Difference between revisions of "Team:UCL/Results"

 
(One intermediate revision by the same user not shown)
Line 2: Line 2:
 
{{UCL/BootstrapCSS}}
 
{{UCL/BootstrapCSS}}
 
{{UCL/Style}}
 
{{UCL/Style}}
 +
  
 
<html lang="en">
 
<html lang="en">
Line 8: Line 9:
 
     <meta name="viewport" content="width=device-width, initial-scale=1.0, maximum-scale=1.0">
 
     <meta name="viewport" content="width=device-width, initial-scale=1.0, maximum-scale=1.0">
 
     <meta http-equiv="X-UA-Compatible" content="ie=edge">
 
     <meta http-equiv="X-UA-Compatible" content="ie=edge">
−
     <title>UCL SETA - Model</title>
+
     <title>UCL SETA - Results</title>
 
     <link rel="icon" href="https://static.igem.org/mediawiki/2018/7/73/T--UCL--Logo2.png">
 
     <link rel="icon" href="https://static.igem.org/mediawiki/2018/7/73/T--UCL--Logo2.png">
−
      
+
     <link rel="stylesheet" href="../resources/css/bootstrap1.css"  />
 +
    <link rel="stylesheet" href="../style.css"  />
 
</head>
 
</head>
  
Line 16: Line 18:
 
<script type="text/javascript" src="https://2018.igem.org/Template:UCL/JQuery?action=raw&ctype=text/javascript"></script>
 
<script type="text/javascript" src="https://2018.igem.org/Template:UCL/JQuery?action=raw&ctype=text/javascript"></script>
 
<script type="text/javascript" src="https://2018.igem.org/Template:UCL/WowJS?action=raw&ctype=text/javascript"></script>
 
<script type="text/javascript" src="https://2018.igem.org/Template:UCL/WowJS?action=raw&ctype=text/javascript"></script>
−
 
+
<script type="text/javascript" src="../resources/js/bootstrapscript.js"></script>
 +
<script type="text/javascript" src="../resources/js/jquery-3.3.1.min.js"></script>
  
 
<script>
 
<script>
Line 33: Line 36:
 
});
 
});
 
</script>
 
</script>
−
 
+
<script src="../resources/js/wow1.js"></script>
 
     <script>
 
     <script>
 
         new WOW().init();
 
         new WOW().init();
Line 86: Line 89:
 
     <div class="dropdown-content_igem"id="dropdownmodelling_igem">
 
     <div class="dropdown-content_igem"id="dropdownmodelling_igem">
 
         <a href="https://2018.igem.org/Team:UCL/Model">Modelling</a>
 
         <a href="https://2018.igem.org/Team:UCL/Model">Modelling</a>
−
        <a href="https://2018.igem.org/Team:UCL/Model#Intein">Intein</a>
 
−
        <a href="https://2018.igem.org/Team:UCL/Model#Intein_Polymerisation">Intein Polymerisation</a>
 
−
        <a href="https://2018.igem.org/Team:UCL/Model#LAMMPS">LAMMPS</a>
 
−
        <a href="https://2018.igem.org/Team:UCL/Model#Scale_Up">Scale Up</a>
 
 
     </div>
 
     </div>
 
   </div>
 
   </div>
Line 96: Line 95:
  
 
     </button>
 
     </button>
−
     <div class="dropdown-content_igem"id"dropdownhumanpractices_igem">
+
     <div class="dropdown-content_igem"id="dropdownhumanpractices_igem">
 
         <a href="https://2018.igem.org/Team:UCL/Human_Practices">Human Practices</a>
 
         <a href="https://2018.igem.org/Team:UCL/Human_Practices">Human Practices</a>
 
         <a href="https://2018.igem.org/Team:UCL/Public_Engagement">Public Engagement</a>
 
         <a href="https://2018.igem.org/Team:UCL/Public_Engagement">Public Engagement</a>
Line 138: Line 137:
 
}
 
}
 
</script>
 
</script>
−
 
−
 
  
 
<script>
 
<script>
Line 171: Line 168:
 
</script>
 
</script>
  
−
 
+
<script type="text/javascript" src="../resources/js/scrolltofixed.js"></script>
 
<script type="text/javascript" src="https://2018.igem.org/Template:UCL/ScrollToFixed?action=raw&ctype=text/javascript"></script>
 
<script type="text/javascript" src="https://2018.igem.org/Template:UCL/ScrollToFixed?action=raw&ctype=text/javascript"></script>
  
Line 180: Line 177:
 
</script>
 
</script>
  
 +
<!--BODY==================================================================== -->
 +
<body>
 +
        <div class="container container_igem">
 +
            <div class="container container_igem">
 +
                <div class="h1_igem_subpage1">Results</div>
 +
            </div>
  
−
<!-- CONTENT******************************************************************************************************************************************************* -->
+
            <div class="container container_igem">
−
<div class="container">
+
                <div id="sidenav" class="sidenav wow fadeInUp" data-wow-duration="2s" data-wow-delay="0.1s">
 +
                    <a href="#MP">Modular Platform</a>
 +
                    <a href="#BB">BioBrick 2.0</a>
 +
                    <a href="#IEP">Integrated Human Practices</a>
 +
                    <a href="#IM">Integrated Modelling</a>
 +
                  </div>
  
−
    <div class="container">
 
−
        <img class="wow fadeInUp subpage_img_igem" src="https://static.igem.org/mediawiki/2018/a/a0/T--UCL--model.svg" data-wow-duration="2s" data-wow-delay="0.1s"/>
 
−
        <div class="h1_igem_subpage">Models</div>
 
−
    </div>
 
−
    <div class="container containter-fluid container_igem">
 
−
        <div id="sidenav" class="sidenav wow fadeInUp" data-wow-duration="2s" data-wow-delay="0.1s">
 
−
          <a href="#Overview">Overview</a>
 
−
          <a href="#CGE">Cell Growth and Expression</a>
 
−
          <a href="#Protein_Polymerisation">Protein Polymerisation</a>
 
−
          <a href="#WORM">W.O.R.M.</a>
 
−
          <a href="#Mech">Mechanical Simulation</a>
 
−
          <a href="#Bibliography">Bibliography</a>
 
−
        </div>
 
  
−
        <section id="Overview">
+
                <section id="MP">
−
            <div class="card card_igem wow fadeInUp" data-wow-duration="2s" data-wow-delay="0.1s">
+
                    <div class="card card_igem wow fadeInUp" data-wow-duration="2s" data-wow-delay="0.1s">
−
                <div class="card-title text-center">
+
                        <div class="card-title text-center">
−
                    <h4>Overview</h4>
+
                            <h4>Modular Platform </h4>
−
                </div>
+
−
                <div class="card-body text-justify">
+
−
                    <div class="container">
+
−
                        <div class="row">
+
−
                            <div class="mmmm col-sm-10">
+
−
                                <p class="card-text card-text_igem text-justify">
+
−
                                  One of the challenges for biomaterial application is to manufacture large scale protein production economically and efficiently. In our model, we integrated several levels of modelling to optimise the productivity of the whole system.
+
−
<br><br>
+
−
For our biomaterial production, we use split intein technology instead of genetic modification to gain longer protein polymers. Split inteins have a specific working temperature and pH condition for sufficient reaction rate. In our models, in addition to basic cell growth and protein expression model, we introduce intein polymerisation model (WORM) and fiber mechanical property simulation. Integrating these models, we can estimate the time required for desired protein polymerisation and thus provide the fiber with strong mechanical properties.
+
−
                                </p>
+
−
                            </div>
+
 
                         </div>
 
                         </div>
−
                    </div>
+
                        <div class="card-body text-justify">
−
                </div>
+
                            <div class="container">
−
            </div>
+
                                <div class="row">
−
        </section>
+
                                    <div class="col-sm-10 mmmm mrfont">
−
 
+
<br>
−
 
+
<p>Starting from the an idea centered around spider silk and its applications, after an insightful conversation with the CSO of Bolt Threads, we devised and developed a tunable construct which brings modular protein engineering in the realm of biomaterials. First, we considered using the spytag-spycatcher system, but we realised that there would have been a much better method to reach our objective. Spliced inteins contribute to the assembly of fusion proteins through directed covalent bonding, in the same fashion as spytag and spycatcher[1]. However, spytag and spycatcher risk to influence the folding and polymerisation process of biomaterials regulated by tight structural constraints through steric hindrance due to their size. Split-inteins avoid this type of issue by splicing out and leaving a permanent covalent bond in a short time, which varies slightly according to literature[2]. In order to simulate the association and potential polymerization through split inteins, we designed a green-red split-inteins system which alternates well characterised chromogenic proteins: GFP, and RFP [3]. Additionally, we devised an Intein Passenger construct to host any peptide of interest to functionalise the biopolymer characterised by an mScarlet report. We obtained plate reader data on the fluorescence of our mScarlet, which confirmed its superiority as an efficient reporter, especially if compared with another intein reporter construct uploaded on the registry by Heidelberg’s iGEM team in 2014.</p>
−
        <section id="CGE">
+
−
            <div class="card card_igem wow fadeInUp" data-wow-duration="2s" data-wow-delay="0.1s">
+
−
                <div class="card-title text-center">
+
−
                    <h4>Cell Growth and Expression</h4>
+
−
                </div>
+
−
                <div class="card-body text-justify">
+
−
                    <div class="container">
+
−
                        <div class="row">
+
−
                            <div class="col-md-10 mmmm mrfont">
+
−
                                <p class="card-text card-text_igem text-justify">
+
−
                              <p>
+
−
 
+
−
                                Verhulst and Pearl and Reed played a huge part in developing a theory on logistic population growth. The rate considers an additional inhibition factor proportional to <i>x<sup>2</sup></i> from the Malthus population model.
+
−
 
+
−
</p>
+
−
                                    <center>
+
−
 
+
−
 
+
−
<br />
+
−
                              <img src="https://latex.codecogs.com/gif.latex?\frac{dy}{dx}=kx(1-bx)" title="\frac{dy}{dx}=kx(1-bx)" /></center>
+
−
<br />
+
−
                              <center>
+
−
                                where
+
−
                                <img src="https://latex.codecogs.com/gif.latex?x(0)=x_{{o}}" title="x(0)=x_{{o}}" />
+
−
</center>
+
−
<br /><br /><br />
+
−
                            <p>
+
−
 
+
−
                      This Riccati equation can then be solved analytically into:
+
−
                    </p>
+
−
                              <br />
+
−
                              <center>
+
−
 
+
−
 
+
−
                              <img src="https://latex.codecogs.com/gif.latex?x=\frac{x_{{o}}e^{kt}}{1-bx_{{o}}(1-e^{kt})}" title="x=\frac{x_{{o}}e^{kt}}{1-bx_{{o}}(1-e^{kt})}" />                        </center>
+
−
                              <br /><br /><br /><p>
+
−
 
+
−
 
+
−
                              We fit our data into the first-order ordinary differential equation model, and the sigmoidal curve that we get has a property of stationary area which is determined by 1/b.</p>
+
−
 
+
−
 
+
−
                              <table class="table ooooo">
+
−
                                  <thead>
+
−
                                      <tr>
+
−
                                          <th class="tableleft">Symbols</th>
+
−
                                          <th class="tablecenter">Values and Units</th>
+
−
                                          <th class="tablecenter">Descriptions</th>
+
−
                                      </tr>
+
−
                                  </thead>
+
−
                                  <tbody>
+
−
                                      <tr>
+
−
                                          <td class="tableleft"><i>t</i></td>
+
−
                                          <td class="tablecenter"><i>hr</i></td>
+
−
                                          <td class="tablecenter">Time</td>
+
−
                                      </tr>
+
−
                                      <tr>
+
−
                                          <td class="tableleft"><i>x</i></td>
+
−
                                          <td class="tablecenter">dimensionless</td>
+
−
                                          <td class="tablecenter">Optical density of a cell at 600 nm wavelength</td>
+
−
                                      </tr>
+
−
                                      <tr>
+
−
                                          <td class="tableleft"><i>b</i></td>
+
−
                                          <td class="tablecenter">dimensionless</td>
+
−
                                          <td class="tablecenter">
+
−
Inhibiting factor, which is inversely proportional to maximum cell population size.
+
−
</td>
+
−
                                      </tr>
+
−
                                      <tr>
+
−
                                          <td class="tableleft"><i>k</i></td>
+
−
                                          <td class="tablecenter">
+
−
                                          <img src="https://latex.codecogs.com/gif.latex?hr^{-1}" title="hr^{-1}" /></td>
+
−
                                          <td class="tablecenter">
+
−
Population growth rate
+
−
</td>
+
−
                                      </tr>
+
−
                                  </tbody>
+
−
                              </table>
+
−
                              <center>
+
−
                              <figcaption>
+
−
                                Table 01: Additional information.
+
−
                              </figcaption>
+
−
                              </center>
+
−
<br /><br>
+
−
<p>We determined the better growth media from the two broth we had available, which were Terrific Broth (TB) and Lysogeny Broth (LB).
+
−
 
+
−
</p>
+
 
<br>
 
<br>
−
<center>
 
−
<img class="m1"src="https://static.igem.org/mediawiki/2018/a/a0/T--UCL--m1.png"alt="oops! You can't see our image.">
 
−
</center>
 
−
<center>
 
−
 
−
 
−
<figcaption>
 
−
  Figure 01. Comparing TB and LB as E. coli growth medium.
 
−
</figcaption>
 
−
</center>
 
−
<br><br />
 
−
<p>The fitted equations tells us the inhibiting factor <i>b</i> for TB and LB, which are 0.3 and 0.4077, respectively. This is as predicted because TB has less inhibition compared to LB because of the nutrient abundance, carrier capacity, and toxicity level caused by the waste side-products of the cells. The same trend is also observed in maximum population, that TB provides a better environment for larger cell population in longer cultivations. Meanwhile for earlier growth rate <i>k</i>, cells in LB grow quicker before achieving stationary phase, which is 0.44hr<sup>-1</sup> compared with 0.24hr<sup>-1</sup> in TB.
 
−
</p>
 
 
<br><br>
 
<br><br>
 
<center>
 
<center>
−
<img class="m1"src="https://static.igem.org/mediawiki/2018/4/44/T--UCL--m2.png"alt="oops! You can't see our image."></center>
+
  <img class="m1"src="https://static.igem.org/mediawiki/2018/f/f0/T--UCL--r11.png"alt="oops!You can't load our image.">
−
<center>
+
−
<figcaption>
+
−
  Figure 02. Optical density measurements of each cell induction.
+
−
</figcaption>
+
 
</center>
 
</center>
−
<br><br>
 
 
<center>
 
<center>
−
<img class="m1"src="https://static.igem.org/mediawiki/2018/6/64/T--UCL--m3.png"alt="oops! You can't see our image."></center>
+
   <figcaption>
−
<center>
+
     Figure 1: Fluorescence comparison between BBa_K284269 and
−
<figcaption>
+
BBa_K1362101 showing substantially higher fluorescence of
−
   Figure 03. Population growth rate (<i>k</i>) for each IPTG concentration.
+
BBa_K284269N0
−
</figcaption>
+
  </figcaption>
−
</center>
+
−
 
+
−
<br><br><br>
+
−
 
+
−
<table class="table ooooo">
+
−
     <thead>
+
−
        <tr>
+
−
            <th class="tableleft">IPTG Conc.</th>
+
−
            <th class="tablecenter">400mM</th>
+
−
            <th class="tablecenter">500mM</th>
+
−
            <th class="tablecenter">600mM</th>
+
−
            <th class="tablecenter">700mM</th>
+
−
            <th class="tablecenter">800mM</th>
+
−
        </tr>
+
−
    </thead>
+
−
    <tbody>
+
−
        <tr>
+
−
            <td class="tableleft"><i>k</i></td>
+
−
            <td class="tablecenter">0.4215</td>
+
−
            <td class="tablecenter">0.4649</td>
+
−
            <td class="tablecenter">0.4735</td>
+
−
            <td class="tablecenter">0.4773</td>
+
−
            <td class="tablecenter">0.4759</td>
+
−
        </tr>
+
−
        <tr>
+
−
            <td class="tableleft"><i>b</i></td>
+
−
            <td class="tablecenter">0.4361</td>
+
−
            <td class="tablecenter">0.4227</td>
+
−
            <td class="tablecenter">0.4208</td>
+
−
            <td class="tablecenter">0.4097</td>
+
−
            <td class="tablecenter">0.4069</td>
+
−
        </tr>
+
−
    </tbody>
+
−
</table>
+
−
 
+
−
<center>
+
−
<figcaption>
+
−
  Table 02: the growth rate (k) and inhibitor factor (b) corresponding to different IPTG induction concentration.
+
−
</figcaption>
+
−
</center>
+
−
 
+
−
 
+
−
<br><br><p>
+
−
  We then build up the kinetics inside a cell and integrate it with lab data analysis to understand protein production and get an effective lab plan for our E. coli system. GFP, RFP, and Intein Passenger fluorescence data of 544/590 nm filtering wavelengths were then extracted and processed. Each dependent value is described as protein-fold change post induction, which is normalised with the same system but uninduced, based on the fraction below:
+
−
 
+
−
</p>
+
−
<center>
+
−
 
+
−
 
+
−
<br /><img src="https://latex.codecogs.com/gif.latex?p=\frac{F}{F_{{o}}}" title="p=\frac{F}{F_{{o}}}" />
+
−
</center>
+
−
<br><br><br>
+
−
<p>The first-order system of linear differential equations are:
+
−
</p>
+
−
<br>
+
−
<center>
+
−
<img src="https://latex.codecogs.com/gif.latex?\frac{dm}{dt}=\alpha&space;x-\beta&space;m" title="\frac{dm}{dt}=\alpha x-\beta m" />
+
 
</center>
 
</center>
 
<br><br>
 
<br><br>
−
<center><img src="https://latex.codecogs.com/gif.latex?\frac{dp}{dt}=\gamma&space;m-\delta&space;p" title="\frac{dp}{dt}=\gamma m-\delta p" /></center>
 
−
<br><br><br>
 
−
<p>The first equation represents the mRNA system in a cell with the assumption of constant gene concentration in each cell. Meanwhile the latter equation is the protein system which is dependent on the previous equation, and fitted to the data from the plate reader, using the Trust Region Algorithm with non-negative floating number as the boundary in each parameter.
 
  
−
</p>
 
−
<br>
 
−
<center>
 
−
<img class="m1"src="https://static.igem.org/mediawiki/2018/3/33/T--UCL--f4.png"alt="oops! You can't see our image."></center>
 
−
<center>
 
−
<figcaption>
 
−
  Figure 04: Fluorescence of Intein Passenger induced with 400 mM IPTG and normalised in each ​ E. coli ​cell. The rest of induced cultures are fitted in the same equation.
 
−
</figcaption>
 
−
</center>
 
−
<br><br><br>
 
−
<table class="table ooooo">
 
−
    <tdead>
 
−
        <tr>
 
−
            <th class="tableleft">IPTG Conc.(mM)</th>
 
−
            <th class="tablecenter">400</th>
 
−
            <th class="tablecenter">500</th>
 
−
            <th class="tablecenter">600</th>
 
−
            <th class="tablecenter">700</th>
 
−
            <th class="tablecenter">800</th>
 
−
        </tr>
 
−
    </tdead>
 
−
    <tbody>
 
−
      <tr>
 
−
          <td class="tableleft"><i>x</i></td>
 
−
          <td class="tablecenter">200</td>
 
−
          <td class="tablecenter">200</td>
 
−
          <td class="tablecenter">200</td>
 
−
          <td class="tablecenter">200</td>
 
−
          <td class="tablecenter">200</td>
 
−
      </tr>
 
−
      <tr>
 
−
          <td class="tableleft"><i>&alpha;</i></td>
 
−
          <td class="tablecenter">0.02073</td>
 
−
          <td class="tablecenter">0.02332</td>
 
−
          <td class="tablecenter">0.02348</td>
 
−
          <td class="tablecenter">0.02156</td>
 
−
          <td class="tablecenter">0.02264</td>
 
−
      </tr>
 
−
      <tr>
 
−
          <td class="tableleft"><i>&beta;</i></td>
 
−
          <td class="tablecenter">0.2482</td>
 
−
          <td class="tablecenter">0.2644</td>
 
−
          <td class="tablecenter">0.2642</td>
 
−
          <td class="tablecenter">0.2665</td>
 
−
          <td class="tablecenter">0.2777</td>
 
−
      </tr>
 
−
      <tr>
 
−
          <td class="tableleft"><i>&gamma;</i></td>
 
−
          <td class="tablecenter">0.6564</td>
 
−
          <td class="tablecenter">0.6799</td>
 
−
          <td class="tablecenter">0.6668</td>
 
−
          <td class="tablecenter">0.7346</td>
 
−
          <td class="tablecenter">0.7449</td>
 
−
      </tr>
 
−
      <tr>
 
−
          <td class="tableleft"><i>&delta;</i></td>
 
−
          <td class="tablecenter">0.2563</td>
 
−
          <td class="tablecenter">0.2744</td>
 
−
          <td class="tablecenter">0.2719</td>
 
−
          <td class="tablecenter">0.2738</td>
 
−
          <td class="tablecenter">0.2827</td>
 
−
      </tr>
 
−
    </tbody>
 
−
</table>
 
−
<center>
 
−
<figcaption>
 
−
  Table 03: Parameter values estimation using Trust Region Algorithm of curve fitting.
 
−
</figcaption>
 
−
</center>
 
  
−
<br><br><br>
+
                                    </div>
−
<table class="table ooooo">
+
                                 </div>
−
    <thead>
+
−
        <tr>
+
−
            <th class="tableleft">Symbols</th>
+
−
            <th class="tablecenter">Units</th>
+
−
            <th class="tablecenter">Descriptions</th>
+
−
        </tr>
+
−
    </thead>
+
−
    <tbody>
+
−
        <tr>
+
−
            <td class="tableleft"><i>t</i></td>
+
−
            <td class="tablecenter">hours</td>
+
−
            <td class="tablecenter">Time</td>
+
−
        </tr>
+
−
        <tr>
+
−
            <td class="tableleft"><i>x</i></td>
+
−
            <td class="tablecenter">200</td>
+
−
            <td class="tablecenter">
+
−
Plasmid copy number of pSB1C3 in each cell
+
−
</td>
+
−
        </tr>
+
−
        <tr>
+
−
            <td class="tableleft"><i>m</i></td>
+
−
            <td class="tablecenter">dimensionless</td>
+
−
            <td class="tablecenter">
+
−
Respective normalised mRNA concentration in each cell
+
−
</td>
+
−
        </tr>
+
−
        <tr>
+
−
            <td class="tableleft"><i>p</i></td>
+
−
            <td class="tablecenter">
+
−
            dimensionless</td>
+
−
            <td class="tablecenter">
+
−
            Respective normalised protein concentration in each cell
+
−
</td>
+
−
        </tr>
+
−
        <tr>
+
−
            <td class="tableleft">&alpha;</td>
+
−
            <td class="tablecenter"><i>hr<sup>-1</sup></i></td>
+
−
            <td class="tablecenter">
+
−
Gene transcription rate or mRNA production rate
+
−
 
+
−
</td>
+
−
        </tr>
+
−
        <tr>
+
−
            <td class="tableleft"><i>&beta;</i></td>
+
−
            <td class="tablecenter"><i>hr<sup>-1</sup></i></td>
+
−
            <td class="tablecenter">
+
−
mRNA degradation rate and dilution factor
+
−
 
+
−
</td>
+
−
        </tr>
+
−
        <tr>
+
−
            <td class="tableleft">&gamma;</td>
+
−
            <td class="tablecenter"><i>hr<sup>-1</sup></i></td>
+
−
            <td class="tablecenter">
+
−
Protein translation rate
+
−
 
+
−
</td>
+
−
        </tr>
+
−
        <tr>
+
−
            <td class="tableleft">&delta;</td>
+
−
            <td class="tablecenter"><i>hr<sup>-1</sup></i></td>
+
−
            <td class="tablecenter">
+
−
Protein degradation rate and dilution factor
+
−
 
+
−
</td>
+
−
        </tr>
+
−
    </tbody>
+
−
</table>
+
−
<center>
+
−
<figcaption>
+
−
  Table 03: Additional information.
+
−
</figcaption>
+
−
</center>
+
−
<br><br>
+
−
<p>
+
−
  The <i>&alpha;</i> and <i>&beta;</i> terms generally stay constant without a trend between IPTG concentrations which is explainable even though IPTG affects cell transcription activity. Since mRNA has a high rate of synthesis and degradation, it reaches equilibrium immediately as its stability closely follows inversely proportional to the mRNA concentration in the cell. The parameter <i>&gamma;</i> and <i>&delta;</i> give a small yet noticeable increasing trend in higher IPTG level, this is related to protein concentration equilibrium, but not to the extent of mRNA degradation rate since protein relatively ‘lives’ longer than mRNA.
+
−
</p>
+
−
<br>
+
−
<center>
+
−
<img class="m1"src="https://static.igem.org/mediawiki/2018/0/0e/T--UCL--f5.png"alt="oops! You can't see our image."></center>
+
−
<center>
+
−
<figcaption>
+
−
  Figure 05: Fluorescence model of Intein Passenger induced with IPTG 400 mM. Fold-change in protein concentration reaches maximum at 42.8 ratio in longer duration of cell cultivation.
+
−
 
+
−
</figcaption>
+
−
</center>
+
−
<br><br>
+
−
<p>Generally IPTG does not give significant differences in high concentrations, so we chose 500 mM of IPTG which the model suggests as the most efficient with the similar expression level compared to the cells with higher concentration of IPTG. Further improvements of this model might include larger choices of medium and wider range of IPTG concentrations, together with cost analysis as it is an important factor in the larger scale production.
+
−
</p>
+
−
                                 </p>
+
 
                             </div>
 
                             </div>
 
                         </div>
 
                         </div>
 
                     </div>
 
                     </div>
−
                 </div>
+
                 </section>
−
            </div>
+
−
        </section>
+
  
−
 
+
                <section id="BB">
−
 
+
                    <div class="card card_igem wow fadeInUp" data-wow-duration="2s" data-wow-delay="0.1s">
−
 
+
                        <div class="card-title text-center">
−
        <section id="Protein_Polymerisation">
+
                            <h4>BioBrick 2.0</h4>
−
            <div class="card card_igem wow fadeInUp" data-wow-duration="2s" data-wow-delay="0.1s">
+
                        </div>
−
                <div class="card-title text-center">
+
                        <div class="card-body text-justify">
−
                    <h4>Protein Polymerisation</h4>
+
                            <div class="container">
−
                </div>
+
                                <div class="row">
−
                <div class="card-body text-justify">
+
                                    <div class="col-sm-10 mmmm mrfont">
−
                    <div class="container">
+
<br>
−
                        <div class="row">
+
<p>Having singled out time constraints as the most concerning issue for the majority of iGEM teams, we designed a shortcut to solve this problem and increase design flexibility.  Our proposed BioBrick2.0 enables cloning through BioBrick, Golden Gate and Gibson assembly. We demonstrated this through a colony forming unit experiment comparing the average number of white to blue colonies between BioBrick and Golden Gate assembly (Table 1).
−
                            <div class="mmmm col-sm-10 mrfont">
+
</p><br><br>
−
                              <br><br>
+
−
<p>To better accommodate the integrated model and identify bottlenecks, we further develop a model entirely of in vitro polymerisation as it is indispensable to the overall process of biopolymer production.
+
−
 
+
−
</p><br>
+
 
<center>
 
<center>
−
<img src="https://latex.codecogs.com/gif.latex?\frac{dP_{n}}{dt}=\sum_{i=1}^{n} \kappa\left [ P_{i} \right ]\left [ P_{n-1} \right ]-\sum_{i=1}^{n_{max}-n}\kappa\left [ P_{n} \right ]\left [ P_{i} \right ]" title="\frac{dP_{n}}{dt}=\sum_{i=1}^{n} \kappa\left [ P_{i} \right ]\left [ P_{n-1} \right ]-\sum_{i=1}^{n_{max}-n}\kappa\left [ P_{n} \right ]\left [ P_{i} \right ]" />
+
   <img class="m1"src="https://static.igem.org/mediawiki/2018/2/28/T--UCL--rrr111.png"alt="oops!You can't load our image.">
−
</center>
+
−
<br><br>
+
−
<p>Each single protein is interpreted as a monomer with an irreversible polymerising capacity until it reaches an assumed maximum size. Polymer formation rate of each species (P<sub>n</sub>) were then modelled in Python and plotted. The first term describes the polymer species formation from the sum of all reactions in the range of its possible constituents, and the second term represents the further polymerisation of the respective species as a substrate, which build up into larger repeats.
+
−
</p>
+
−
<br><br>
+
−
<center>
+
−
   <img class="m1"src="https://static.igem.org/mediawiki/2018/7/78/T--UCL--m11.png"alt="oops!You can't load our image.">
+
 
</center>
 
</center>
 
<center>
 
<center>
 
   <figcaption>
 
   <figcaption>
−
     Figure 06. Monomer distribution in each species as a function of time for <i>Npu</i> DnaE(N) + <i>Ssp</i> DnaE(C) split inteins. Starting solution only consists of monomers, modeled as they merge and accumulate into larger species of polymer.
+
     Table 1: A comparison of the Colony forming units (CFU) between the two assembly techniques standard biobrick assembly, and Golden gate using Biobrick2.0.
 
   </figcaption>
 
   </figcaption>
 
</center>
 
</center>
 
<br><br>
 
<br><br>
 
<center>
 
<center>
−
   <img class="m1"src="https://static.igem.org/mediawiki/2018/4/40/T--UCL--m12.png"alt="oops!You can't load our image.">
+
   <img class="m1"src="https://static.igem.org/mediawiki/2018/3/3c/T--UCL--rr22.png"alt="oops!You can't load our image.">
 
</center>
 
</center>
 
<center>
 
<center>
 
   <figcaption>
 
   <figcaption>
−
     Figure 07. Monomer distribution in each species as a function of time <i>Ssp</i> DnaE inteins. Starting solution only consists of monomers, modeled as they merge and accumulate into larger species of polymer.
+
     Figure 2: Representative Plates of BBa_K2842669 cloned into BioBrick2.0 <br>
 +
(1) Golden Gate Assembly undiluted <br>
 +
(2) BioBrick Assembly undiluted <br>
 +
(3) Golden Gate Assembly 10x dilution <br>
 +
(4) BioBrick Assembly 10x dilution <br>
 +
(5) Golden Gate Assembly 100x dilution <br>
 +
(6) BioBrick Assembly 100x dilution <br>
  
 
   </figcaption>
 
   </figcaption>
Line 611: Line 264:
 
<br><br>
 
<br><br>
 
<center>
 
<center>
−
   <video width="100%" controls>
+
   <img class="m1"src="https://static.igem.org/mediawiki/2018/d/d8/T--UCL--rr33.png"alt="oops!You can't load our image.">
−
    <source src="https://static.igem.org/mediawiki/2018/3/30/T--UCL--abc.mp4" type="video/mp4" />
+
−
    Your browser does not support this video.
+
−
  </video>
+
 
</center>
 
</center>
 
<center>
 
<center>
 
   <figcaption>
 
   <figcaption>
−
     Figure 8. Ratio of monomer distribution species over time in Npu DnaE(N) + <i>Ssp</i> DnaE(C) split inteins combination. Frame rates do not represent real-time.
+
     Figure 3: Digestion of pSB1C3 BioBrick2.0 to show correct functioning of the new restriction sites. <br>
 +
(1) BsaI digested BioBrick2.0 (Golden Gate Assembly) <br>
 +
(2) EcoRI+PstI digested BioBrick2.0 (BioBrick Assembly) <br>
 +
(3) undigested BioBrick2.0 <br>
  
 
   </figcaption>
 
   </figcaption>
 
</center>
 
</center>
−
<br><br>
+
                                    </div>
−
<center>
+
                                </div>
−
  <video width="100%" controls>
+
                            </div>
−
    <source src="https://static.igem.org/mediawiki/2018/3/31/T--UCL--cbb.mp4" type="video/mp4" />
+
                        </div>
−
    Your browser does not support this video.
+
                    </div>
−
  </video>
+
                </section>
−
</center>
+
                <section id="IEP">
−
<center>
+
                    <div class="card card_igem wow fadeInUp" data-wow-duration="2s" data-wow-delay="0.1s">
−
  <figcaption>
+
                        <div class="card-title text-center">
−
    Figure 9. Ratio of monomer distribution species over time in <i>Ssp</i> DnaE inteins. Frame rates do not represent real-time.
+
                            <h4>Integrated Human Practices</h4>
−
 
+
                        </div>
−
  </figcaption>
+
                        <div class="card-body text-justify">
−
</center>
+
                            <div class="container">
−
<br><br>
+
                                <div class="row">
−
 
+
                                    <div class="col-sm-10 mmmm mrfont">
−
<table class="table ooooo">
+
−
    <thead>
+
−
        <tr>
+
−
            <th class="tableleft">Symbols</th>
+
−
            <th class="tablecenter">Values and Units</th>
+
−
            <th class="tablecenter">Descriptions</th>
+
−
        </tr>
+
−
    </thead>
+
−
    <tbody>
+
−
        <tr>
+
−
            <td class="tableleft"><i>t</i></td>
+
−
            <td class="tablecenter">s</td>
+
−
            <td class="tablecenter">Time</td>
+
−
        </tr>
+
−
        <tr>
+
−
            <td class="tableleft"><i>n</i></td>
+
−
            <td class="tablecenter">dimensionless</td>
+
−
            <td class="tablecenter">
+
−
Number of repeats, defining a certain species of polymer
+
−
</td>
+
−
        </tr>
+
−
        <tr>
+
−
            <td class="tableleft"><i>P<sub>n</sub></i></td>
+
−
            <td class="tablecenter">mol L<sup>-1</sup></td>
+
−
            <td class="tablecenter">
+
−
              Polymer species <i>n</i>
+
−
</td>
+
−
        </tr>
+
−
        <tr>
+
−
            <td class="tableleft"><i>k</i></td>
+
−
            <td class="tablecenter">
+
−
          L mol<sup>-1</sup> S<sup>-1></sup></td>
+
−
            <td class="tablecenter">
+
−
              Rate constants of Intein activity of a certain intein
+
−
</td>
+
−
        </tr>
+
−
        <tr>
+
−
            <td class="tableleft"><i>n<sub>max</sub></i></th>
+
−
            <td class="tablecenter">dimensionless</th>
+
−
            <td class="tablecenter">
+
−
Maximum repeat number in a polymer</th>
+
−
        </tr>
+
−
    </tbody>
+
−
</table>
+
−
<center>
+
−
<figcaption>
+
−
  Table 04: Additional information.
+
−
</figcaption>
+
−
</center>
+
−
<br><br><br>
+
−
<p>Basically each intein rate of reaction (<i>k</i>) differs with each intein system we use.</p>
+
−
<br />
+
−
<table class="table ooooo">
+
−
    <thead>
+
−
        <tr>
+
−
            <th class="tableleft">Intein</th>
+
−
            <th class="tablecenter">Rate of reaction (s<sup>-1</sup>)</th>
+
−
        </tr>
+
−
    </thead>
+
−
    <tbody>
+
−
        <tr>
+
−
            <td class="tableleft"><i>Npu</i> DNA-E</td>
+
−
            <td class="tablecenter">1.1x10<sup>-2</sup></td>
+
−
 
+
−
        </tr>
+
−
        <tr>
+
−
            <td class="tableleft"><i>Ssp</i>DNA-E</td>
+
−
            <td class="tablecenter">3.3x10<sup>-4</sup></td>
+
−
 
+
−
</td>
+
−
        </tr>
+
−
        <tr>
+
−
            <td class="tableleft"><i>Npu</i> DNA-E(N) + <i>Ssp</i>DNA-E</td>
+
−
            <td class="tablecenter">2.8x10<sup>-5</sup></td>
+
−
 
+
−
 
+
−
</td>
+
−
        </tr>
+
−
        <tr>
+
−
            <td class="tableleft"><i>Sce</i>VMA</td>
+
−
            <td class="tablecenter">
+
−
        2.0x10<sup>-3</sup></td>
+
−
        </tr>
+
−
        <tr>
+
−
            <td class="tableleft"><i>Ssp</i>DNA-B</th>
+
−
            <td class="tablecenter">9.9x10<sup>-4</sup></th>
+
−
        </tr>
+
−
    </tbody>
+
−
</table>
+
−
<center>
+
−
<figcaption>
+
−
  Table 05. Specific activity of each inteins. Rate of reaction are collected from the references mentioned in the bottom of the page.
+
−
</figcaption>
+
−
</center>
+
−
<br><br>
+
−
<p>
+
−
  The results we get from the example runs of <i>Npu</i> DnaE(N) and <i>Ssp</i> DnaE(C) intein combinations are after 10 minutes of the simulated experiment, only 3.45% of monomers are in the form of the top 25% longest polymer species. However for <i>Ssp</i> DnaE inteins, in the course of 10 minutes, 72.3% of the monomers are polymerised into the same top 25% of the longest polymer region. This really shows how the intein polymerisation will require much less time compared to the cell culture and protein production. Further improvements will be introducing more parameters into the estimation function to represent more accurate results.
+
−
</p>
+
−
<br><br><br>
+
−
<div id="WORM"class="card-title text-center">
+
−
    <h4>Intein Polymerisation</h4>
+
−
    <br>
+
−
    <h3 class="orange mrfont2">W.O.R.M.</h3>
+
−
</div>
+
−
<br><br>
+
−
<p>
+
−
  To further model the polymerisation of our proteins, we devised a simplified proof of concept of our project using reporter proteins RFP and GFP to predict the polymerisation capabilities of split inteins. Each reporter protein is flanked by orthogonal inteins, which match up to the other reporter’s inteins to allow RFP and GFP to continuously link up into various lengths of polymers.
+
−
</p>
+
 
<br>
 
<br>
−
<center>
+
<p>The benefits of our project on society would come from direct interactions with the manufacturing industry. If spider silk polymerisation and functionalisation could be made possible but also cheaper, it would reflect on the cost of production and hence selling. This is why we decided to target Bolt Threads first which gave us a huge insight on the biomaterials industry. This impacted on the way we thought about and designed our project. Our experiments were based on spider silk alone initially but we ended up inventing a modular platform for all biomaterials in the end. This tunable platform is made of 3 main parts which can be made to plug-and-play with different biomaterials. We changed our experiments to polymerise other proteins rather than spider silk alone. Other conversations made us look into the way we’d apply a spider silk filter in a community and other functionalizing proteins we could apply to our biomaterials.
−
  <img class="m1"src="https://static.igem.org/mediawiki/2018/c/c5/T--UCL--iii1.png"alt="oops!You can't load our image.">
+
−
</center>
+
−
<center>
+
−
  <figcaption>
+
−
    Figure 10: Polymerisation of intein flanked reporter proteins. <br>
+
−
Npu-N intein on GFP splices out with the Npu-C intein on RFP. Similarly, the TerL-N intein on the RFP splices out with the TerL-C intein on the GFP. This reaction results in the polymerisation of GFP and RFP.
+
−
 
+
−
  </figcaption>
+
−
</center>
+
−
<br>
+
−
We used the python libraries PyGame and PyMunk to create an animated and interactive agent-based model of intein polymerisation. Acronymed WORM, this model depicts a Wonderful Oligomerisation Reaction Model.
+
−
<br><br>
+
−
 
+
−
<video width="100%" controls>
+
−
  <source src="https://static.igem.org/mediawiki/2018/6/61/T--UCL--worm.mp4" type="video/mp4" />
+
−
  Your browser does not support this video.
+
−
</video>
+
−
<br><br>
+
−
<p>Depicted in the video is a snapshot of the model WORM that shows intein polymerisation. The yellow and orange rectangles represent an split intein pair, as do the blue and cyan rectangles. The red and green rectangles represent the reporter proteins, RFP and GFP, respectively. </p>
+
−
<br>
+
−
<p>As an agent-based mode, WORM was created based on the following rules: <br>
+
−
  1)Particles diffuse with random angular and lateral velocity, <br>
+
−
  2)Velocity increases with temperature (based on average kinetic energy)
+
−
<br>
+
−
<center><img src="https://latex.codecogs.com/gif.latex?(\frac{1}{2}mv^{2})=\frac{3}{2}kT" title="(\frac{1}{2}mv^{2})=\frac{3}{2}kT" /></center>
+
−
<p>  Where: <br>
+
−
m= mass (kg) <br>
+
−
v= velocity <br>
+
−
k= Boltzmann constant <br>
+
−
    T= temperature (K) <br>
+
−
 
+
 
</p>
 
</p>
−
 
+
<br><br />
−
 
+
                                    </div>
−
 
+
                                </div>
−
<br><br>
+
−
3)Like reacts with unlike (RFP reacts with GFP), <br>
+
−
4)Particles are polarised (Npu-N [A1]→ Npu-C [B3], TerL-N [B1]→ TerL-C [A3]) <br>
+
−
5)Splicing removes 1+3 on reaction (removes the inteins), <br>
+
−
6)Steric hindrance by particles  <br>
+
−
<br><br>
+
−
 
+
−
<center>
+
−
  <img class="m1"src="https://static.igem.org/mediawiki/2018/7/73/T--UCL--f111.png"alt="oops!You can't load our image.">
+
−
</center>
+
−
<center>
+
−
  <figcaption>
+
−
    FIGURE 11: Distribution of polymer lengths after 20 runs. <br>
+
−
WORM was run for 5 minutes, 20 times. The distribution of total polymers for each polymer lengths from 1-20, and lengths over 20, were plotted. The error was plotted as the square root of the total polymers in each polymer length.
+
−
 
+
−
  </figcaption>
+
−
</center>
+
−
<br><br>
+
−
<p>The interactive components of the WORM model include user’s choice of temperature and protein mass to selectively model the reaction conditions and functionalized protein of interest. These applications can be used by running the code found in our GitHub. The combination of predictive experimental data and the visually appealing computational model makes a user friendly platform for the public to better understand biology and modelling.</p>
+
−
<br><br>
+
−
 
+
−
 
+
−
 
+
−
 
+
−
 
+
−
 
+
−
 
+
−
 
+
−
 
+
−
 
+
−
 
+
−
 
+
−
 
+
 
                             </div>
 
                             </div>
−
 
 
                         </div>
 
                         </div>
 
                     </div>
 
                     </div>
−
                 </div>
+
                 </section>
−
            </div>
+
                <section id="IM">
−
        </section>
+
                    <div class="card card_igem wow fadeInUp" data-wow-duration="2s" data-wow-delay="0.1s">
−
 
+
                        <div class="card-title text-center">
−
 
+
                            <h4>Integrated Modelling</h4>
−
 
+
                        </div>
−
 
+
                        <div class="card-body text-justify">
−
 
+
                            <div class="container">
−
 
+
                                <div class="row">
−
        <section id="Mech">
+
                                    <div class="col-sm-10 mmmm mrfont">
−
            <div class="mrfont card card_igem wow fadeInUp" data-wow-duration="2s" data-wow-delay="0.1s">
+
<br>
−
                <div class="card-title text-center">
+
<p>We integrated all of our models into our work - the first kinetic model we looked at how varying IPTG concentrations and cell broth would affect gene and protein expression as we scaled up our project. The result of our first model is using 500 mM of IPTG which the model suggests as the most efficient option, and choosing TB as the medium with better carrying capacity.
−
                    <h4>Mechanical Simulation</h4>
+
−
                </div>
+
−
                <div class="card-body text-justify">
+
−
                    <div class="container">
+
−
                        <div class="row">
+
−
                            <div class="col-sm-10 mmmm mrfont">
+
−
                                <p class="card-text card-text_igem text-justify">
+
−
                                  <br>
+
−
                                    <p>In this part, we also modeled the biomaterials’ micro-structure of the coarse-grained system. We predicted the structure of different spider silk repeats (tetramer, octamer and dodecamer) both after stretching and shearing. This method is a deterministic approach to foresee the network structure of spider silk repeats prior to the actual lab experiment and helps us to understand the design of the actual protein that we are going to produce afterwards.
+
 
</p>
 
</p>
−
<br>
+
<br><br>
−
<p>Here is our initial designed sequence, a spider silk protein in repeats of four:</p>
+
−
<span class="nodec">
+
−
GQGGYGGLGSQGAGRGGLGGQGAGAAAAAAAAGGAGQGGYGGLGSQGAGRGGLGGQGAGAAAAAAAAAGGYGGLGSQGAGRGGLGGQGAGAAAAAAAAGGAGQGGYGGLGSQGAGRGGLGGQGAGAAAAAAAAGGA</span>
+
−
<br><br><br>
+
−
The respective sequence is then quantitatively analysed for their hydrophobicity and hydrophilicity and plotted.
+
−
<br>
+
 
<center>
 
<center>
−
   <img class="m1"src="https://static.igem.org/mediawiki/2018/1/19/T--UCL--m7.png"alt="oops!You can't load our image.">
+
   <img class="m1"src="https://static.igem.org/mediawiki/2018/4/4a/T--UCL--graph1.png"alt="oops!You can't load our image.">
−
</center>
+
−
<center>
+
−
  <figcaption>
+
−
    Figure 11. Kyte-Doolittle-Hydropathy plot. The vertical axis is the measure of hydrophobicity in each amino acid in the sequence, with higher score means greater degree of hydrophobicity. With the help of ProtScale from ExPASy.
+
−
  </figcaption>
+
 
</center>
 
</center>
 
<br><br>
 
<br><br>
−
<p>In this particular sequence, we can observe that the hydrophobic and hydrophilic parts alternate inside the spider silk fiber, which will form aggregates and bridge connections to achieve desired tensile and toughness due to the hydrophobic interactions.
+
<p>We found that the maximum protein fold-change as 42.8 ratio over control. It can be achieved by inoculating the cells just over 25 hours. This is really important to know the maximum yield of the process and even determine the efficiency of the protein extraction in the next steps.</p>
−
</p>
+
 
<br>
 
<br>
−
<p>
+
<p>The second model is the intein polymerisation, it gives us the estimate time required to produce the certain length of polymer before doing the actual experiment. From the results using specifically our intein, we then know 15 minutes are enough time to get about 90% of minimum polymer species 12 (dodecamer). Then molecular dynamics simulations are done to give an insight on the structure of our protein fiber to determine the desired polymer length to achieve good mechanical properties, the result is then fed back into the BioBrick design so we are able to construct the polymer without doing an actual lab experiment and hence reduce unnecessary steps, time and energy spent. </p>
−
  This alternating pattern seen in the figure allows us to simplify proteins as the hydrophobic domains and hydrophilic domains. These domains are then represented as a collection of the so-called ‘beads’, and depending on relative molecular volume of each domain, the coarse-grained model is then scaled and measured into an estimate of three amino acid residues per bead.
+
                                    </div>
−
</p>
+
                                 </div>
−
<br>
+
−
<p>This system is composed from 3 components: water, hydrophilic and hydrophobic beads. Every 9 water molecules is represented as a single bead, so its 270 Å size (each water molecule is about 30 Å) is proportional to the protein beads. </p>
+
−
<br>
+
−
<p>We then introduce LAMMPS as our software, which is an abbreviation of Large-scale Atomic/Molecular Massively Parallel Simulator, to simulate the behaviour of our ‘beads’ collection in a single compartment and thus gave us an insight into the physical property of the recombinant spider silk.</p>
+
−
<br><br>
+
−
 
+
−
<div class="card-title text-center mrfont2">
+
−
  <h1 class="orange">Simulations</h1>
+
−
  <h5>Hover to compare the 2 sides.</h5>
+
−
  <br><br>
+
−
  <h6><span class="marrrgin"style="float:left">Equal</span> <span class="marrgin"style="float:right">Stretch</span></h6>
+
−
</div>
+
−
<br>
+
−
<div class="col-sm-12">
+
−
  <hr />
+
−
  <div class="card-title text-center mrfont2">
+
−
    <center><h5>Tetramer</h5></center>
+
−
  </div>
+
−
  <hr />
+
−
<div class="row yyyyy">
+
−
<div class="col-sm-6">
+
−
    <p class="card-text card-text_igem text-justify">
+
−
      <center>
+
−
        <img class="gifs"src="https://static.igem.org/mediawiki/2018/3/30/T--UCL--4e.gif" />
+
−
      </center>
+
−
    </p>
+
−
</div>
+
−
<div class="col-sm-6">
+
−
    <p class="card-text card-text_igem text-justify">
+
−
      <center>
+
−
        <img class="gifs"src="https://static.igem.org/mediawiki/2018/6/64/T--UCL--4s.gif" />
+
−
      </center>
+
−
    </p>
+
−
</div>
+
−
</div>
+
−
<hr>
+
−
<div class="card-title text-center mrfont2">
+
−
  <center><h5>Octamer</h5></center>
+
−
</div>
+
−
<hr>
+
−
<div class="row yyyyy">
+
−
<div class="col-sm-6">
+
−
    <p class="card-text card-text_igem text-justify">
+
−
      <center>
+
−
        <img class="gifs"src="https://static.igem.org/mediawiki/2018/d/da/T--UCL--8e.gif" />
+
−
      </center>
+
−
    </p>
+
−
</div>
+
−
<div class="col-sm-6">
+
−
    <p class="card-text card-text_igem text-justify">
+
−
      <center>
+
−
        <img class="gifs"src="https://static.igem.org/mediawiki/2018/3/39/T--UCL--8s.gif" />
+
−
      </center>
+
−
    </p>
+
−
</div>
+
−
</div>
+
−
<hr>
+
−
<div class="card-title text-center mrfont2">
+
−
  <center><h5>Dodecamer</h5></center>
+
−
</div>
+
−
<hr>
+
−
<div class="row yyyyy">
+
−
<div class="col-sm-6">
+
−
    <p class="card-text card-text_igem text-justify">
+
−
      <center>
+
−
        <img class="gifs"src="https://static.igem.org/mediawiki/2018/9/9d/T--UCL--12e.gif" />
+
−
      </center>
+
−
    </p>
+
−
</div>
+
−
<div class="col-sm-6">
+
−
    <p class="card-text card-text_igem text-justify">
+
−
      <center>
+
−
        <img class="gifs"src="https://static.igem.org/mediawiki/2018/5/51/T--UCL--12s.gif" />
+
−
      </center>
+
−
    </p>
+
−
</div>
+
−
</div>
+
−
 
+
−
</div>
+
−
 
+
−
<br><br>
+
−
<p>For these simulation results, the configurations of spider silk fiber after applying a constant engineering strain rate (7.5 × 10<sup>-6</sup> τ<sup>-1</sup> along the x-axis) and at equilibrium after shear flow (0.01 τ<sup>-1</sup> along x axis for 420000 shear steps). The red nodes show hydrophobic aggregated protein clusters and the blue lines are the bridge connecting two clusters. Uniform distribution and high density of clusters and bridges represent the better mechanical property, higher tensile and toughness. For stretch testing. we can analyse the tetramer structure which consists of large and frequent cavity in the fiber. The octamer and dodecamer is predicted to be able to tolerate this strain, which means they have a considerable threshold of mechanical strain. In order to examine which structure is the best, we apply shear flow. In this case, it is observed that only the dodecamer still keeps its original structure but others break at different levels. In conclusion, dodecamer is the best configuration required in making desired fibers.
+
−
</p>
+
−
 
+
−
                                 </p>
+
 
                             </div>
 
                             </div>
 
                         </div>
 
                         </div>
 
                     </div>
 
                     </div>
−
                 </div>
+
                 </section>
 
             </div>
 
             </div>
−
         </section>
+
         </div>
 +
        </div>
  
−
        <section id="Bibliography">
+
    </div>
−
            <div class="card card_igem wow fadeInUp" data-wow-duration="2s" data-wow-delay="0.1s">
+
    <!--FOOTER================================================================== -->
−
                <div class="card-title text-center">
+
−
                    <h4>Bibliography</h4>
+
−
                </div>
+
−
                <div class="card-body text-justify">
+
−
                    <div class="container">
+
−
                        <div class="row">
+
−
                            <div class="col-sm-10 mmmm mrfont">
+
−
                                <p class="card-text card-text_igem text-justify">
+
−
                                    <ol>
+
−
                                        <li>
+
−
                                            Verhulst PF. Notice sur la loi que la population poursuit dans son accroissement. Correspondance mathématique et physique. 1838;10:113–21.
+
−
                                        </li>
+
−
                                        <li>
+
−
                                          Pearl R, Reed L. On the Rate of Growth of the Population of the United States since 1790 and Its Mathematical Representation. Proceedings of the National Academy of Sciences. 1920;6(6):275-288.
+
  
−
                                        </li>
+
    <div class="footer">
−
                                        <li>
+
        <div class="container">
−
                                          Bailey J, Ollis D. Biochemical engineering fundamentals. 2nd ed. New York: McGraw-Hill; 1944.
+
            <div class="row">
 +
                <div class="col-lg-4">
 +
                    <center>
 +
                        <div class="h2_footer_igem">Our Location</div>
 +
                        University College London<br />
 +
      Gower St, Bloomsbury<br />London <br />WC1E 6BT
 +
                        <span></span>
  
−
                                        </li>
+
                    </center>
−
                                        <li>
+
                </div>
−
                                            Part:pSB1C3 - parts.igem.org [Internet]. Parts.igem.org. 2008. Available from: http://parts.igem.org/Part:pSB1C3
+
                <div class="col-lg-4">
−
                                        </li>
+
                    <center>
 +
                        <div class="h2_footer_igem">Contact Us</div>
 +
                      ucl.igem@ucl.ac.uk
 +
                        <a class="footer_img_igem" href="https://www.ucl.ac.uk/">
 +
                            <img src="https://static.igem.org/mediawiki/2018/f/ff/T--UCL--UCLLogo.jpeg" height="100rem;" />
 +
                        </a>
 +
                    </center>
 +
                </div>
 +
                <div class="col-lg-4">
 +
                    <center>
 +
                        <div class="h2_footer_igem">Follow Us</div>
 +
                        <a href="https://www.facebook.com/UCLiGEM/"class="contacticons">
 +
                            <img src="https://static.igem.org/mediawiki/2018/c/c4/T--UCL--FBLogo.svg" style="padding-top:1em;" height="62rem;"/>
 +
                        </a>
 +
                        <a href="https://twitter.com/ucligem?lang=en"class="contacticons">
 +
                            <img src="https://static.igem.org/mediawiki/2018/e/ed/T--UCL--TwitterLogo.svg" style="padding-top:1em;" height="62rem;"/>
 +
                        </a>
 +
                        <a href="https://www.instagram.com/ucl.igem/"class="contacticons">
 +
                            <img src="https://static.igem.org/mediawiki/2018/3/31/T--UCL--InstaLogo.png" style="padding-top:1em;" height="62rem;"/>
  
−
                                        <li>
+
                        </a>
−
                                            Zettler J, Schütz V, Mootz HD. The naturally split Npu DnaE intein exhibits an extraordinarily high rate in the protein trans-splicing reaction. FEBS Letters 2009;583:909–14.
+
                     </center>
−
                                        </li>
+
−
                                        <li>
+
−
                                            Nichols NM, Benner JS, Martin DD, Evans TC. Zinc Ion Effects on Individual Ssp DnaE Intein Splicing Steps: Regulating Pathway Progression. Biochemistry 2003;42:5301–11.
+
−
                                        </li>
+
−
                                        <li>
+
−
                                            Al-Ali H, Ragan TJ, Gao X, Harris TK. Reconstitution of Modular PDK1 Functions on Trans-Splicing of the Regulatory PH and Catalytic Kinase Domains. Bioconjugate Chemistry 2007;18:1294–302.
+
−
                                        </li>
+
−
                                        <li>
+
−
                                          Brenzel S, Kurpiers T, Mootz HD. Engineering Artificially Split Inteins for Applications in Protein Chemistry:  Biochemical Characterization of the Split Ssp DnaB Intein and Comparison to the Split Sce VMA Intein. Biochemistry 2006;45:1571–8.
+
−
                                        </li>
+
−
 
+
−
                                        <li>
+
−
                                            Kyte J, Doolittle RFA. simple method for displaying the hydropathic character of a protein. Journal of Molecular Biology. 1982;157(1):105–32.
+
−
                                        </li>
+
−
                                        <li>
+
−
                                            ExPASy - ProtScale [Internet]. Web.expasy.org. Available from: https://web.expasy.org/protscale/
+
−
                                        </li>
+
−
                                        <li>
+
−
                                            Rim N, Roberts E, Ebrahimi D, Dinjaski N, Jacobsen M, Martín-Moldes Z, Buehler M, Kaplan D and Wong J. Predicting Silk Fiber Mechanical Properties through Multiscale Simulation and Protein Design. ACS Biomaterials Science & Engineering. 2017;3(8), pp.1542-1556.
+
−
                                        </li>
+
−
                                        <li>
+
−
                                          Lin S, Ryu S, Tokareva O, Gronau G, Jacobsen M, Huang W, Rizzo D, Li D, Staii C, Pugno N, Wong J, Kaplan D and Buehler M. Predictive modelling-based design and experiments for synthesis and spinning of bioinspired silk fibres. Nature Communications. 2015;6(1).
+
−
 
+
−
                                        </li>
+
−
                                    </ol>
+
−
                                </p>
+
−
                            </div>
+
−
                        </div>
+
−
                     </div>
+
 
                 </div>
 
                 </div>
−
            </div>
 
−
        </section>
 
−
 
−
 
−
 
−
 
−
    </div>
 
−
</div>
 
−
</div>
 
−
<div class="footer">
 
−
    <div class="container">
 
−
        <div class="row">
 
−
            <div class="col-lg-4">
 
−
                <center>
 
−
                    <div class="h2_footer_igem">Our Location</div>
 
−
                    University College London<br />
 
−
  Gower St, Bloomsbury<br />London <br />WC1E 6BT
 
−
                    <span></span>
 
−
 
−
                </center>
 
−
            </div>
 
−
            <div class="col-lg-4">
 
−
                <center>
 
−
                    <div class="h2_footer_igem">Contact Us</div>
 
−
                  ucl.igem@ucl.ac.uk
 
−
                    <a class="footer_img_igem" href="https://www.ucl.ac.uk/">
 
−
                        <img src="https://static.igem.org/mediawiki/2018/f/ff/T--UCL--UCLLogo.jpeg" height="100rem;" />
 
−
                    </a>
 
−
                </center>
 
−
            </div>
 
−
            <div class="col-lg-4">
 
−
                <center>
 
−
                    <div class="h2_footer_igem">Follow Us</div>
 
−
                    <a href="https://www.facebook.com/UCLiGEM/"class="contacticons">
 
−
                        <img src="https://static.igem.org/mediawiki/2018/c/c4/T--UCL--FBLogo.svg" style="padding-top:1em;" height="62rem;"/>
 
−
                    </a>
 
−
                    <a href="https://twitter.com/ucligem?lang=en"class="contacticons">
 
−
                        <img src="https://static.igem.org/mediawiki/2018/e/ed/T--UCL--TwitterLogo.svg" style="padding-top:1em;" height="62rem;"/>
 
−
                    </a>
 
−
                    <a href="https://www.instagram.com/ucl.igem/"class="contacticons">
 
−
                        <img src="https://static.igem.org/mediawiki/2018/3/31/T--UCL--InstaLogo.png" style="padding-top:1em;" height="62rem;"/>
 
−
 
−
                    </a>
 
−
                </center>
 
 
             </div>
 
             </div>
 
         </div>
 
         </div>
 
     </div>
 
     </div>
−
</div>
 
 
</body>
 
</body>
 +
</html>

Latest revision as of 13:32, 8 December 2018


UCL SETA - Results

Results

Modular Platform


Starting from the an idea centered around spider silk and its applications, after an insightful conversation with the CSO of Bolt Threads, we devised and developed a tunable construct which brings modular protein engineering in the realm of biomaterials. First, we considered using the spytag-spycatcher system, but we realised that there would have been a much better method to reach our objective. Spliced inteins contribute to the assembly of fusion proteins through directed covalent bonding, in the same fashion as spytag and spycatcher[1]. However, spytag and spycatcher risk to influence the folding and polymerisation process of biomaterials regulated by tight structural constraints through steric hindrance due to their size. Split-inteins avoid this type of issue by splicing out and leaving a permanent covalent bond in a short time, which varies slightly according to literature[2]. In order to simulate the association and potential polymerization through split inteins, we designed a green-red split-inteins system which alternates well characterised chromogenic proteins: GFP, and RFP [3]. Additionally, we devised an Intein Passenger construct to host any peptide of interest to functionalise the biopolymer characterised by an mScarlet report. We obtained plate reader data on the fluorescence of our mScarlet, which confirmed its superiority as an efficient reporter, especially if compared with another intein reporter construct uploaded on the registry by Heidelberg’s iGEM team in 2014.




oops!You can't load our image.
Figure 1: Fluorescence comparison between BBa_K284269 and BBa_K1362101 showing substantially higher fluorescence of BBa_K284269N0


BioBrick 2.0


Having singled out time constraints as the most concerning issue for the majority of iGEM teams, we designed a shortcut to solve this problem and increase design flexibility. Our proposed BioBrick2.0 enables cloning through BioBrick, Golden Gate and Gibson assembly. We demonstrated this through a colony forming unit experiment comparing the average number of white to blue colonies between BioBrick and Golden Gate assembly (Table 1).



oops!You can't load our image.
Table 1: A comparison of the Colony forming units (CFU) between the two assembly techniques standard biobrick assembly, and Golden gate using Biobrick2.0.


oops!You can't load our image.
Figure 2: Representative Plates of BBa_K2842669 cloned into BioBrick2.0
(1) Golden Gate Assembly undiluted
(2) BioBrick Assembly undiluted
(3) Golden Gate Assembly 10x dilution
(4) BioBrick Assembly 10x dilution
(5) Golden Gate Assembly 100x dilution
(6) BioBrick Assembly 100x dilution


oops!You can't load our image.
Figure 3: Digestion of pSB1C3 BioBrick2.0 to show correct functioning of the new restriction sites.
(1) BsaI digested BioBrick2.0 (Golden Gate Assembly)
(2) EcoRI+PstI digested BioBrick2.0 (BioBrick Assembly)
(3) undigested BioBrick2.0

Integrated Human Practices


The benefits of our project on society would come from direct interactions with the manufacturing industry. If spider silk polymerisation and functionalisation could be made possible but also cheaper, it would reflect on the cost of production and hence selling. This is why we decided to target Bolt Threads first which gave us a huge insight on the biomaterials industry. This impacted on the way we thought about and designed our project. Our experiments were based on spider silk alone initially but we ended up inventing a modular platform for all biomaterials in the end. This tunable platform is made of 3 main parts which can be made to plug-and-play with different biomaterials. We changed our experiments to polymerise other proteins rather than spider silk alone. Other conversations made us look into the way we’d apply a spider silk filter in a community and other functionalizing proteins we could apply to our biomaterials.



Integrated Modelling


We integrated all of our models into our work - the first kinetic model we looked at how varying IPTG concentrations and cell broth would affect gene and protein expression as we scaled up our project. The result of our first model is using 500 mM of IPTG which the model suggests as the most efficient option, and choosing TB as the medium with better carrying capacity.



oops!You can't load our image.


We found that the maximum protein fold-change as 42.8 ratio over control. It can be achieved by inoculating the cells just over 25 hours. This is really important to know the maximum yield of the process and even determine the efficiency of the protein extraction in the next steps.


The second model is the intein polymerisation, it gives us the estimate time required to produce the certain length of polymer before doing the actual experiment. From the results using specifically our intein, we then know 15 minutes are enough time to get about 90% of minimum polymer species 12 (dodecamer). Then molecular dynamics simulations are done to give an insight on the structure of our protein fiber to determine the desired polymer length to achieve good mechanical properties, the result is then fed back into the BioBrick design so we are able to construct the polymer without doing an actual lab experiment and hence reduce unnecessary steps, time and energy spent.

Our Location
University College London
Gower St, Bloomsbury
London
WC1E 6BT
Contact Us
ucl.igem@ucl.ac.uk
Follow Us