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

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                        <h2>Introduction</h2>
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                         <p>Natural systems are highly complex to understand as well as to experiment with. To make predictions of the outcomes,  
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                             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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                                <h3 class="card-title text-center mb-3">INDEX</h3>
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                                 <li>GMO delivery</li>
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                                <section id="Index1">
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                                 <li>GMO growth and colonisation</li>
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                                  <ul>
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                                 <li>Protease production</li>
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                                    <li><h5><a class="index_link" href="#1">Wetlab Calander</a></h5></li>
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                                <li>Diffusion and ovum Hardening</li>
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                                    <li><h5><a class="index_link" href="#2">Protocols</a></h5></li>
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                                <li>Kill switch for reversibility</li>
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                                    </ul>                         
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                                <li>Kill switch for Biosafety</li>
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                                    January
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                                  <p><strong>Week 1</strong> (1 Jan 2021 -3 Jan 2021) :</p>
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                                  <p>A literature review of the female reproductive tract, found that ZP2 protein mutation in females result in infertility, literature review of the microbiome of the reproductive tract&nbsp;</p>
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                                  <p><strong>Week 2</strong> (4 Jan, 2021 -10 Jan 2021):&nbsp;</p>
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                                  <p>A literature review of the targets of the ovum, ruled out producing non-specific enzymes from the bacteria because of potential harm&nbsp;</p>
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                                  <p><strong>Week 3 </strong>(11 Jan 2021 - 17 Jan 2021) :&nbsp;</p>
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                                  <p>Learning about iGEM parts, kits and registry to get an idea of available things.&nbsp;</p>
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                                  <p>Categorized the species of bacteria in both fallopian tubes, found ovastacin.</p>
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                                  <p><strong>Week 4 </strong>(18 Jan 2021 - 24 Jan 2021) :&nbsp;</p>
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                                  <p>&nbsp;Studied more on lactobacillus, met with experts in our institute to discuss the idea, looked into lex A system for progesterone sensing, completely ruled out production of NAG enzyme by bacteria&nbsp;</p>
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                                  <p><br /><br /><strong>Week 5 (</strong>24 Jan 2021 - 31 Jan 2021) : A literature review about the immune system of the reproductive system, started review on a kill switch for reversibility.</p>
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                                  <p><strong>Week 1 </strong>(1 Feb 2021 - 7 Feb 2021 )<strong>:</strong></p>
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                                  <p>Found more about iGEM competition deliverables and had a meeting with our PIs. Literature review on Zone hardening&nbsp;</p>
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                                  <p><strong>Week 2 </strong>( 8 Feb 2021 - 14 Feb 2021)<strong>:</strong></p>
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                                  <p>&nbsp;A literature review on the ZP2 cleavage model by ovastacin and learnt more about the strucutre of ovastacin&nbsp;</p>
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                                  <p><strong>Week 3 </strong>(15 Feb 2021 - 21 Feb 2021)<strong>:</strong></p>
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                                  <p>&nbsp;Searched for commensal for implementation of our idea and an appropriate model organism to use as chassis by looking into secretion systems, looked into progesterone sensing by the SRTF transcription factor.&nbsp;</p>
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                                  <p><br /><strong>Week 4 </strong>(22 Feb 2021 - 28 Feb 2021)<strong>:</strong> Finalised model organism as <em>B. subtilis</em> for proof of concept and <em>Lactobacillus acidophilus</em> as the commensal to modify, finalized SRTF transcription factor for progesterone inducible system</p>                                </div>
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                                  March
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                                  <p><strong>Week 1 </strong><span style="font-weight: 400;">(1 March 2021 -7 March 2021)</span><strong>:&nbsp;</strong></p>
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                                  <p><span style="font-weight: 400;">Looked at the HGT aspects of the genetically modified bacteria through literature review and found out more about the safety aspects.&nbsp;</span></p>
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                                  <p><strong>Week 2 </strong><span style="font-weight: 400;">(7 March 2021 - 14 March 2021)</span><strong>:&nbsp;</strong></p>
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                                  <p><span style="font-weight: 400;">Finalized the different modules of our project, started in-silico work for proof of concept. Looked at different competition deliverables from the wet lab point of view and planned how to achieve them.&nbsp;</span></p>
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                                  <p><strong>Week 3</strong><span style="font-weight: 400;"> (15 March 2021 - 21 March 2021) </span><strong>:&nbsp;</strong></p>
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                                  <p><span style="font-weight: 400;">Looked into more toxin-antitoxin systems for the kill switch, started designing preliminary experiments for the proof of concept&nbsp;</span></p>
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                                  <p><strong>Week 4 </strong><span style="font-weight: 400;">(22 March 2021 - 31 March 2021)</span><strong> :</strong></p>
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                                  <p><span style="font-weight: 400;">Met Prof Satish K Gupta for getting feedback on our idea, agreement to procure clones of Human ZP2 in </span><em><span style="font-weight: 400;">E.coli</span></em><em><span style="font-weight: 400;">DH5&alpha;</span></em><span style="font-weight: 400;"> from his lab at NII, Delhi. Continued work on the toxin-antitoxin system and started looking for more models for the hormonal sensing system </span></p>                                </div>
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                                  April
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                                  <p><strong>Week 1 </strong>(1 April 2021- 3 April 2021)<strong> :</strong></p>
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                                  <p>Procured clones of hZP2 from Prof Satish K Gupta and shipped them to our lab at IISER Tirupati. Continued the work on designing experiments for the proof of concept of our project.&nbsp;</p>
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                                  <p><strong>Week 2 </strong>(4 April 2021 - 10 April 2021)<strong>:&nbsp;</strong></p>
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                                  <p>Decided to work with yeast as a model organism too and started designing experiments for yeast species. Started looking at resources to procure strains of <em>Bacillus subtilis.</em></p>
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                                  <p><strong>Week 3 </strong>(11 April 2021 -17 April 2021)<strong>:&nbsp;</strong></p>
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                                  <p>Meeting with experts working on yeast systems from our institute, started looking for protocols for different experiments, realized the limitations of progesterone inducible systems, and started looking for alternative hormone sensing models in prokaryotes.&nbsp;</p>
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                                  <p><strong>Week 4 </strong>(18 April 2021 - 24 April 2021)<strong> :</strong>&nbsp;</p>
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                                  <p>Realized the sensitivity issue of the estrogen sensing system in prokaryotes, looked into delayed expression and oscillatory circuits, Started simulating the systems to get an idea about these systems. Found out delayed expression and oscillation would not work for cycles on a monthly basis.&nbsp;</p>                                </div>
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                                  May
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                                  <p><strong>Week 1 </strong>(1 May 2021 - 8 May 2021) <strong>:&nbsp;</strong></p>
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                                  <p>Worked on finding alternatives for the progesterone inducible system, literature review on kill switches resumed and protocol review process started&nbsp;</p>
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                                  <p><strong>Week 2 </strong>(9 May 2021 - 15 May 2021)<strong>:</strong></p>
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                                  <p>End Semester exams&nbsp;</p>
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                                  <p><strong>Week 3 </strong>(16 May 2021- 22 May 2021) <strong>:&nbsp;</strong></p>
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                                  <p>Started looking into growth kinetics for reducing ovastacin production time, Heterologous gene expression in <em>Bacillus subtilis</em> on different pH, temperatures, and concentrations. Ruled out light selected Dnase based toxins for kill switch and biosafety.&nbsp;</p>
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                                  <p><strong>Week 4 </strong>(23 May 2021- 31 May 2021) :&nbsp;</p>
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                                  <p>Yeast workflow finalized. Started looking into the ytvA system for biosafety and Decided which promoters to take for experiments, looked for finalized parts in the iGEM parts registry.</p>
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                                  June
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                                  <p><strong>Week 1</strong> ( 1 June 2021 - 5 June 2021)<strong> :&nbsp;</strong></p>
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                                  <p>Started making the <em>Bacillus subtilis</em> workflow, finalized the experiments, Calculated d-score for terminator efficiency, and selected terminator for making improvement to part.&nbsp;</p>
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                                  <p><strong>Week 2 </strong>( 6 June 2021 - 12 June 2021)<strong> :&nbsp;</strong></p>
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                                  <p>Worked on Genetic circuits for all the experiments, finalized the protocols for genome integration in <em>Bacillus subtilis</em>.&nbsp;</p>
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                                  <p><strong>Week 3 </strong>(13 June 2021 - 19 June 2021)<strong> :&nbsp;</strong></p>
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                                  <p>Started looking for protocols of protein purification in the yeast and <em>Bacillus subtilis </em>and discussed them with various experts from our institute. Found out the reagents required for carrying out the experiments.&nbsp;</p>
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                                  <p><strong>Week 4 </strong>(20 June 2021 - 26 June 2021):</p>
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                                  <p>Met with Professor Sabri from IISER Trivandrum to discuss <em>Bacillus subtilis </em>experiments and had an agreement with him to ship the clones to our laboratory at IISER Tirupati. Did an in-depth analysis of transformation and protein purification protocols for <em>Bacillus subtilis </em>and looked for reagents for the same.&nbsp;</p>                                </div>
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                                  July
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                                  <p><strong>Week 1</strong> (1 July 2021 - 10 July 2021) :</p>
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                                  <p>Discussions on experiments and protocols continued and sent a list of required reagents and consumables to our laboratory and placed initial orders for reagents and consumables.&nbsp;</p>
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                                  <p><strong>Week 2</strong> (11 July 2021 - 17 July 2021) :&nbsp;</p>
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                                  <p>Talked with PhDs and seniors on performing the experiments upon return to campus, ordered gene fragments from IDT for <em>Bacillus subtilis</em>, and ordered yeast gene fragments from Twist Bioscience&nbsp;</p>
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                                  <p><strong>Week 3</strong> (18 July 2021 - 24 July 2021) :&nbsp;</p>
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                                  <p>Returned to IISER Tirupati campus and Quarantined for a week. Compiled final protocols for the experiments and placed more orders for experiments.&nbsp;</p>
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                                  <p><strong>Week 4</strong> (25 July - 31 July 2021) :&nbsp;</p>
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                                  <p>We received our iGEM distribution kit and NEB molecular biology kits. We presented the wet lab plan to our guide PhDs and Professors. Had our lab safety training to start the experiments. Prepared experimental things to start our initial experimental journey.</p>                                </div>
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                                  August
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                                  <p><strong>Week 1</strong> (1 Aug 2021 - 7 Aug 2021):&nbsp;</p>
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                                  <p>Started our experiments by culturing the <em>E. coli</em> containing the plasmid pDR111 and pDR110 and purified them. Thereafter we did restriction digestion of purified plasmids with BamHI and EcoRI. Performed a growth curve of <em>E Coli DH5&alpha;</em> and <em>Bacillus subtilis 168</em> in LB media and performed <em>Saccharomyces cerevisiae</em> strain <em>2158</em> and strain <em>860</em> in YPD media.</p>
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                                  <p><strong>Week 2 </strong>(8 Aug 2021 - 14 Aug 2021) :&nbsp;</p>
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                                  <p>Received orders from Himedia. Did Miniprep for the plasmid pDR111 stock preparation. Prepared the stock solutions for the competency of<em> E Coli DH5&alpha;</em> and made <em>E Coli DH5&alpha;</em> cells competent followed by storing them at -80 degrees C. Performed gram staining to check the bacterial contamination on the plates. Had a demo session for Agarose Gel preparation with our seniors.&nbsp;</p>
 +
                                                                 
 +
                                  <p><strong>Week 3</strong> (15 Aug 2021 - 21 Aug 2021) :&nbsp;</p>
 +
                                  <p>Transformed the <em>E Coli DH5&alpha; </em>cells with pDR111 to check the quality of the prepared competent cells. Received IDT primers and stock solutions were prepared for SDS PAGE. Worked on making the lab setting more inclusive through LabEyes. Performed Restriction digestion on the plasmid pDR111 with BamHI- hf, and EcoRI- hf.&nbsp;</p>
 +
                                                                 
 +
                                  <p><strong>Week 4</strong> (22 Aug 2021 - 31 Aug 2021) :&nbsp;</p>
 +
                                  <p>Performed sequential digestion with EcoRI and BamHI and performed gel extraction with the digested vector. Received Twist order for the yeast fragments. Streaked the <em>E. Coli DH5&alpha;</em> with hZP2 clones and expressed the hZP2 protein by IPTG induction. Stored the extracted protein in -20 degree C. Received the pRS424, pRS426, pRS425 yeast shuttle vector plasmids from Dr Viji Subramanian lab.</p>
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                                 </div>
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                                  September
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                                  <p><strong>Week 1</strong> ( 1 Sept 2021 - 11 Sept 2021):&nbsp;</p>
 +
                                  <p>Did protein purification and ran SDS PAGE multiple times for standardization of ZP2 clones with different IPTG concentrations (0.5mM and 1mM). Started working on oligonucleotides received from TWIST (yeast construct) and did not get satisfactory results. Performed Miniprep with the pRS426 plasmid (for <em>S Cerevisiae</em> vector)and pRSETb plasmid (for ZP2 in <em>E. Coli </em>). Purified the pRS426 plasmid and transformed the<em> E Coli DH5&alpha;</em> with pRSETb and got positive results. Prepared stock solutions for Bacillus subtilis transformation and competency.</p>
 +
                                  <p>&nbsp;</p>
 +
                                  <p><strong>Week 2 </strong>(12 Sept 2021 - 18 Sept 2021) :&nbsp;</p>
 +
                                  <p>Transformed the <em>Bacillus subtilis</em> and observed the growth of cells on plates in 9hrs. Prepared competent cells of <em>E. Coli BL21</em> strain and transformed them with pRSETb for ZP2 expression. Golden Gate trials for hyperspank-ovastacin-ZP2 and yeast construct were unsuccessful. Ran SDS for purified hZP2.&nbsp;</p>
 +
                                  <p>&nbsp;</p>
 +
                                  <p><strong>Week 3</strong> (19 Sept 2021 - 25 Sept 2021) :&nbsp;</p>
 +
                                  <p>Did IMAC(Ni-NTA) for hZP2 purification and Western Blot. Started golden gate trials with Improvement 1 and p22-srtf cassette. Did transformation of the ligated product of yeast construct with pRS426 plasmid and hyperspank-ovastacin-ZP2 cassette with pDR111 plasmid in NEB 10&beta; cells and got positive results.&nbsp;</p>
 +
                                  <p>&nbsp;</p>
 +
                                  <p><strong>Week 4 </strong>(26 Sept 2021 - 30 Sept 2021) :&nbsp;</p>
 +
                                  <p>Mid sem exams&nbsp;</p>                                </div>
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                            </div>
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                                  October
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                                  <p><strong>Week 1 </strong>(1 Oct 2021 - 9 Oct 2021)<strong>:&nbsp;</strong></p>
 +
                                  <p><strong>Week 2 (</strong>10 Oct 2021 - 16 Oct 2021)<strong> :&nbsp;</strong></p>
 +
                                  <p><strong>Week 3 </strong>(17 Oct 2021 - 23 Oct 2021)<strong> :&nbsp;</strong></p>
 +
                                  <p><strong>Week 4 </strong>(24 Oct 2021 - 31 Oct 2021)<strong> :&nbsp;</strong></p>                                </div>
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                            <h2 id="2" class="pt-5 pb-3">Protocols</h2>
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                                  INITIAL SETUP
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                              <div class="accordion-body">
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                                <ol>
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                                  <li><h5 class="pt-3">Growth Curve</h5>
 +
                                    <ol>
 +
                                      <li><h6 class="pt-2">Bacillus subtilis 168</h6>
 +
                                        <p>Click here on button to download the PDF file.</p>
 +
                                        <a class="btn btn-primary" href="PDFLINK" download>
 +
                                        <i class="fa fa-file-pdf-o" aria-hidden="true"></i>Download</a></li>
 +
                                      <li><h6 class="pt-2">E. coli DH5α</h6>
 +
                                        <p>Click here on button to download the PDF file.</p>
 +
                                        <a class="btn btn-primary" href="PDFLINK" download>
 +
                                        <i class="fa fa-file-pdf-o" aria-hidden="true"></i>Download</a></li>
 +
                                      <li><h6 class="pt-2">Yeast (Saccharomyces cerevisiae strain S288C)</h6>
 +
                                        <p>Click here on button to download the PDF file.</p>
 +
                                        <a class="btn btn-primary" href="PDFLINK" download>
 +
                                        <i class="fa fa-file-pdf-o" aria-hidden="true"></i>Download</a></li>
 +
                                    </ol>
 +
                                  </li>
 +
                                  <li>
 +
                                    <h5 class="pt-2">Culture preparation (Streaking, Spreading, Inoculation)</h5>
 +
                                    <p>Click here on button to download the PDF file.</p>
 +
                                    <a class="btn btn-primary" href="PDFLINK" download>
 +
                                    <i class="fa fa-file-pdf-o" aria-hidden="true"></i>Download</a></li>
 +
                                  </li>
 +
                                  <li>
 +
                                    <h5 class="pt-2">Revival from glycerol stock AND preparation of glycerol stock</h5>
 +
                                    <p>Click here on button to download the PDF file.</p>
 +
                                    <a class="btn btn-primary" href="PDFLINK" download>
 +
                                    <i class="fa fa-file-pdf-o" aria-hidden="true"></i>Download</a></li>
 +
                                  </li>
 
                                 </ol>
 
                                 </ol>
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                            <h2>Delivery & Colonisation</h2><br>
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                             <h3>INTRODUCTION: THE JOURNEY OF GMO BEGINS</h3><br>
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                            <h4>PART A: Delivery</h4><br>
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                            <h5>OVERVIEW</h5>
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                            <p>Delivery is an essential part of our project as it determines the audience we reach. We tried to develop multiple
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                             <h2 class="accordion-header" id="heading14">
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                                ways to deliver GMO in a user-friendly way with minimum invasion. Minimum invasion means it should not colonize
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                                the whole reproductive tract or reach the ovaries. The device should ensure bacterial colonization in the ampulla
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                                 CLONING
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                                of the fallopian tube.
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                                One of the constraints that we faced is the high viscosity of the oviductal fluid.</p><P>
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                                After calculating the time taken for delivery in each method, talking to a couple of IVF experts, and getting their
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                                inputs into it,we thought hysteroscopic techniques would be the best for our purpose. This method gives us an advantage
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                              <div class="accordion-body">
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                                by delivering the bacteria directly into the ampulla region.</p>
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                             <h5>DEVICE DESIGNING (How to deliver?)</h5>
+
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                            <p>GMO will be delivered directly to the ampulla by a process called Hysteroscopy. It’s done with medical assistance and requires
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                                constant X-Ray monitoring to ensure the catheter is inserted without any issues. The reason is that the Ostia and the Fallopian
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                                tube are dynamic structures and that there is no chemical or significant physical difference between the ampulla and the other
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                                components of the Fallopian tube.</p>
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                            <p>A solution of GMO of a specific concentration is prepared. Then the catheter is inserted under medical guidance up to the ampulla.
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                                Then an injector is projected with narrow long incisions on both sides of it, which ensures that the GMO forms a ring along the
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                                circumference of the tube. Ovum is now
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                                360° surrounded by the GMO. </p>
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                            <p>The GMO Lactobacillus Acidophilus has pili which expectantly forms hydrogen bonds at the tube circumference with mucin found in mucus.
+
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                                The GMO is now adhered to the walls and multiplies once it adjusts to the introduced environment (lag phase) </p>
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                            <h5>FUTURE ASPECTS:</h5>
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                            <p>Since we aim to reach a larger population, the hysteroscopic technique, we believe, is too expensive.
+
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                                We also considered using hydrogels for the delivery of bacteria at a pH-specific region.</p><P>
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                                Why is this system compatible with the delivery of bacteria in the ampulla of the Fallopian tube region?</P><P>
+
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                                We found out that the pH in the oviductal Ampulla region is close to 8.0.
+
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                                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>
+
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−
                                Amongst the stimuli-responsive hydrogels, pH-sensitive hydrogels are the most studied hydrogels. The rate at
+
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                                which hydrogels respond depends upon their size, shape, cross-linking density, number of ionic groups, and composition,
+
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                                which can be tailored by varying these factors. The response rate increases with increasing pore size and number of ionic
+
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                                groups and decreasing their size and cross-linking density.</P><P>
+
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+
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                                For a delivery in a basic medium, we planned to use anionic hydrogels, such as carboxymethyl chitosan, which swell at higher
+
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                                pH (basic medium) due to ionization of the acidic groups. As a result, the ionized negatively charged pendant groups on the
+
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                                polymer chains cause repulsion leading to swelling. This property of hydrogels can be exploited for GMO delivery at pH 7.4
+
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                                in the ampulla of the fallopian tube. </p>
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                            <h5>CELL ENCAPSULATION (What to deliver?)</h5>
+
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                            <p>Our goal is to design a live-bacteria entrapment system. More than just encapsulating bacteria, we want to entirely prevent
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                                their escape from the bead body into the surroundings.</p>
+
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                            <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.
+
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                                Potential candidates for hydrogel materials include chitosan, guar gum, and xanthan. Chitosan forms Hydrogen bonds with the
+
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                                mucin protein in the mucus allowing for the anchoring at the walls and preventing the hydrogels from getting washed away.</p>
+
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                            <h4>PART B: Colonization</h4><br>
+
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                            <h5>OVERVIEW (why is it necessary to study growth and colonization)</h5>
+
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                            <p>In biosynthesis, growth kinetics is a crucial study to be conducted. The growth of a cell comprises both the size and the number.
+
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                                These growths are affected by external factors such as temperature, the chemical composition of the growth nutrient, etc., and by
+
−
                                different physiological factors such as growth factor proteins[1][2]. The cells in a particular environment extract the nutrients
+
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                                from the growth media and produce biomolecules, which humans utilize for different purposes. This phenomenon has applications, from
+
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                                producing delicious Italian wine to getting antibiotics which saves millions of lives every year. We will be using this simple
+
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                                formula to reach the goals of our project. To have a qualitative understanding of the protein production by the Genetically Modified
+
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                                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,
+
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                                will help us figure out protein production. This detailed study of growth kinetics will help us calculate the initial inoculation
+
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                                required for the production of a protein that we need.</p>
+
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                            <p>For a better understanding, we will be considering two cases. The first comprises the study of the growth of Bacillus Subtilis in
+
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                                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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                                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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                                Acidophilus in the fallopian tube, which is our target region.  </p>
+
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                            <h6>GROWTH MODELS</h6><br>
+
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                            <p>PETRI DISH MODEL (Bacillus Subtilis, curve fitting)</p><br>
+
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                            <p>CONTINUOUS CULTURE MODEL (lactobacillus acidophilus + fallopian tube environment)</p><br>
+
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                            <h5>INOCULUM CALCULATIONS (connection with 2nd module)</h5><br>
+
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                            <h2>Protease production</h2><br>
+
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                            <h3>OVERVIEW ( overall picture) </h3>
+
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                            <p>Well known for extracellular protease production[1], Bacillus subtilis is our model organism for the proof of concept experiments. Moreover,
+
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                                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
+
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                                Acidophilus. This module deals with the whole mechanism of action ideally the GMO must follow for contraception to be attained. To know how
+
−
                            this module developed to what it is, please refer to the engineering success (hyperlink).</p>
+
−
                            <p>Let's create the flow, you must know by now that we plan to produce the protease known as ovastacin,
+
−
                                 an indigenous protease[2] to the human ovum. It is known to cause Zona pellucida hardening naturally
+
−
                                used by the ovum to prevent polyspermy[3]. To see the mechanism of zona pellucida hardening click here
+
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                                (goes to project overview where it is explained).</p>
+
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−
                           
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                             <h5> 1] Total amount of ovastacin required: </h5>
+
−
                           
+
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                            <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>
+
−
                            <br>
+
−
                            <h6>Constants and calculations:</h6><br>
+
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                             <div class="table-responsive-md">
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                                <table class="table">
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                                    <thead>
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                                        <tr>
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                                          <th>Parameter (need alternative) </th>
+
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                                          <th>Value</th>
+
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                                          <th>References</th>
+
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                                        </tr>
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                                    </thead>
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                                    <tbody>
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                                        <tr>
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                                          <td>Zona pellucida thickness</td>
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                                          <td>18.9 μm</td>
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                                          <td>Does zona pellucida thickness influence the fertilization rate? E. Bertrand1 , M.Van Den Bergh and Y.Englert</td>
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                                        </tr>
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                                        <tr>
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                                          <td>Oocyte diameter</td>
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                                          <td>123.5 μm</td>
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                                          <td>Does zona pellucida thickness influence the fertilization rate? E. Bertrand1 , M.Van Den Bergh and Y.Englert</td>
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                                        </tr>
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                                        <tr>
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                                          <td>Size of ZP2</td>
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                                          <td>90–110 kDa</td>
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                                          <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>
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                                        </tr>
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                                        <tr>
+
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                                          <td>Diameter of ZP2r</td>
+
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                                          <td>0.0092 μm</td>
+
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                                          <td>Zetasizer Nano Sensitivity Calculator (Classic)</td>
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                                        </tr>
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                                    </tbody>
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                                </table>
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                            </div>                           
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                            <p>Assuming cuboid with all spheres of size same as ZP2 to
+
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                            get volume of one unit = 3.1 e-6 μm3 </p>
+
−
                            <p>Volume occupied by whole ZP matrix = 12.4 e5 μm3 </p>
+
−
                            <p>Calculated the number of ZP2 glycoproteins present in the ZP matrix = 3.92 x 1011 molecules</p>
+
−
                            <p>Assuming 1 ovastacin cleaves 1 ZP2</p>
+
−
                            <p>No. of ovastacin = No. of ZP2 = 12.4 e5 / 3.1 e-6 = 3.92 x 1011  molecules ]</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>
+
−
                            <h5>2] Production and transport of ovastacin by GMO</h5> 
+
−
                            <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> 
+
−
                            <h4>GENETIC CIRCUIT</h4>
+
−
                            <p>Progesterone repressible system</p>
+
−
                            <h4>JOURNEY OF OVASTACIN</h4>
+
−
                            <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>
+
−
                            <ul>
+
−
                                <li>< r <sup>2</sup> >= 4Dt (for 2-D)</li>
+
−
                                <li>< r <sup>2</sup> >= 6Dt (for 3-D)</li>
+
−
                                <p>Where,</p>
+
−
                                <li>< r <sup>2</sup>  > → mean squared (radial) distance travelled by the molecule</li>
+
−
                                <p>using,</p>
+
 
                                 <ol>
 
                                 <ol>
−
                                    <li>k<sub>B</sub> = 1.38064852 ×10<sup>23</sup> m<sup>2</sup> kg<sup>-2</sup> s<sup>-1</sup> K  [Boltzmann constant]</li>
+
                                  <li class="pt-3"> <h5>Plasmid purification (Miniprep)</h5>                               <p>Click here on button to download the PDF file.</p>
−
                                    <li>η = 0.799 Pa·s [Viscosity of Oviductal Fluid]</li>
+
                                <a class="btn btn-primary" href="PDFLINK" download>
−
                                    <li>T = 35.5 + 273.15 = 308.65 K [Temperature of Oviduct]</li>
+
                                <i class="fa fa-file-pdf-o" aria-hidden="true"></i>Download</a></li>
−
                                    <li>r = 22kDa = 2.43 nm [Radius of ovastacin molecule]</li>
+
                                  <li class="pt-3"> <h5>Nanodrop</h5>                               <p>Click here on button to download the PDF file.</p>
−
                                    <li>a = 61.7 μm [Radius of the ovum]</li>
+
                                <a class="btn btn-primary" href="PDFLINK" download>
−
                                    <li>s = 5 mm [Radius of the ampulla region of the oviduct]</li>
+
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−
                                    <li>N<sub>0</sub> = 3.92 ×10<sup>11</sup> molecules = 0.000650946529392 nanomoles [Number of ovastacin to react with all ZP2]</li>
+
                                  <li class="pt-3"> <h5>Agarose gel electrophoresis</h5>                              <p>Click here on button to download the PDF file.</p>
−
                                     <p>
+
                                <a class="btn btn-primary" href="PDFLINK" download>
−
                                        For, Ovastacin D=1.1644194699 ×10<sup>-13</sup>   m<sup>2</sup> s<sup>-1</sup>
+
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−
                                        </p>
+
                                  <li class="pt-3"> <h5>Agarose gel extraction</h5>                              <p>Click here on button to download the PDF file.</p>
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                                  <li class="pt-3"> <h5>Restriction digestion</h5>                              <p>Click here on button to download the PDF file.</p>
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                                  <li class="pt-3"> <h5>PCR amplification</h5>                              <p>Click here on button to download the PDF file.</p>
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                                  <li class="pt-3"> <h5>PCR cleanup</h5>                              <p>Click here on button to download the PDF file.</p>
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                                  <li class="pt-3"> <h5>Golden Gate assembly</h5>                              <p>Click here on button to download the PDF file.</p>
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                                  <li class="pt-3"> <h5>Speedvac</h5>                              <p>Click here on button to download the PDF file.</p>
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                                <a class="btn btn-primary" href="PDFLINK" download>
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                                  <li class="pt-3"> <h5>Ligation</h5>                              <p>Click here on button to download the PDF file.</p>
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                                  <li class="pt-3"> <h5>Patching and Colony PCR</h5>                               <p>Click here on button to download the PDF file.</p>
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                                <a class="btn btn-primary" href="PDFLINK" download>
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                                  <li class="pt-3"> <h5>Replica plating and Starch test</h5>                              <p>Click here on button to download the PDF file.</p>
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                                <a class="btn btn-primary" href="PDFLINK" download>
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 +
                                  <li class="pt-3"> <h5>Competency and transformation</h5>
 +
                                  <ol>
 +
                                    <li class="pt-3"><h6>E. coli</h6>                                   
 +
                                      <p>Click here on button to download the PDF file.</p>
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                                    </li>
 +
                                     <li class="pt-3"><h6>Bacillus subtilis</h6>                                   
 +
                                      <p>Click here on button to download the PDF file.</p>
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 +
                                    </li>
 +
                                    <li class="pt-3"><h6>S. Cerevisiae</h6>                                
 +
                                      <p>Click here on button to download the PDF file.</p>
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                                    </li>
 +
                                  </ol>
 +
                                </li>
 
                                 </ol>
 
                                 </ol>
−
                                 <li>t → Time elapsed</li>
+
 
−
               
+
                              </div>
−
                            </ul>
+
                              </div>
 +
                          </div>
 +
                          <div class="accordion-item">
 +
                            <h2 class="accordion-header" id="heading15">
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 +
                                 PROTEIN PURIFICATION AND ANALYSIS
 +
                              </button>
 +
                            </h2>
 +
                            <div id="collapse15" class="accordion-collapse collapse" aria-labelledby="heading15" data-bs-parent="#accordionExample">
 +
                              <div class="accordion-body">
 +
                                <ol>
 +
                                  <li class="pt-3"> <h5>Protein induction</h5>                             
 +
                                    <p>Click here on button to download the PDF file.</p>
 +
                                    <a class="btn btn-primary" href="PDFLINK" download>
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 +
                                  </li>
 +
                                  <li class="pt-3"> <h5>Ni-NTA </h5>
 +
                                    <p>Click here on button to download the PDF file.</p>
 +
                                    <a class="btn btn-primary" href="PDFLINK" download>
 +
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 +
                                  </li>
 +
                                  <li class="pt-3"> <h5>Western blotting</h5>
 +
                                    <ul>
 +
                                      <li>   
 +
                                        <h6>E. coli - BL21 strain  - ZP2 analysis</h6>                             
 +
                                        <p>Click here on button to download the PDF file.</p>
 +
                                        <a class="btn btn-primary" href="PDFLINK" download>
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                                        <i class="fa fa-file-pdf-o" aria-hidden="true"></i>Download</a></li>
 +
                                    </ul>
 +
                                  </li>
 +
                                  <li class="pt-3"> <h5>SDS </h5>
 +
                                    <p>Click here on button to download the PDF file.</p>
 +
                                    <a class="btn btn-primary" href="PDFLINK" download>
 +
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 +
                                  </li>
 +
                                  <li class="pt-3"> <h5>Western blotting</h5>
 +
                                    <ul>
 +
                                      <li>   
 +
                                        <h6>E. coli - BL21 strain  - ZP2 analysis</h6>                             
 +
                                        <p>Click here on button to download the PDF file.</p>
 +
                                        <a class="btn btn-primary" href="PDFLINK" download>
 +
                                        <i class="fa fa-file-pdf-o" aria-hidden="true"></i>Download</a></li>
 +
                                    </ul>
 +
                                  </li>
 +
                                  <li class="pt-3"> <h5>Plate Reader</h5>                             
 +
                                    <p>Click here on button to download the PDF file.</p>
 +
                                    <a class="btn btn-primary" href="PDFLINK" download>
 +
                                    <i class="fa fa-file-pdf-o" aria-hidden="true"></i>Download</a>
 +
                                  </li>
 +
                                </ol>
 +
                              </div>
 +
                            </div>
 +
                          </div>
 +
 
 +
                      </div>
 +
 
 
                     </div>
 
                     </div>
 
                 </div>
 
                 </div>
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                <div class="card d-none d-md-block" style="max-width: 18rem;width: 25%; border: none !important;" id="index">
 
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                    <div class="card-body">
 
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                      <h5 class="card-title">INDEX</h5>
 
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                      <h6 class="card-subtitle mb-2 text-muted">Card subtitle</h6>
 
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                      <p class="card-text">Some quick example text to build on the card title and make up the bulk of the card's content.</p>
 
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                      <a href="#" class="card-link">Card link</a>
 
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                      <a href="#" class="card-link">Another link</a>
 
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                      <ul>
 
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                          <li><a href="#waste1">waste 1</a></li>
 
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                          <li><a href="#waste2">waste 2</a></li>
 
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                          <li><a href="#waste3">waste 3</a></li>
 
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                          <li><a href="#waste4">waste 4</a></li>
 
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                          <li><a href="#waste1">waste 1</a></li>
 
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                          <li><a href="#waste2">waste 2</a></li>
 
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                          <li><a href="#waste3">waste 3</a></li>
 
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                          <li><a href="#waste4">waste 4</a></li>
 
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                          <li><a href="#waste1">waste 1</a></li>
 
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                          <li><a href="#waste2">waste 2</a></li>
 
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                          <li><a href="#waste3">waste 3</a></li>
 
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                          <li><a href="#waste4">waste 4</a></li>
 
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                      </ul>
 
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                    </div>
 
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Revision as of 20:53, 12 October 2021


Ovi-Cloak

SCROLL

Wetlab Calender

Week 1 (1 Jan 2021 -3 Jan 2021) :

A literature review of the female reproductive tract, found that ZP2 protein mutation in females result in infertility, literature review of the microbiome of the reproductive tract 

Week 2 (4 Jan, 2021 -10 Jan 2021): 

A literature review of the targets of the ovum, ruled out producing non-specific enzymes from the bacteria because of potential harm 

Week 3 (11 Jan 2021 - 17 Jan 2021) : 

Learning about iGEM parts, kits and registry to get an idea of available things. 

Categorized the species of bacteria in both fallopian tubes, found ovastacin.

Week 4 (18 Jan 2021 - 24 Jan 2021) : 

 Studied more on lactobacillus, met with experts in our institute to discuss the idea, looked into lex A system for progesterone sensing, completely ruled out production of NAG enzyme by bacteria 



Week 5 (24 Jan 2021 - 31 Jan 2021) : A literature review about the immune system of the reproductive system, started review on a kill switch for reversibility.

Week 1 (1 Feb 2021 - 7 Feb 2021 ):

Found more about iGEM competition deliverables and had a meeting with our PIs. Literature review on Zone hardening 

Week 2 ( 8 Feb 2021 - 14 Feb 2021):

 A literature review on the ZP2 cleavage model by ovastacin and learnt more about the strucutre of ovastacin 

Week 3 (15 Feb 2021 - 21 Feb 2021):

 Searched for commensal for implementation of our idea and an appropriate model organism to use as chassis by looking into secretion systems, looked into progesterone sensing by the SRTF transcription factor. 


Week 4 (22 Feb 2021 - 28 Feb 2021): Finalised model organism as B. subtilis for proof of concept and Lactobacillus acidophilus as the commensal to modify, finalized SRTF transcription factor for progesterone inducible system

Week 1 (1 March 2021 -7 March 2021): 

Looked at the HGT aspects of the genetically modified bacteria through literature review and found out more about the safety aspects. 

Week 2 (7 March 2021 - 14 March 2021): 

Finalized the different modules of our project, started in-silico work for proof of concept. Looked at different competition deliverables from the wet lab point of view and planned how to achieve them. 

Week 3 (15 March 2021 - 21 March 2021) : 

Looked into more toxin-antitoxin systems for the kill switch, started designing preliminary experiments for the proof of concept 

Week 4 (22 March 2021 - 31 March 2021) :

Met Prof Satish K Gupta for getting feedback on our idea, agreement to procure clones of Human ZP2 in E.coliDH5α from his lab at NII, Delhi. Continued work on the toxin-antitoxin system and started looking for more models for the hormonal sensing system

Week 1 (1 April 2021- 3 April 2021) :

Procured clones of hZP2 from Prof Satish K Gupta and shipped them to our lab at IISER Tirupati. Continued the work on designing experiments for the proof of concept of our project. 

Week 2 (4 April 2021 - 10 April 2021): 

Decided to work with yeast as a model organism too and started designing experiments for yeast species. Started looking at resources to procure strains of Bacillus subtilis.

Week 3 (11 April 2021 -17 April 2021): 

Meeting with experts working on yeast systems from our institute, started looking for protocols for different experiments, realized the limitations of progesterone inducible systems, and started looking for alternative hormone sensing models in prokaryotes. 

Week 4 (18 April 2021 - 24 April 2021) : 

Realized the sensitivity issue of the estrogen sensing system in prokaryotes, looked into delayed expression and oscillatory circuits, Started simulating the systems to get an idea about these systems. Found out delayed expression and oscillation would not work for cycles on a monthly basis. 

Week 1 (1 May 2021 - 8 May 2021) : 

Worked on finding alternatives for the progesterone inducible system, literature review on kill switches resumed and protocol review process started 

Week 2 (9 May 2021 - 15 May 2021):

End Semester exams 

Week 3 (16 May 2021- 22 May 2021) : 

Started looking into growth kinetics for reducing ovastacin production time, Heterologous gene expression in Bacillus subtilis on different pH, temperatures, and concentrations. Ruled out light selected Dnase based toxins for kill switch and biosafety. 

Week 4 (23 May 2021- 31 May 2021) : 

Yeast workflow finalized. Started looking into the ytvA system for biosafety and Decided which promoters to take for experiments, looked for finalized parts in the iGEM parts registry.

Week 1 ( 1 June 2021 - 5 June 2021) : 

Started making the Bacillus subtilis workflow, finalized the experiments, Calculated d-score for terminator efficiency, and selected terminator for making improvement to part. 

Week 2 ( 6 June 2021 - 12 June 2021) : 

Worked on Genetic circuits for all the experiments, finalized the protocols for genome integration in Bacillus subtilis. 

Week 3 (13 June 2021 - 19 June 2021) : 

Started looking for protocols of protein purification in the yeast and Bacillus subtilis and discussed them with various experts from our institute. Found out the reagents required for carrying out the experiments. 

Week 4 (20 June 2021 - 26 June 2021):

Met with Professor Sabri from IISER Trivandrum to discuss Bacillus subtilis experiments and had an agreement with him to ship the clones to our laboratory at IISER Tirupati. Did an in-depth analysis of transformation and protein purification protocols for Bacillus subtilis and looked for reagents for the same. 

Week 1 (1 July 2021 - 10 July 2021) :

Discussions on experiments and protocols continued and sent a list of required reagents and consumables to our laboratory and placed initial orders for reagents and consumables. 

Week 2 (11 July 2021 - 17 July 2021) : 

Talked with PhDs and seniors on performing the experiments upon return to campus, ordered gene fragments from IDT for Bacillus subtilis, and ordered yeast gene fragments from Twist Bioscience 

Week 3 (18 July 2021 - 24 July 2021) : 

Returned to IISER Tirupati campus and Quarantined for a week. Compiled final protocols for the experiments and placed more orders for experiments. 

Week 4 (25 July - 31 July 2021) : 

We received our iGEM distribution kit and NEB molecular biology kits. We presented the wet lab plan to our guide PhDs and Professors. Had our lab safety training to start the experiments. Prepared experimental things to start our initial experimental journey.

Week 1 (1 Aug 2021 - 7 Aug 2021): 

Started our experiments by culturing the E. coli containing the plasmid pDR111 and pDR110 and purified them. Thereafter we did restriction digestion of purified plasmids with BamHI and EcoRI. Performed a growth curve of E Coli DH5α and Bacillus subtilis 168 in LB media and performed Saccharomyces cerevisiae strain 2158 and strain 860 in YPD media.

Week 2 (8 Aug 2021 - 14 Aug 2021) : 

Received orders from Himedia. Did Miniprep for the plasmid pDR111 stock preparation. Prepared the stock solutions for the competency of E Coli DH5α and made E Coli DH5α cells competent followed by storing them at -80 degrees C. Performed gram staining to check the bacterial contamination on the plates. Had a demo session for Agarose Gel preparation with our seniors. 

Week 3 (15 Aug 2021 - 21 Aug 2021) : 

Transformed the E Coli DH5α cells with pDR111 to check the quality of the prepared competent cells. Received IDT primers and stock solutions were prepared for SDS PAGE. Worked on making the lab setting more inclusive through LabEyes. Performed Restriction digestion on the plasmid pDR111 with BamHI- hf, and EcoRI- hf. 

Week 4 (22 Aug 2021 - 31 Aug 2021) : 

Performed sequential digestion with EcoRI and BamHI and performed gel extraction with the digested vector. Received Twist order for the yeast fragments. Streaked the E. Coli DH5α with hZP2 clones and expressed the hZP2 protein by IPTG induction. Stored the extracted protein in -20 degree C. Received the pRS424, pRS426, pRS425 yeast shuttle vector plasmids from Dr Viji Subramanian lab.

Week 1 ( 1 Sept 2021 - 11 Sept 2021): 

Did protein purification and ran SDS PAGE multiple times for standardization of ZP2 clones with different IPTG concentrations (0.5mM and 1mM). Started working on oligonucleotides received from TWIST (yeast construct) and did not get satisfactory results. Performed Miniprep with the pRS426 plasmid (for S Cerevisiae vector)and pRSETb plasmid (for ZP2 in E. Coli ). Purified the pRS426 plasmid and transformed the E Coli DH5α with pRSETb and got positive results. Prepared stock solutions for Bacillus subtilis transformation and competency.

 

Week 2 (12 Sept 2021 - 18 Sept 2021) : 

Transformed the Bacillus subtilis and observed the growth of cells on plates in 9hrs. Prepared competent cells of E. Coli BL21 strain and transformed them with pRSETb for ZP2 expression. Golden Gate trials for hyperspank-ovastacin-ZP2 and yeast construct were unsuccessful. Ran SDS for purified hZP2. 

 

Week 3 (19 Sept 2021 - 25 Sept 2021) : 

Did IMAC(Ni-NTA) for hZP2 purification and Western Blot. Started golden gate trials with Improvement 1 and p22-srtf cassette. Did transformation of the ligated product of yeast construct with pRS426 plasmid and hyperspank-ovastacin-ZP2 cassette with pDR111 plasmid in NEB 10β cells and got positive results. 

 

Week 4 (26 Sept 2021 - 30 Sept 2021) : 

Mid sem exams 

Week 1 (1 Oct 2021 - 9 Oct 2021): 

Week 2 (10 Oct 2021 - 16 Oct 2021) : 

Week 3 (17 Oct 2021 - 23 Oct 2021) : 

Week 4 (24 Oct 2021 - 31 Oct 2021) : 

Protocols

  1. Growth Curve
    1. Bacillus subtilis 168

      Click here on button to download the PDF file.

      Download
    2. E. coli DH5α

      Click here on button to download the PDF file.

      Download
    3. Yeast (Saccharomyces cerevisiae strain S288C)

      Click here on button to download the PDF file.

      Download
  2. Culture preparation (Streaking, Spreading, Inoculation)

    Click here on button to download the PDF file.

    Download
  3. Revival from glycerol stock AND preparation of glycerol stock

    Click here on button to download the PDF file.

    Download

  1. Plasmid purification (Miniprep)

    Click here on button to download the PDF file.

    Download
  2. Nanodrop

    Click here on button to download the PDF file.

    Download
  3. Agarose gel electrophoresis

    Click here on button to download the PDF file.

    Download
  4. Agarose gel extraction

    Click here on button to download the PDF file.

    Download
  5. Restriction digestion

    Click here on button to download the PDF file.

    Download
  6. PCR amplification

    Click here on button to download the PDF file.

    Download
  7. PCR cleanup

    Click here on button to download the PDF file.

    Download
  8. Golden Gate assembly

    Click here on button to download the PDF file.

    Download
  9. Speedvac

    Click here on button to download the PDF file.

    Download
  10. Ligation

    Click here on button to download the PDF file.

    Download
  11. Patching and Colony PCR

    Click here on button to download the PDF file.

    Download
  12. Replica plating and Starch test

    Click here on button to download the PDF file.

    Download
  13. Competency and transformation
    1. E. coli

      Click here on button to download the PDF file.

      Download
    2. Bacillus subtilis

      Click here on button to download the PDF file.

      Download
    3. S. Cerevisiae

      Click here on button to download the PDF file.

      Download

  1. Protein induction

    Click here on button to download the PDF file.

    Download
  2. Ni-NTA

    Click here on button to download the PDF file.

    Download
  3. Western blotting
    • E. coli - BL21 strain - ZP2 analysis

      Click here on button to download the PDF file.

      Download
  4. SDS

    Click here on button to download the PDF file.

    Download
  5. Western blotting
    • E. coli - BL21 strain - ZP2 analysis

      Click here on button to download the PDF file.

      Download
  6. Plate Reader

    Click here on button to download the PDF file.

    Download
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