image:none;list-style-type:none;color:#000000"><li><a
 +
style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Project">Project Overview</a>
 +
</li><li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Project/Catalyst ">Module
 +
Engineering</a></li><li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Project/Protein_Engineering
 +
">Protein Engineering</a></li>
 +
<li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Project/Strain ">Chassis
 +
Engineering</a></li>
 +
  <li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Project/Directed_Evolution ">
 +
- Directed Evolution</a></li>
 +
  <li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Project/Our_Strain "> -
 +
Rational Engineering</a></li>
 +
<li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Criteria">Critera</a>
 +
</li></ul> </li>
-
<li style="float:left ;margin:0 10px;"><a href="https://2012.igem.org/Team:UC_Davis/Project "><p>Project</p></a> <ul style="text-indent:-15px;list-style-image:none;list-style-type:none;color:#000000"><li><a style="color:#000000 " href="https://2012.igem.org/Team:UC_Davis/Project">Project Overview</a> </li><li><a style="color:#000000 "  href="https://2012.igem.org/Team:UC_Davis/Project/Strain ">Modular Engineering</a></li><li><a style="color:#000000 " href="https://2012.igem.org/Team:UC_Davis/Project/Catalyst ">Chassis Engineering</a></li>
+
<li style="float:left ;margin:0 10px"><a
-
<li><a style="color:#000000 " href="https://2012.igem.org/Team:UC_Davis/Project/Directed_Evolution "> - Directed Evolution</a></li>
+
href="https://2012.igem.org/Team:UC_Davis/Safety "> <p>Safety</p></a>
-
<li><a style="color:#000000 " href="https://2012.igem.org/Team:UC_Davis/Project/Our_Strain "> - Rational Engineering</a></li>
+
<a style="color:#000000 "
-
<li><a style="color:#000000 " href="https://2012.igem.org/Team:UC_Davis/Project/Protein_Engineering ">Protein Engineering</a></li><li><a style="color:#000000 " href="https://2012.igem.org/Team:UC_Davis/Parts ">Parts</a></li> </ul> </li>
+
href="https://2012.igem.org/Team:UC_Davis/Safety "> Safety</a> </li>
-
<li style="float:left ;margin:0 10px"><a href="https://2012.igem.org/Team:UC_Davis/Safety "> <p>Safety</p></a> <a style="color:#000000 " href="https://2012.igem.org/Team:UC_Davis/Safety "> Safety</a> </li>
+
<li style="float:left ;margin:0 10px;"><a
 +
href="https://2012.igem.org/Team:UC_Davis/Notebook ">
 +
<p>Notebook</p></a> <ul
 +
style="text-indent:-15px;list-style-image:none;list-style-type:none;color:#000000
 +
"><li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Notebook">Notebook</a>
 +
</li><li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Notebook/Protocols
 +
">Protocols</a> </li><li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Notebook/Gallery">Gallery</a>
 +
</li><li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Criteria">Critera</a> </li>
 +
</ul> </li>
-
<li style="float:left ;margin:0 10px;"><a href="https://2012.igem.org/Team:UC_Davis/Notebook "> <p>Notebook</p></a> <ul style="text-indent:-15px;list-style-image:none;list-style-type:none;color:#000000  "><li><a style="color:#000000 " href="https://2012.igem.org/Team:UC_Davis/Notebook">Overview</a> </li><li><a style="color:#000000 " href="https://2012.igem.org/Team:UC_Davis/Notebook/Protocols ">Protocols</a> </li><li><a style="color:#000000 " href="https://2012.igem.org/Team:UC_Davis/Notebook ">Notebook</a> </li><li><a style="color:#000000 " href="https://2012.igem.org/Team:UC_Davis/Notebook/Gallery ">Gallery</a> </li> </ul> </li>
+
<li style="float:left ;margin:0 10px;"><a
 +
title="https://2012.igem.org/Team:UC_Davis/Data "> <p>Data </p></a> <ul
 +
style="text-indent:-15px;list-style-image:none;list-style-type:none;color:#000000
 +
  "><li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Data/Cutinase_Activity ">
 +
Cutinase Activity</a> </li><li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Data/Ethylene_Glycol ">
 +
Ethylene Glycol</a> </li><li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Data/Modeling ">
 +
Modeling</a> </li><li><a style="color:#000000 "
-
<li style="float:left ;margin:0 10px;"><a href="https://2012.igem.org/Team:UC_Davis/Data "> <p>Data </p><ul style="text-indent:-15px;list-style-image:none;
+
href="https://2012.igem.org/Team:UC_Davis/Parts ">Parts</a></li> </ul>
-
list-style-type:none;color:#000000"><li>Data One </li><li>Data Two </li><li>Data Three </li></ul></a> </li>
+
-
<li style="float:left ;margin:0 10px"><a href="https://2012.igem.org/Team:UC_Davis/Attributions "> <p>Attribution </p></a><a style="color:#000000 " href="https://2012.igem.org/Team:UC_Davis/Attributions "> Attribution</a></li>
+
<li style="float:left ;margin:0 10px"><a
 +
href="https://2012.igem.org/Team:UC_Davis/Attributions ">
 +
<p>Attribution </p></a><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Attributions ">
 +
Attribution</a></li>
-
<li style="float:left ;margin:0 10px"><a href="https://2012.igem.org/Main_Page "> <p>iGEM </p></a><a style="color:#000000 " href="https://2012.igem.org/Main_Page " > iGEM</a></li>
+
<li style="float:left ;margin:0 10px"><a
-
</ul>
+
href="https://2012.igem.org/Main_Page "> <p>iGEM </p></a><ul
 +
style="text-indent:-15px;list-style-image:none;list-style-type:none;color:#000000
 +
"><li><a style="color:#000000 " href="https://2012.igem.org/Main_Page
 +
">Main iGEM</a> </li><li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Criteria "> Criteria</a>
 +
</li><li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Human_Practices ">Human
 +
Practices</a></li> </ul>
</div>
</div>
<!-- site map ends here -->
<!-- site map ends here -->

Latest revision as of 01:27, 27 October 2012

Team:UC Davis - 2012.igem.org

UCDavis iGEM Tweets

Our Sponsors

Directed Evolution

Evolution occurs naturally by selection pressure, but in an overall slow pace. To speed up the process for certain desired traits, it is possible to re-passage cells to grow on a certain type of media, or expose certain mutagens to increase the variation in the cells to select for surviving mutants. Our team has carried out both of these selection pressures in hopes to isolate an ethylene glycol utilizing bacterium.

History

Mutants of E. coli able to grow on propylene glycol were selected for ethylene glycol enzymatic breakdown after ethyl methanesulfonate (EMS) mutagenesis by the University of Barcelona [1]. The culture conditions strongly selected for ethylene glycol utilizing mutants, by having ethylene glycol as the sole carbon source in liquid culture. Through directed evolution, colonies were continuously streaked on ethylene glycol plates for three more generations, in order to isolate ethylene glycol degrading mutants. Through spectrophotometric assays, increased activities of glycolaldehyde reductase and glycolaldehyde dehydrogenase were observed in the ethylene glycol isolates.


Genome Sequencing

We are sequencing the genome of Strain E-15 EG3 so that we can see what changes are present between the MG1655 and Strain E-15 EG3. Once sequenced, we will be able to compare the sequences between each strain which would show single nucleotide polymorphisms (SNPs), as well as any deletions or insertions. From this, we will be able to apply the knowledge to a hybrid approach with the rational engineering. Using the transposase, Tn5, we have constructed an Illumina compatible library that we plan to submit to our Sequencing Core Facilities. Unfortunately, the sequencing run on the MiSeq takes around one week and we were not able to get sequence data before the competition.

Tecan Experiments

Re-streaks

After receiving Strain E-15 EG3, the stab culture was re-streaked on ethylene glycol (EG) agar for three generations to ensure that the strain retained the two functional enzymes key for ethylene glycol utilization after thirty years of storage. Colonies became defined on the EG plates every three to four days, and were immediately re-streaked onto a new EG plate. With this selection pressure to choose the fastest developing colony, our hope is to isolate an evolved version of Strain E-15 EG3, that is capable of replicating on EG at a faster pace.
We are comparing the most recent re-streak (re-streak #12) with the earliest re-streak (re-streak #3) to test if constantly selecting Strain E-15 EG3 improves the overall growth rate of the strain.

Liquid Cultures

We made liquid cultures of the re-streaked Strain E-15 EG3 and re-passaged the strain in new ethylene glycol media for twenty-five generation cycles each time the tubes reached an OD 600 of 0.2. By repeating this process, we were constantly selecting the fastest growing cells on ethylene glycol. We hoped to create a population whose growth rate was even greater than the original Strain E-15 EG3. (Click here for data relating to the repassaging.) Since the liquid culture contains a mixed population of EG utilizing cells, it is important to first plate the cells and select for the fastest developing colonies. After observing the presence of colonies, the first fifty-five colonies were selected, tested for rate of growth in EG media, and compared to the standard, Strain E-15 EG3 from re-streak #3.













EMS mutagenesis

Ethyl methanesulfonate (EMS) is a liquid mutagen that favors a G/C to A/T transition. We are employing the EMS to both the MG1655 and E-15 EG3 liquid cultures in different concentrations (28 μL, 56 μL, 84 μL to 4 mL of cells) for different amounts of time (0 minute, 15 minutes, 30 minutes, and 45 minutes) [3]. However, we are not focusing on a specific gene with a high G/C content, so the EMS just serves the purpose of adding variation to the population. We wanted to compare the effects of EMS and whether mutagenesis introduced any variation in the utilization of ethylene glycol at different rates. To set up the comparison across plates for relative growth rates, a set of Strain E-15 EG3 standards were included in each plate. We expect that the EMS caused some variation in each of the populations. However, they are probably minute variations, and are likely to be similar in efficiency.













The cultures that were exposed to EMS for 45 minutes still showed colonies, meaning that the EMS was not strong enough to kill off the cells. Therefore, these colonies should theoretically show the most variation. The first fifty-five colonies that developed in both MG1655 and E-15 EG13 were compared at the 0 minute time point and the 45 minute time point using the Tecan (here's our data).
Given more time, these colonies would be sequenced and compared to the E-15 EG13 to see how any base changes introduced by EMS will affect the efficiency of the enzymes.

References

1. Boronat, Albert, Caballero, Estrella, and Juan Aguilar. “Experimental Evolution of a Metabolic Pathway for Ethylene Glycol Utilization by Escherichia coli.” Journal of Bacteriology, Vol. 153 No. 1, pp. 134-139, January 1983.
2. Andrianantoandro, Ernesto, Subhayu Basu, David K. Karig, and Ron Weiss. "Synthetic biology: new engineering rules for an emerging discipline." Nature - Molecular Systems Biology. (2006): n. page. Web. 29 Aug. 2012. .
3. Cupples, Claire, and Jeffrey Miller. "A set of lacZ mutations in Escherichia coli that allow rapid detection of each of the six base substitutions." Proceedings of the National Academy of Sciences. 86. (1989): 5345-5349. Print.

Retrieved from "http://2012.igem.org/Team:UC_Davis/Project/Directed_Evolution"