Difference between revisions of "Team:IISER-Tirupati India/Contribution"

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                         <h2>Introduction</h2>
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                     <div class="col-md-4 col-lg-3 p-2 d-none d-md-block" style="background-color: #FFBD59 !important; color: #8D1063;">
                        <p>Natural systems are highly complex to understand as well as to experiment with. To make predictions of the outcomes,
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                         <div class="card" style=" max-width: 15.5rem; border: none !important;background-color:#FFBD59;" id="index">
                            we use the available information and the knowledge of physics, mathematics, chemistry, and computer science to build a theoretical model. This page deals with the models built on the different aspects of our project namely :</p>
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                             <div class="card-body">
                             <ol>
+
                                 <h3 class="card-title text-center mb-3">INDEX</h3>
                                 <li>GMO delivery</li>
+
                                 <section id="Index1">  
                                 <li>GMO growth and colonisation</li>
+
                                    <h5><a class="index_link" href="#1">New Parts From Literature</a></h5>
                                <li>Protease production</li>
+
                                    <ul>
                                <li>Diffusion and ovum Hardening</li>
+
                                        <li><a class="index_link" href="#11">Promoters</a></li>
                                <li>Kill switch for reversibility</li>
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                                        <li><a class="index_link" href="#12">P22 Operator Library</a></li>
                                <li>Kill switch for Biosafety</li>
+
                                        <li><a class="index_link" href="#13">Coding sequences</a></li>
                                </ol>
+
                                        <li><a class="index_link" href="#14">Device</a></li>
                            <h2>Delivery & Colonisation</h2><br>
+
                                        <li><a class="index_link" href="#15">References</a></li>  
                            <h3>INTRODUCTION: THE JOURNEY OF GMO BEGINS</h3><br>
+
                                    </ul>                        
                            <h4>PART A: Delivery</h4><br>
+
                                 </section>
                            <h5>OVERVIEW</h5>
+
                            <p>Delivery is an essential part of our project as it determines the audience we reach. We tried to develop multiple
+
                                ways to deliver GMO in a user-friendly way with minimum invasion. Minimum invasion means it should not colonize
+
                                the whole reproductive tract or reach the ovaries. The device should ensure bacterial colonization in the ampulla
+
                                of the fallopian tube.
+
                                One of the constraints that we faced is the high viscosity of the oviductal fluid.</p><P>
+
                                After calculating the time taken for delivery in each method, talking to a couple of IVF experts, and getting their
+
                                inputs into it,we thought hysteroscopic techniques would be the best for our purpose. This method gives us an advantage
+
                                by delivering the bacteria directly into the ampulla region.</p>
+
                            <h5>DEVICE DESIGNING (How to deliver?)</h5>
+
                            <p>GMO will be delivered directly to the ampulla by a process called Hysteroscopy. It’s done with medical assistance and requires
+
                                 constant X-Ray monitoring to ensure the catheter is inserted without any issues. The reason is that the Ostia and the Fallopian
+
                                tube are dynamic structures and that there is no chemical or significant physical difference between the ampulla and the other
+
                                components of the Fallopian tube.</p>
+
 
                                  
 
                                  
                            <p>A solution of GMO of a specific concentration is prepared. Then the catheter is inserted under medical guidance up to the ampulla.
+
                                <section id="Index2">  
                                Then an injector is projected with narrow long incisions on both sides of it, which ensures that the GMO forms a ring along the
+
                                    <h5><a class="index_link" href="#2">Gene Gala</a></h5>                       
                                 circumference of the tube. Ovum is now
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                                 </section>
                                360° surrounded by the GMO. </p>
+
   
                               
+
                             </div>
                             <p>The GMO Lactobacillus Acidophilus has pili which expectantly forms hydrogen bonds at the tube circumference with mucin found in mucus.
+
                          </div>
                                The GMO is now adhered to the walls and multiplies once it adjusts to the introduced environment (lag phase) </p>
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                    </div>
                            <h5>FUTURE ASPECTS:</h5>  
+
 
                            <p>Since we aim to reach a larger population, the hysteroscopic technique, we believe, is too expensive.
+
                    <div class="col-md-8 p-5">
                                We also considered using hydrogels for the delivery of bacteria at a pH-specific region.</p><P>
+
                        <h2 id="1">New Parts from literature:</h2>
                                Why is this system compatible with the delivery of bacteria in the ampulla of the Fallopian tube region?</P><P>
+
                        <h3 id="11">Promoters:</h3>
                                We found out that the pH in the oviductal Ampulla region is close to 8.0.
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                        <p>In order to achieve robustness in the system, it is necessary to have a library of promoters with a wide range of transcription rates. One such library of synthetic promoters from Liu et al. (2018) consisted of 214 synthetic promoters with consensus sequence as shown below <a style="color: #8d1063;" href="#ref5">[1]</a>:</p>
                                That’s why we considered using hydrogels for bacterial delivery, which swells up at a pH range of 7.8 to 8.0. </P><P>
+
                        <div class="trable-responsive py-3" style="overflow-x: scroll;">
                               
+
                            <img src="./assets/SP Backbone.png" alt="Trulli">
                                Amongst the stimuli-responsive hydrogels, pH-sensitive hydrogels are the most studied hydrogels. The rate at
+
                        </div>
                                which hydrogels respond depends upon their size, shape, cross-linking density, number of ionic groups, and composition,
+
                        <p class="text-center">Fig. 1 SP Backbone</p>
                                which can be tailored by varying these factors. The response rate increases with increasing pore size and number of ionic
+
 
                                groups and decreasing their size and cross-linking density.</P><P>
+
                        <p>All these promoters are constitutive hence can be used for general protein production. From this library we used SP126, SP146 and SP200 having relative activity with respect to <a href="http://parts.igem.org/Part:BBa_K143013">P43</a> as follows:</p>
                               
+
                        <div class="table-responsive">
                                For a delivery in a basic medium, we planned to use anionic hydrogels, such as carboxymethyl chitosan, which swell at higher
+
                            <table class="table table-striped">
                                pH (basic medium) due to ionization of the acidic groups. As a result, the ionized negatively charged pendant groups on the
+
                            <thead>
                                polymer chains cause repulsion leading to swelling. This property of hydrogels can be exploited for GMO delivery at pH 7.4
+
                            <tr>
                                in the ampulla of the fallopian tube. </p>
+
                            <th>
                             <h5>CELL ENCAPSULATION (What to deliver?)</h5>  
+
                            <p>Promotor</p>
                             <p>Our goal is to design a live-bacteria entrapment system. More than just encapsulating bacteria, we want to entirely prevent
+
                            </th>
                                their escape from the bead body into the surroundings.</p>
+
                            <th>
                             <p>The Oviductal Fluid is slightly alkaline (ph 7.4 to 7.7), so we need our encapsulating membrane to dissolve away at this pH.
+
                            <p>Sequence 5&lsquo; 3&lsquo;</p>
                                Potential candidates for hydrogel materials include chitosan, guar gum, and xanthan. Chitosan forms Hydrogen bonds with the
+
                             </th>
                                mucin protein in the mucus allowing for the anchoring at the walls and preventing the hydrogels from getting washed away.</p>
+
                            <th>
                               
+
                             <p>Relative activity wrt<a href="http://parts.igem.org/Part:BBa_K143013"><strong> P43 </strong></a><strong>- GFP (%)</strong></p>
                               
+
                             </th>
                               
+
                            <th>
                             <h4>PART B: Colonization</h4><br>
+
                            <p>Standard deviation</p>
                             <h5>OVERVIEW (why is it necessary to study growth and colonization)</h5>  
+
                            </th>
                             <p>In biosynthesis, growth kinetics is a crucial study to be conducted. The growth of a cell comprises both the size and the number.
+
                             </tr>
                                These growths are affected by external factors such as temperature, the chemical composition of the growth nutrient, etc., and by
+
                            </thead>
                                different physiological factors such as growth factor proteins[1][2]. The cells in a particular environment extract the nutrients
+
                            <tbody>
                                from the growth media and produce biomolecules, which humans utilize for different purposes. This phenomenon has applications, from
+
                             <tr>
                                producing delicious Italian wine to getting antibiotics which saves millions of lives every year. We will be using this simple
+
                            <td>
                                formula to reach the goals of our project. To have a qualitative understanding of the protein production by the Genetically Modified
+
                             <p><a href="http://parts.igem.org/Part:BBa_K3889010">SP126</a></p>
                                anism(GMO), we need to have a theoretical approach. We need to understand how we can calculate the growth of GMOs [3]. This, in turn,
+
                            </td>
                                will help us figure out protein production. This detailed study of growth kinetics will help us calculate the initial inoculation
+
                            <td>
                                required for the production of a protein that we need.</p>
+
                            <p>AAAAATTATAAAAATGTGTTGACAAAGGGGGTCCTGTATGTTATAATAGCTT</p>
                             <p>For a better understanding, we will be considering two cases. The first comprises the study of the growth of Bacillus Subtilis in
+
                            </td>
                                normal petri dish conditions. This is the known environment and we can have this condition easily in computer simulation and/or lab
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                            <td>
                                and reconfirm our model. The second case of study is the continuous culture model where we study the growth of Lactobacillus
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                            <p>29.07</p>
                                Acidophilus in the fallopian tube, which is our target region. </p>
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                            </td>
                              
+
                            <td>
                              
+
                             <p>0.23</p>
                             <h6>GROWTH MODELS</h6><br>
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                            </td>
                             <p>PETRI DISH MODEL (Bacillus Subtilis, curve fitting)</p><br>
+
                            </tr>
                             <p>CONTINUOUS CULTURE MODEL (lactobacillus acidophilus + fallopian tube environment)</p><br>
+
                            <tr>
                             <h5>INOCULUM CALCULATIONS (connection with 2nd module)</h5><br>
+
                            <td>
                             <h2>Protease production</h2><br>
+
                            <p><a href="http://parts.igem.org/Part:BBa_K3889011">SP146</a></p>
                             <h3>OVERVIEW ( overall picture) </h3>
+
                             </td>
                             <p>Well known for extracellular protease production[1], Bacillus subtilis is our model organism for the proof of concept experiments. Moreover,
+
                             <td>
                                it is a gram-positive bacteria which even if not too close but is closer than e coli (gram-negative) to our proposed bacteria lactobacillus
+
                             <p>AAAAATAACAAAAACGTGTTGACAATAAAGATTAACCGTGATATAATTAAAT</p>
                                Acidophilus. This module deals with the whole mechanism of action ideally the GMO must follow for contraception to be attained. To know how
+
                            </td>
                             this module developed to what it is, please refer to the engineering success (hyperlink).</p>  
+
                            <td>
                             <p>Let's create the flow, you must know by now that we plan to produce the protease known as ovastacin,
+
                             <p>40.39</p>
                                an indigenous protease[2] to the human ovum. It is known to cause Zona pellucida hardening naturally
+
                            </td>
                                used by the ovum to prevent polyspermy[3]. To see the mechanism of zona pellucida hardening click here
+
                            <td>
                                (goes to project overview where it is explained).</p>
+
                             <p>0.69</p>
                              
+
                            </td>
                              
+
                             </tr>
                             <h5> 1] Total amount of ovastacin required: </h5>
+
                            <tr>
                           
+
                            <td>
                            <p>[We assume one active ovastacin cleaves one ZP2 glycoprotein present in the Zona pellucida matrix. To get the number of molecules of ovastacin needed, we calculated the number of ZP2 glycoproteins present on the surface of the ovum. For this, we reached out to studies that looked at the thickness of the ovum with and without the zona pellucida layer to find its overall thickness and the radius of the ovum[4]. </p>
+
                             <p><a href="http://parts.igem.org/Part:BBa_K3889012">SP200</a></p>
                            <br>
+
                             </td>
                            <h6>Constants and calculations:</h6><br>
+
                            <td>
                            <div class="table-responsive-md">
+
                             <p>AAAAATTAGAAAAATGTGTTGACACTCGGACGAAACAATGGTATAATGGCAA</p>
                                <table class="table">
+
                            </td>
                                    <thead>
+
                            <td>
                                        <tr>
+
                             <p>76.82</p>
                                          <th>Parameter (need alternative) </th>
+
                             </td>
                                          <th>Value</th>
+
                            <td>
                                          <th>References</th>
+
                            <p>0.9</p>
                                        </tr>
+
                             </td>
                                    </thead>
+
                             </tr>
                                    <tbody>
+
                             </tbody>
                                        <tr>
+
                            </table>
                                          <td>Zona pellucida thickness</td>
+
                        </div>
                                          <td>18.9 μm</td>
+
 
                                          <td>Does zona pellucida thickness influence the fertilization rate? E. Bertrand1 , M.Van Den Bergh and Y.Englert</td>
+
                        <h3 id="12">P22 Operator Library:</h3>
                                        </tr>
+
 
                                        <tr>
+
                        <p>P22 repressor (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K3889020">BBa_K3889020</a>) binds to this sequence as a dimer. This inhibits the enzymes from transcripting the genes on whose promoter this operator site is fused with. Hence this could be used with any promoter in order to form a repressible system. Different binding affinities of a repressor provides a variable system that can be used for different expression levels of the target thereby enabling its in a variety of systems.Optimization and tweaking of a system can be done by varying the operator sites as well.</p>
                                          <td>Oocyte diameter</td>
+
 
                                          <td>123.5 μm</td>
+
                        <div class="table-responsive">
                                          <td>Does zona pellucida thickness influence the fertilization rate? E. Bertrand1 , M.Van Den Bergh and Y.Englert</td>
+
                            <table class="table table-striped">
                                        </tr>
+
                            <thead>
                                        <tr>
+
                            <tr>
                                          <td>Size of ZP2</td>
+
                            <th>
                                          <td>90–110 kDa</td>
+
                            <p>Part Name</p>
                                          <td>Characterization of human zona pellucida glycoproteins A R Bauskin 1, D R Franken, U Eberspaecher, P Donner DOI: 10.1093/molehr/5.6.534</td>
+
                            </th>
                                        </tr>
+
                            <th>
                                        <tr>
+
                            <p>Sequence</p>
                                          <td>Diameter of ZP2r</td>
+
                            </th>
                                          <td>0.0092 μm</td>
+
                            <th>
                                          <td>Zetasizer Nano Sensitivity Calculator (Classic)</td>
+
                            <p>Rel KD</p>
                                        </tr>
+
                            </th>
                                    </tbody>
+
                            <th>
                                </table>
+
                            <p>KD (in M)</p>
                             </div>                          
+
                            </th>
                             <p>Assuming cuboid with all spheres of size same as ZP2 to
+
                            </tr>
                             get volume of one unit = 3.1 e-6 μm3 </p>
+
                            </thead>
                             <p>Volume occupied by whole ZP matrix = 12.4 e5 μm3 </p>
+
                            <tbody>
                             <p>Calculated the number of ZP2 glycoproteins present in the ZP matrix = 3.92 x 1011 molecules</p>
+
                            <tr>
                             <p>Assuming 1 ovastacin cleaves 1 ZP2</p>
+
                            <td>
                             <p>No. of ovastacin = No. of ZP2 = 12.4 e5 / 3.1 e-6 = 3.92 x 1011  molecules ]</p>
+
                            <p><a href="http://parts.igem.org/Part:BBa_K3889080">P22 binding site A</a></p>
                             <p>In order to cause complete hardening __ number of molecules are required to reach the ovum. The next question that arises is how much is produced and how much of produced ovastacin will reach the ovum. So we have two major things left to look at: </p>
+
                            </td>
                             <h5>2] Production and transport of ovastacin by GMO</h5>  
+
                            <td>
                             <p>The production of ovastacin is required for three days pre and post ovulation, please go to “Genetic Circuits” to learn details regarding the genetic circuit. </p>  
+
                            <p>ATTTAAGATATCTTAAAT</p>
                             <h4>GENETIC CIRCUIT</h4>
+
                            </td>
                             <p>Progesterone repressible system</p>
+
                            <td>
                             <h4>JOURNEY OF OVASTACIN</h4>
+
                            <p>1</p>
                             <p>From the papers, we know that the ovum is propelled by the ciliary motion away from the uterus and that means the ovum is in contact with the wall. Thesize ampulla region of the Fallopian tube (2.5 mm ≤ radius ≤ 5 mm ) is much larger than the radius of the ovum (61.7μm) A molecule under the influence of Brownian motion move according to the equation</p>
+
                            </td>
                             <ul>
+
                            <td>
                                <li>< r <sup>2</sup> >= 4Dt (for 2-D)</li>
+
                            <p>1.6 &times; 10<sup>&minus;8</sup></p>
                                <li>< r <sup>2</sup> >= 6Dt (for 3-D)</li>
+
                            </td>
                                <p>Where,</p>
+
                            </tr>
                                <li>< r <sup>2</sup> > → mean squared (radial) distance travelled by the molecule</li>
+
                            <tr>
                                <p>using,</p>
+
                            <td>
                                <ol>
+
                            <p><a href="http://parts.igem.org/Part:BBa_K3889081">P22 binding site B</a></p>
                                    <li>k<sub>B</sub> = 1.38064852 ×10<sup>23</sup> m<sup>2</sup> kg<sup>-2</sup> s<sup>-1</sup> [Boltzmann constant]</li>
+
                            </td>
                                    <li>η = 0.799 Pa·s [Viscosity of Oviductal Fluid]</li>
+
                            <td>
                                    <li>T = 35.5 + 273.15 = 308.65 K [Temperature of Oviduct]</li>
+
                            <p>AATTAAGATATCTTAATT</p>
                                    <li>r = 22kDa = 2.43 nm [Radius of ovastacin molecule]</li>
+
                            </td>
                                    <li>a = 61.7 μm [Radius of the ovum]</li>
+
                            <td>
                                    <li>s = 5 mm [Radius of the ampulla region of the oviduct]</li>
+
                            <p>1.8</p>
                                    <li>N<sub>0</sub> = 3.92 ×10<sup>11</sup> molecules = 0.000650946529392 nanomoles [Number of ovastacin to react with all ZP2]</li>
+
                            </td>
                                    <p>
+
                            <td>
                                        For, Ovastacin D=1.1644194699 ×10<sup>-13</sup>   m<sup>2</sup> s<sup>-1</sup>
+
                            <p>2.88 &times; 10-8</p>
                                        </p>
+
                            </td>
                                </ol>
+
                            </tr>
                                <li>t → Time elapsed</li>
+
                            <tr>
               
+
                            <td>
                            </ul>
+
                            <p><a href="http://parts.igem.org/Part:BBa_K3889082">P22 binding site C</a></p>
 +
                            </td>
 +
                            <td>
 +
                            <p>ATTTAAGAATTCTTAAAT</p>
 +
                            </td>
 +
                            <td>
 +
                            <p>2</p>
 +
                            </td>
 +
                            <td>
 +
                            <p>3.2 &times; 10<sup>&minus;8</sup></p>
 +
                            </td>
 +
                            </tr>
 +
                            <tr>
 +
                            <td>
 +
                            <p><a href="http://parts.igem.org/Part:BBa_K3889083">P22 binding site D</a></p>
 +
                            </td>
 +
                            <td>
 +
                            <p>AGTTAAGATATCTTAACT</p>
 +
                            </td>
 +
                            <td>
 +
                            <p>2.6</p>
 +
                            </td>
 +
                            <td>
 +
                            <p>4.16 &times; 10<sup>&minus;8</sup></p>
 +
                            </td>
 +
                            </tr>
 +
                            <tr>
 +
                            <td>
 +
                            <p><a href="http://parts.igem.org/Part:BBa_K3889084">P22 binding site E</a></p>
 +
                             </td>
 +
                            <td>
 +
                             <p>ATTAAAGATATCTTTAAT</p>
 +
                             </td>
 +
                            <td>
 +
                            <p>3.8</p>
 +
                            </td>
 +
                            <td>
 +
                            <p>6.08 &times; 10<sup>&minus;8</sup></p>
 +
                            </td>
 +
                            </tr>
 +
                            <tr>
 +
                            <td>
 +
                             <p><a href="http://parts.igem.org/Part:BBa_K3889085">P22 binding site F</a></p>
 +
                            </td>
 +
                            <td>
 +
                            <p>ACTTAAGATATCTTAAGT</p>
 +
                            </td>
 +
                            <td>
 +
                            <p>4.3</p>
 +
                            </td>
 +
                            <td>
 +
                             <p>6.88 &times; 10<sup>&minus;8</sup></p>
 +
                            </td>
 +
                            </tr>
 +
                            <tr>
 +
                            <td>
 +
                            <p><a href="http://parts.igem.org/Part:BBa_K3889086">P22 binding site G</a></p>
 +
                            </td>
 +
                            <td>
 +
                             <p>ATTCAAGATATCTTGAAT</p>
 +
                            </td>
 +
                            <td>
 +
                             <p>5</p>
 +
                            </td>
 +
                            <td>
 +
                            <p>8.0 &times; 10<sup>&minus;8</sup></p>
 +
                            </td>
 +
                            </tr>
 +
                            <tr>
 +
                            <td>
 +
                            <p><a href="http://parts.igem.org/Part:BBa_K3889087">P22 binding site H</a></p>
 +
                            </td>
 +
                            <td>
 +
                            <p>ATTGAAGATATCTTCAAT</p>
 +
                            </td>
 +
                            <td>
 +
                            <p>7.6</p>
 +
                            </td>
 +
                            <td>
 +
                            <p>1.216 &times; 10<sup>&minus;7</sup></p>
 +
                            </td>
 +
                            </tr>
 +
                            <tr>
 +
                            <td>
 +
                             <p><a href="http://parts.igem.org/Part:BBa_K3889088">P22 binding site I</a></p>
 +
                             </td>
 +
                            <td>
 +
                             <p>ATTTAAGAGCTCTTAAAT</p>
 +
                             </td>
 +
                            <td>
 +
                            <p>10</p>
 +
                            </td>
 +
                            <td>
 +
                             <p>1.6 &times; 10<sup>&minus;7</sup></p>
 +
                             </td>
 +
                            </tr>
 +
                            <tr>
 +
                            <td>
 +
                             <p><a href="http://parts.igem.org/Part:BBa_K3889089">P22 binding site J</a></p>
 +
                             </td>
 +
                            <td>
 +
                            <p>ATTTAAGACGTCTTAAAT</p>
 +
                            </td>
 +
                            <td>
 +
                            <p>10</p>
 +
                            </td>
 +
                            <td>
 +
                            <p>1.6 &times; 10<sup>&minus;7</sup></p>
 +
                            </td>
 +
                            </tr>
 +
                            <tr>
 +
                            <td>
 +
                            <p><a href="http://parts.igem.org/Part:BBa_K3889090">P22 binding site K</a></p>
 +
                            </td>
 +
                            <td>
 +
                            <p>ATTTACGATATCGTAAAT</p>
 +
                            </td>
 +
                            <td>
 +
                            <p>30</p>
 +
                            </td>
 +
                            <td>
 +
                            <p>4.8 &times; 10<sup>&minus;7</sup></p>
 +
                            </td>
 +
                            </tr>
 +
                            <tr>
 +
                            <td>
 +
                            <p><a href="http://parts.igem.org/Part:BBa_K3889091">P22 binding site L</a></p>
 +
                            </td>
 +
                            <td>
 +
                            <p>ATTTAAAATATTTTAAAT</p>
 +
                            </td>
 +
                            <td>
 +
                            <p>55</p>
 +
                            </td>
 +
                            <td>
 +
                            <p>8.8 &times; 10<sup>&minus;7</sup></p>
 +
                            </td>
 +
                            </tr>
 +
                            </tbody>
 +
                            </table>
 +
                        </div>
 +
 
 +
                        <figure class="col-12 col-sm-10 col-md-8 m-auto">
 +
                            <img src="./assets/p22_binding_site.png" alt="Trulli" style="width:100%">
 +
                            <figcaption class="text-center p-3">
 +
                                Fig.2 - Kd values of P22 binding site
 +
                            </figcaption>
 +
                        </figure>
 +
 
 +
                        <h3 id="13">Coding sequences:</h3>
 +
                        <p><a href="http://parts.igem.org/Part:BBa_K3889021">SRTF1</a> or steroid responsive transcription factor 1 can negatively regulate any promoter activity with which it is fused with. SRTF1 binds to its binding site(<a href="http://parts.igem.org/Part:BBa_K3889030">BBa_K3889030</a>) as done in <a href="http://parts.igem.org/BBa_K3889150">BBa_K3889150</a>. Presence of progesterone causes unbinding of SRTF thereby releasing it from the DNA, inducing the target gene.Thus,progesterone acts as an inducer and can be used in a progesterone inducible system by other teams as well.<a style="color: #8d1063;" href="#ref5">[4]</a></p>
 +
                        <p><br /><br /></p>
 +
                        <h3 id="14">Device:</h3>
 +
                        <p>Terminator checking device (<a href="http://parts.igem.org/BBa_K3889140">BBa_K3889140</a>): In order to check terminator efficiency a simple reference circuit was used similar to what used by Gale et al. (2021)<a style="color: #8d1063;" href="#ref5">[5]</a> as shown below:</p>
 +
                        <figure class="col-12 col-sm-10 col-md-8 m-auto">
 +
                            <img src="./assets/Termcheckdevice.jpg" alt="Trulli" style="width:100%">
 +
                            <figcaption class="text-center p-3">
 +
                                Fig.3 - Terminator Check Device
 +
                            </figcaption>
 +
                        </figure>
 +
 
 +
                        <p>Now spacer can be replaced with any terminator in order to see the expression of sfGFP and mCherry.</p>
 +
 
 +
                        <figure class="col-12 col-sm-10 col-md-8 m-auto">
 +
                            <img src="./assets/Term to be checked.jpg" alt="Trulli" style="width:100%">
 +
                            <figcaption class="text-center p-3">
 +
                                Fig.4 - Terminator to be checked
 +
                            </figcaption>
 +
                        </figure>
 +
                        <div class="table-responsive">
 +
                        <p>Formulae for terminator efficiency<a style="color: #8d1063;" href="#ref5">[5]</a></p>
 +
 
 +
                        $TE_{Device}=\frac{mCherry_{0}}{sfGFP_{0}}$
 +
                        <br>
 +
                        where,
 +
                        <br>
 +
                        <br>
 +
                        $mCherry_{0}\rightarrow$  mCherry produced by device without terminator<br><br>
 +
                        $sfGFP_{0}\rightarrow$ sfGFP produced by device without terminator<br><br>
 +
                        Using the device without any changes, $TE_{Device}$  can be calculated which gives the expression of <br>$mCherry$ in absence of a terminator.<br><br>
 +
                        $TE=100-\left[\left(\frac{mCherry}{sfGPF}\right)\times\left(\frac{1}{TE_{Device}}\right)\times100\right]$        (2)<br><br>
 +
                        where, <br><br>
 +
                        $mCherry$ $\rightarrow$ mCherry produced by device with the terminator that needs to checked<br><br>
 +
                        $sfGFP$ $\rightarrow$ sfGFP produced by device with the terminator that needs to checked<br><br>
 +
                        </div>
 +
                       
 +
                       
 +
 
 +
                        <h3 class="p-3" id="15">REFERENCES</h3>
 +
                        <p id="ref1">[1] Liu, D., Mao, Z., Guo, J., Wei, L., Ma, H., Tang, Y., Chen, T., Wang, Z., &amp; Zhao, X. (2018). Construction, Model-Based Analysis, and Characterization of a Promoter Library for Fine-Tuned Gene Expression in Bacillus subtilis. ACS Synthetic Biology, 7(7), 1785&ndash;1797. https://doi.org/10.1021/acssynbio.8b00115&nbsp;</p>
 +
 
 +
                        <p id="ref2">[2] Yang, S., Du, G., Chen, J., &amp; Kang, Z. (2017). Characterization and application of endogenous phase-dependent promoters in Bacillus subtilis. Applied Microbiology and Biotechnology, 101(10), 4151&ndash;4161. https://doi.org/10.1007/s00253-017-8142-7</p>
 +
 
 +
                        <p id="ref3">[3] Watkins, D., Hsiao, C., Woods, K. K., Koudelka, G. B., &amp; Williams, L. D. (2008). P22 c2 Repressor&minus;Operator Complex:&thinsp; Mechanisms of Direct and Indirect Readout. Biochemistry, 47(8), 2325&ndash;2338. https://doi.org/10.1021/bi701826f</p>
 +
 
 +
                        <p id="ref4">[4] Baer, R. Cooper (2020). Discovery, characterization, and ligand specificity engineering of a novel bacterial transcription factor inducible by progesterone Boston University School of Medicine, 801 Massachusetts Avenue Suite 400 Boston, MA 02118 Retrieved from : https://hdl.handle.net/2144/41109</p>
 +
 
 +
                        <p id="ref5">[5] Gale, G. A. R., Wang, B., &amp; McCormick, A. J. (2021). Evaluation and Comparison of the Efficiency of Transcription Terminators in Different Cyanobacterial Species. Frontiers in Microbiology, 11. https://doi.org/10.3389/fmicb.2020.624011&nbsp;</p>
 +
 
 +
                        <h2 id="2">Gene Gala&nbsp;</h2>
 +
 
 +
                        <p>We held a Mini-Summer school in collaboration with the iGEM 2021 team of IISER Kolkata. It was a 5 day Mini-Summer School for Girl students studying in 12th Standards of the schools under the Directorate of Education, GNCT Delhi. As part of the summer school, the two teams together prepared a 5 day lesson plan, 2 quiz sessions and a day-to -day handbook made for reference for the students. We would like to present these resources as a contribution to iGEM.&nbsp;</p>
 +
 
 +
                        <p>Future iGEM teams can use them directly for conducting similar programmes in their regions/countries to the relevant audiences giving proper attributions to both the contributing teams. These resources will be extremely useful for teams who are preparing for similar education events. Conducting classes for 5 day enriched with activities and quiz sessions can be a daunting task for teams. The lesson plan provided here was able to keep the students engaged throughout the 5 days and it was easy for the team members to present as well. These content handbooks, lesson plans and quizzes will come in handy for future iGEM teams to prepare for such an event and take their public engagement to the next level.&nbsp;</p>
 +
 
 +
                        <p>The content is relevant for introducing high school seniors to Synthetic Biology, while giving them a holistic and application based view of the biology courses taught at the high school level.&nbsp;</p>
 +
 
 +
                        <p>Drive link to the handbook, quizzes and lesson plan -&nbsp;</p>
 +
                        <p><strong>Zip File Name : Mini Summer school Resources -</strong><a href="https://drive.google.com/folderview?id=1LkMO9n0KYhkKXA8lggWYrLmLnC2MjK4c">https://drive.google.com/folderview?id=1LkMO9n0KYhkKXA8lggWYrLmLnC2MjK4c</a>&nbsp;</p>
 +
 
 +
                        <p>Note : It will be helpful if 2 people present the content, which will stop the lesson from becoming monotonous and keep students engaged.</p>  
 
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Revision as of 13:16, 13 October 2021


Ovi-Cloak

SCROLL

New Parts from literature:

Promoters:

In order to achieve robustness in the system, it is necessary to have a library of promoters with a wide range of transcription rates. One such library of synthetic promoters from Liu et al. (2018) consisted of 214 synthetic promoters with consensus sequence as shown below [1]:

Trulli

Fig. 1 SP Backbone

All these promoters are constitutive hence can be used for general protein production. From this library we used SP126, SP146 and SP200 having relative activity with respect to P43 as follows:

Promotor

Sequence 5‘ 3‘

Relative activity wrt P43 - GFP (%)

Standard deviation

SP126

AAAAATTATAAAAATGTGTTGACAAAGGGGGTCCTGTATGTTATAATAGCTT

29.07

0.23

SP146

AAAAATAACAAAAACGTGTTGACAATAAAGATTAACCGTGATATAATTAAAT

40.39

0.69

SP200

AAAAATTAGAAAAATGTGTTGACACTCGGACGAAACAATGGTATAATGGCAA

76.82

0.9

P22 Operator Library:

P22 repressor (BBa_K3889020) binds to this sequence as a dimer. This inhibits the enzymes from transcripting the genes on whose promoter this operator site is fused with. Hence this could be used with any promoter in order to form a repressible system. Different binding affinities of a repressor provides a variable system that can be used for different expression levels of the target thereby enabling its in a variety of systems.Optimization and tweaking of a system can be done by varying the operator sites as well.

Part Name

Sequence

Rel KD

KD (in M)

P22 binding site A

ATTTAAGATATCTTAAAT

1

1.6 × 10−8

P22 binding site B

AATTAAGATATCTTAATT

1.8

2.88 × 10-8

P22 binding site C

ATTTAAGAATTCTTAAAT

2

3.2 × 10−8

P22 binding site D

AGTTAAGATATCTTAACT

2.6

4.16 × 10−8

P22 binding site E

ATTAAAGATATCTTTAAT

3.8

6.08 × 10−8

P22 binding site F

ACTTAAGATATCTTAAGT

4.3

6.88 × 10−8

P22 binding site G

ATTCAAGATATCTTGAAT

5

8.0 × 10−8

P22 binding site H

ATTGAAGATATCTTCAAT

7.6

1.216 × 10−7

P22 binding site I

ATTTAAGAGCTCTTAAAT

10

1.6 × 10−7

P22 binding site J

ATTTAAGACGTCTTAAAT

10

1.6 × 10−7

P22 binding site K

ATTTACGATATCGTAAAT

30

4.8 × 10−7

P22 binding site L

ATTTAAAATATTTTAAAT

55

8.8 × 10−7

Trulli
Fig.2 - Kd values of P22 binding site

Coding sequences:

SRTF1 or steroid responsive transcription factor 1 can negatively regulate any promoter activity with which it is fused with. SRTF1 binds to its binding site(BBa_K3889030) as done in BBa_K3889150. Presence of progesterone causes unbinding of SRTF thereby releasing it from the DNA, inducing the target gene.Thus,progesterone acts as an inducer and can be used in a progesterone inducible system by other teams as well.[4]



Device:

Terminator checking device (BBa_K3889140): In order to check terminator efficiency a simple reference circuit was used similar to what used by Gale et al. (2021)[5] as shown below:

Trulli
Fig.3 - Terminator Check Device

Now spacer can be replaced with any terminator in order to see the expression of sfGFP and mCherry.

Trulli
Fig.4 - Terminator to be checked

Formulae for terminator efficiency[5]

$TE_{Device}=\frac{mCherry_{0}}{sfGFP_{0}}$
where,

$mCherry_{0}\rightarrow$ mCherry produced by device without terminator

$sfGFP_{0}\rightarrow$ sfGFP produced by device without terminator

Using the device without any changes, $TE_{Device}$ can be calculated which gives the expression of
$mCherry$ in absence of a terminator.

$TE=100-\left[\left(\frac{mCherry}{sfGPF}\right)\times\left(\frac{1}{TE_{Device}}\right)\times100\right]$ (2)

where,

$mCherry$ $\rightarrow$ mCherry produced by device with the terminator that needs to checked

$sfGFP$ $\rightarrow$ sfGFP produced by device with the terminator that needs to checked

REFERENCES

[1] Liu, D., Mao, Z., Guo, J., Wei, L., Ma, H., Tang, Y., Chen, T., Wang, Z., & Zhao, X. (2018). Construction, Model-Based Analysis, and Characterization of a Promoter Library for Fine-Tuned Gene Expression in Bacillus subtilis. ACS Synthetic Biology, 7(7), 1785–1797. https://doi.org/10.1021/acssynbio.8b00115 

[2] Yang, S., Du, G., Chen, J., & Kang, Z. (2017). Characterization and application of endogenous phase-dependent promoters in Bacillus subtilis. Applied Microbiology and Biotechnology, 101(10), 4151–4161. https://doi.org/10.1007/s00253-017-8142-7

[3] Watkins, D., Hsiao, C., Woods, K. K., Koudelka, G. B., & Williams, L. D. (2008). P22 c2 Repressor−Operator Complex:  Mechanisms of Direct and Indirect Readout. Biochemistry, 47(8), 2325–2338. https://doi.org/10.1021/bi701826f

[4] Baer, R. Cooper (2020). Discovery, characterization, and ligand specificity engineering of a novel bacterial transcription factor inducible by progesterone Boston University School of Medicine, 801 Massachusetts Avenue Suite 400 Boston, MA 02118 Retrieved from : https://hdl.handle.net/2144/41109

[5] Gale, G. A. R., Wang, B., & McCormick, A. J. (2021). Evaluation and Comparison of the Efficiency of Transcription Terminators in Different Cyanobacterial Species. Frontiers in Microbiology, 11. https://doi.org/10.3389/fmicb.2020.624011 

Gene Gala 

We held a Mini-Summer school in collaboration with the iGEM 2021 team of IISER Kolkata. It was a 5 day Mini-Summer School for Girl students studying in 12th Standards of the schools under the Directorate of Education, GNCT Delhi. As part of the summer school, the two teams together prepared a 5 day lesson plan, 2 quiz sessions and a day-to -day handbook made for reference for the students. We would like to present these resources as a contribution to iGEM. 

Future iGEM teams can use them directly for conducting similar programmes in their regions/countries to the relevant audiences giving proper attributions to both the contributing teams. These resources will be extremely useful for teams who are preparing for similar education events. Conducting classes for 5 day enriched with activities and quiz sessions can be a daunting task for teams. The lesson plan provided here was able to keep the students engaged throughout the 5 days and it was easy for the team members to present as well. These content handbooks, lesson plans and quizzes will come in handy for future iGEM teams to prepare for such an event and take their public engagement to the next level. 

The content is relevant for introducing high school seniors to Synthetic Biology, while giving them a holistic and application based view of the biology courses taught at the high school level. 

Drive link to the handbook, quizzes and lesson plan - 

Zip File Name : Mini Summer school Resources -https://drive.google.com/folderview?id=1LkMO9n0KYhkKXA8lggWYrLmLnC2MjK4c 

Note : It will be helpful if 2 people present the content, which will stop the lesson from becoming monotonous and keep students engaged.

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