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Description and lab notebook done

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padding: 8px;
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/* Front Page */
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<li class="dropdown">
<a href="#" class="dropdown_toggle">Wetlab &#9662;</a>
<ul class="dropdown_menu">
<li><a href="#">Kidney Stones?</a></li>
<li><a href="#">Kidney Stones</a></li>
<li><a href="{{ url_for('pages', page='experiments') }}">Experiments</a></li>
<li><a href="{{ url_for('pages', page='notebook') }}">Lab Notebook</a></li>
<li><a href="{{ url_for('pages', page='results1') }}">Wetlab Results</a></li>
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<div class="content">
<div class="toc-scroll" id="fixedContainer">
<nav id="toc">
<a href="#section1" class=""><span class="dot">●</span>Why and how?</a>
<a href="#section2" class=""><span class="dot">●</span>Peroxide</a>
<a href="#section3" class=""><span class="dot">●</span>References</a>
<a href="#section1" class=""><span class="dot">●</span>Abstract</a>
<a href="#section2" class=""><span class="dot">●</span>Kidney Stones</a>
<a href="#section3" class=""><span class="dot">●</span>Current Treatments</a>
<a href="#section4" class=""><span class="dot">●</span>Our solution</a>
<a href="#section5" class=""><span class="dot">●</span>The product</a>
<a href="#section6" class=""><span class="dot">●</span>Wetlab experiments</a>
<a href="#section7" class=""><span class="dot">●</span>Drylab simulations</a>
<a href="#section8" class=""><span class="dot">●</span>References</a>
</nav>
</div>
<div class="paragraphs">
<div class="desc">
<section id="section1">
<h3 class="sub-title">Why and how?</h3>
<p>Kidney stones are one of the most painful conditions a human can suffer from, primarily because most stones need to exit the body with the urine. Most kidney stones (>65%) are hard stones consisting of calcium oxalate crystals that form in the renal pelvis. Smaller stones are able to move down the ureters into the bladder and out with the urine. Treatment consists of pain-relieving medication, an excess of water, and movement. Large stones that get stuck, often in the ureters, are either removed by surgery or broken down with sound waves before being excreted from the body naturally.
Our goal is to use synthetic biology to build the stepping stones for a potential enzyme-based treatment against kidney stones. In our project, we will research whether you can break down oxalate, and potentially calcium oxalate crystals, using E. coli (K-12) producing the enzyme oxalate oxidase. We aspire to find an alternative, less painful way of removing kidney stones. Furthermore, a large majority of kidney stone patients experience a recurrence of kidney stones, and we hope that the enzyme produced by the bacteria will be able to lower the levels of oxalate, as kidney stones are often caused by hyperoxaluria.
</p>
</section>
</div>
<div class="desc">
<section id="section2">
<h3 class="sub-title">The peroxide problem</h3>
<p>
Oxalate oxidase is a useful enzyme that is seen in a lot of organisms, however that does not mean that it is perfect. It does reduce oxalate, although it reduces it to hydrogen peroxide. This could potentially be dangerous if the enzyme increases the concentration of peroxide and thereby the oxidative stress of the system. This is why we are also working on a system to increase the concentration of bacterial catalase, as a response to increased levels of hydrogen peroxide through a promotor with a peroxide transcription factor. Thereby we will be able to ensure safe levels of oxidative stress even with the added hydrogen peroxide produced by the reduction of oxalate.
</p>
</section>
</div>
<div class="bibliography">
<div class="references">
<section id="section3">
<h4>References</h4>
<div>
<span class="author">Doe, J., & Smith, A.</span>
<span class="year">(2020).</span>
<span class="title">An example scientific article.</span>
<span class="journal">Journal of Examples,</span>
<span class="volume">15</span>
<span class="issue">(3),</span>
<span class="pages">123-134.</span>
<span class="doi">https://doi.org/10.1234/example.2020.56789</span>
</div>
<div>
<span class="author">Doe, J., & Smith, A.</span>
<span class="year">(2020).</span>
<span class="title">An example scientific article.</span>
<span class="journal">Journal of Examples,</span>
<span class="volume">15</span>
<span class="issue">(3),</span>
<span class="pages">123-134.</span>
<span class="doi">https://doi.org/10.1234/example.2020.56789</span>
</div>
<div>
<span class="author">Doe, J., & Smith, A.</span>
<span class="year">(2020).</span>
<span class="title">An example scientific article.</span>
<span class="journal">Journal of Examples,</span>
<span class="volume">15</span>
<span class="issue">(3),</span>
<span class="pages">123-134.</span>
<span class="doi">https://doi.org/10.1234/example.2020.56789</span>
</div>
</section>
<section id="section1">
<div class="desc">
<h3>Abstract</h3>
<i>Over 1 out of 10 people are expected to experience kidney stones in their lifetime. The pain has been compared to childbirth. That is why we aim to ease the pain with the use of enzymes. The problem with current treatments is their invasive and sometimes painful nature and the need for stones of specific sizes, making doctor visits a must. </i>
<p class="indent"><i>Moreover, fifty percent of people get kidney stones again after having experienced them once. To avoid people having to endure having kidney stones multiple times. We propose using GMO Lactic acid bacteria, engineered to express the enzyme oxalate oxidase when oxalate is present. The bacteria will be administered through yogurt since it is a widely consumed beverage in arid parts of the world where low water quality and high temperatures lead to increased kidney stone formation. </i></p>
<p class="indent"><i>Furthermore we successfully transformed our bacteria with the enzyme and proved its ability to break down oxalate. More so we designed four new programmes to ease the process of conducting docking simulations for future iGEM’ers.</i></p>
</div>
</div>
</section>
<section id="section2">
<div class="desc">
<h3>The issue - Kidney stones</h3>
<p>Kidney stones can affect anyone, with over 1 in 10 people expected to experience them at some point in their lives. The pain is often compared to the intensity of childbirth. Moreover, individuals who have had kidney stones are 30% more likely to develop another. This issue is especially pronounced in arid regions, which led us to visit Morocco. However, kidney stones are still commonplace in northern countries; in Denmark, for instance, 10% of men and 5% of women will endure the pain of kidney stones during their lifetime [1]. Kidney stones form when the concentration of certain waste products, most commonly oxalate, increases in the bladder, normally due to insufficient hydration [2]. These crystals develop in the kidneys and cause significant discomfort as they must pass through the ureter, a narrow tube with an average diameter of 3 to 4 mm [3].Regarding the typical size of kidney stones is the data unreliable since most kidney stones are passed at home and not in hospitals.</p>
</div>
</section>
<section id="section3">
<div class="desc">
<h3>The problems with current treatments</h3>
<p>There are already ways to treat kidney stones, nonetheless, they all have some vital flaws, from being extremely painful to invasive.</p>
<ul>
<li><p><b>The painkillers and pray: </b>The most common way to treat kidney stones is to simply drink a large amount of water and take over-the-counter painkillers. This can however be extremely painful. This is the primary treatment for stones from less than 2mm to 7mm. Which means that the other treatments (that try to make the stones smaller) usually also ends with the “painkillers and pray”-method, since their goal is to simply make this step less painful.</p></li>
<li><p><b>Ureteroscopy: </b>Works by inserting a small ureteroscope through the urethra, the bladder and the ureter up to where the stone is located. If it's small it can be snared with a basket device and removed. If it's bigger it will first need to be fragmented with a laser, before being removed [4]. This approach is normally done on stones from 7-10 mm</p></li>
<li><p><b>Shock-wave lithotripsy: </b>Works by blasting stones into fragments with the use of high-energy sound waves, to make them easier to pass. This means that this still has the same end as the “natural” treatment, however, the crystals may be smaller after the shock-wave lithotripsy making it less painful. This approach is normally done with stones around 10-30mm.</p></li>
<li><p><b>Percutaneous nephrolithotomy: </b>Works by surgery removing the stones, this does however have the same problems as normal surgery with the risk of infections and so on. That is also the reason why this approach is normally only done on stones bigger than 30mm [5][6].</p></li>
</ul>
</div>
</section>
<section id="section4">
<div class="desc">
<h3>Our solution</h3>
<p>Our initial idea was to develop a product aimed at breaking down the most common type of kidney stone: calcium oxalate. The goal was to create a less painful treatment option for kidney stone sufferers.</p>
<p class="indent">With this in mind, we began researching enzymes capable of breaking down calcium oxalate. During this process, we discovered an enzyme from barley (Hordeum Vulgare), which can degrade oxalate through the following reaction [7]:</p>
<p class="indent" style="text-align: center; font-size:20px;">Oxalate + O<sub>2</sub> + 2H<sup>+</sup> &#8652; 2CO<sub>2</sub> + H<sub>2</sub>O<sub>2</sub></p>
<p class="indent">There are two main problems with this reaction; the first is that it breaks down oxalate and not necessarily calcium oxalate. The second is that it creates hydrogen peroxide. Furthermore, if we had to create a therapeutic that worked directly on the kidney stones, we needed to be able to deliver it to the kidneys. While methods for drug delivery in the kidneys are still researched, targeting a medicine for the kidney is an extensive procedure, and something we did not attend the main focus of our project to be [8].This meant we had to go back to the drawing board and revise our ideas. </p>
<p class="indent">We went through revisions for our final product and landed on the final concept being genetically modified lactic acid bacteria expressing the enzyme oxalate oxidase. Bacteria naturally have pathways that would be able to break down the hydrogen peroxide, making it much safer [9].</p>
<p class="indent">Articles showed that lowering the concentration of oxalate in the gut largely reduces the risk of getting kidney stones [10]. So therefore we should be able to create a bacteria that breaks down the oxalate in the gut and prevents kidney stones in this way. Here we also solve the problem of delivery by making our product into a probiotic. While avoiding having to research whether oxalate oxidase can break down oxalate in the form calcium oxalate. </p>
<p class="indent">Further evidence that this product may work is the fact that species of bacteria with the capability to break down oxalate in the gut, are often lacking in individuals suffering from kidney stones. For example Oxalobacter Formigenes [11]. This is a specialist oxalotroph bacteria living in the gut. Meaning that the bacterias only source of energy is oxalate. One might think, oh lets just introduce this bacterium as a probiotic. but the fact that they only depend on oxalate, and are very sensitive to antibiotics [12] means that they may very easily be wiped out from the microbiome. in the practicals they seem to be hard to culture, and oral administration seems improbable cause the bacteria are sensitive to pH and most likely will be killed by the gastric acid in the stomach. (though some strains may be more pH tolerant) [13]. Therefore purely introducing Oxalobacter Formigenes will not suffice. and our project still has merit. </p>
</div>
</section>
<section id="section5">
<div class="desc">
<h3>The product - a yogurt drink</h3>
<p>We wanted the product to be accessible and cheap, so a large number of people would benefit from the solution. That's why we landed on a yogurt drink (more specifically Ayran since it's common in the drier parts of the world). This would make our idea a drink that everyone could enjoy while giving it the possibility of easing the pain involved with kidney stones by making them smaller. This would work by choosing a promoter that would react when oxalate was present, and therefore not activating, (or activating in smaller amounts) if no kidney stones would be present.</p>
<p class="indent">Our solution is better than the current treatments, even though it still ends with the “painkillers and pray” method since it can be introduced and potentially help with stones of all sizes. Furthermore, it could have a longer-lasting effect if the bacteria we introduce can become a part of the microbiome and thereby continue to produce the enzyme whenever oxalate is available. This is however not a burden and the product would most likely need to have been consumed over a large amount of time before, to make sure the bacteria have a chance to become a part of the microbiome. The bacteria could however at one point lose the plasmid due to factors such as horizontal gene transfer which would make it “lighter” and thereby overpower the bacteria that still have the plasmid taking over their part of the microbiome. This is why it would not be a permanent solution respectfully. </p>
</div>
</section>
<section id="section6">
<div class="desc">
<h3>The Wetlab experiments</h3>
<p>To make our proof of concept we attempted to transform E. Coli. Even though our final concept was GMO of lactic acid bacteria since E.coli is a standard in biotechnology, which meant that all our members that were in the GMO lab, had already worked with E.coli before, making it safer and a more secure choice for our project. The transformation was successful and the enzymes were isolated for testing which showed that they were able to break down oxalate 7, 9, 13,</p>
</div>
</section>
<section id="section7">
<div class="desc">
<h3>The Drylab simulations</h3>
<p>In dry lab, we assessed the possibilities of molecular interactions with naturally present compounds in the body inhibiting the activity of our main enzyme, oxalate oxidase. To run the docking we used AutoDock4 which enables the docking of metalloenzymes such as main enzyme. To complete the screening process of ligand-enzyme interactions a total of four individual python programs were developed to streamline the individual steps of the docking process. The docking simulations showed that histidine could bind in the active site leading to problems. This realization led us to use GST-tag instead of 6xHis-tag. Additionally, we employed AlphaFold from Google DeepMind to predict the final protein structure of the chain of GST-tag, TEV-site and oxalate oxidase. This provided us with theoretical proof that the enzyme’s functionality remained intact with the purification tag.</p>
</div>
</section>
<section id="section9">
<div class="desc">
<h3>References</h3>
<ol>
<li>Admin, & Admin. (2024, August 8). 10 Surprising Facts about Kidney Stones. Keck Medicine of USC. https://www.keckmedicine.org/blog/10-surprising-facts-about-kidney-stones/</li>
<li>Kidney stones. (n.d.). National Kidney Foundation. https://www.kidney.org/kidney-health/kidney-stones</li>
<li>Rossiaky, D. (2022, October 13). What are the different types of kidney stones? Healthline. https://www.healthline.com/health/types-of-kidney-stones-chart#treatment</li>
<li>Ureteroscopy. (2021, August 8). Johns Hopkins Medicine. https://www.hopkinsmedicine.org/health/treatment-tests-and-therapies/ureteroscopy</li>
<li>Kidney Stones. (n.d.). National Kidney Foundation. https://www.kidney.org/kidney-topics/kidney-stones</li>
<li>Mathis, A., PhD. (2023, June 28). Kidney stone size chart for determining treatment in 2024. Healthcanal.com. https://www.healthcanal.com/health/kidney-stones/kidney-stone-size-chart</li>
<li>Bank, R. P. D. (n.d.). RCSB PDB - 1FI2: CRYSTAL STRUCTURE OF GERMIN (OXALATE OXIDASE). https://www.rcsb.org/structure/1FI2</li>
<li>Huang, L., Ye, Q., Chen, X., Huang, X., Zhang, Q., Wu, C., Liu, H., & Yang, C. (2024). Research progress of drug delivery systems targeting the kidneys. Pharmaceuticals, 17(5), 625. https://doi.org/10.3390/ph17050625</li>
<li>Sen, A., & Imlay, J. A. (2021). How microbes defend themselves from incoming hydrogen peroxide. Frontiers in Immunology, 12. https://doi.org/10.3389/fimmu.2021.667343</li>
<li>Chen, T., Qian, B., Zou, J., Luo, P., Zou, J., Li, W., Chen, Q., & Zheng, L. (2023). Oxalate as a potent promoter of kidney stone formation. Frontiers in Medicine, 10. https://doi.org/10.3389/fmed.2023.1159616</li>
<li>Siener, R., Bangen, U., Sidhu, H., Hönow, R., Von Unruh, G., & Hesse, A. (2013). The role of Oxalobacter formigenes colonization in calcium oxalate stone disease. Kidney International, 83(6), 1144–1149. https://doi.org/10.1038/ki.2013.104</li>
<li>Stewart, C. S., Duncan, S. H., & Cave, D. R. (2003). Oxalobacter formigenes and its role in oxalate metabolism in the human gut. FEMS Microbiology Letters, 230(1), 1–7. https://doi.org/10.1016/s0378-1097(03)00864-4</li>
<li>Ellis, M. L., Dowell, A. E., Li, X., & Knight, J. (2016). Probiotic properties of Oxalobacter formigenes: an in vitro examination. Archives of Microbiology, 198(10), 1019–1026. https://doi.org/10.1007/s00203-016-1272-y</li>
</ol>
</div>
</section>
</div>
</div>
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<div>
<section id="section6">
<h4>References</h4>
<div>
<p style="font-size: 16px;">[1] Cassell, A., Jalloh, M., Ndoye, M., Mbodji, M., Gaye, O., Thiam, N. M., Diallo, A., Labou, I., Niang, L., & Gueye, S. (2020). Surgical management of urolithiasis of the upper tract – Current trends. <i>Research and Reports in Urology, 12</i>, 225–238. https://doi.org/10.2147/RRU.S257669 (29-9-2024)</p>
</div>
<ol>
<li><p style="font-size: 16px;">Cassell, A., Jalloh, M., Ndoye, M., Mbodji, M., Gaye, O., Thiam, N. M., Diallo, A., Labou, I., Niang, L., & Gueye, S. (2020). Surgical management of urolithiasis of the upper tract – Current trends. <i>Research and Reports in Urology, 12</i>, 225–238. https://doi.org/10.2147/RRU.S257669 (29-9-2024)</p></li>
</ol>
</section>
</div>
</div>
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{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block title %}Lab Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block page_content %}
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<div class="content">
<div class="toc-scroll" id="fixedContainer">
<nav id="toc">
<a href="#section1" class=""><span class="dot">●</span>Title</a>
<a href="#subsection1_1" class="subsection"><span class="dot">●</span>subsection</a>
<a href="#section1" class=""><span class="dot">●</span>09/09/24</a>
<a href="#section2" class=""><span class="dot">●</span>13/08/24</a>
<a href="#section3" class=""><span class="dot">●</span>17/08/24</a>
<a href="#section4" class=""><span class="dot">●</span>23/09/24</a>
<a href="#section5" class=""><span class="dot">●</span>27/09/24</a>
<a href="#section6" class=""><span class="dot">●</span>30/09/24</a>
<a href="#section7" class=""><span class="dot">●</span>1/10/24</a>
</nav>
</div>
<div class="paragraphs">
<section id="section1">
<div class="desc">
<h3>Title 1</h3>
<h3>09/09/24<br>Goal: Synthesize Calcium Oxalate crystals and prepare LB medium and Agar-plates for further experiments</h3>
<p>Participants: Caitlin</p>
<ul>
<li>The exact measurements of Na2Ox and CaCl2 was 0,839g and 0,696 respectively</li>
<li>The pH of the Tris-HCl buffer was 6,46 - it was made form 0,303g TRIS in 200 mL demineralised water. Then the pH was adjusted using 0,1M HCl and 0,1M NaOH before filling up to 250 mL with demineralised water</li>
<li>There was a crystal formation immediately after adding the two salts, therefore NaCl was not added to the solution</li>
<li>It sat for 2 weeks to allow the crystals to fall to the bottom and the water to evaporate</li>
</ul>
</div>
</section>
<section id="section2">
<div class="desc">
<h3>13/08/24<br>Goal: Make a serial dilution of hydrogen peroxide to test bacterial survival</h3>
<p>Participants: Caitlin, Mads, Anna, Viktor, Jonathan <br> Hydrogen peroxide survivability by use of a growth curve</p>
<ul>
<li>The experiment went smoothly apart from having to remove 50 µl of water from the control tube to assure that the concentration of bacteria was constant</li>
</ul>
</div>
</section>
<section id="section3">
<div class="desc">
<h3>17/08/24 <br>Goal: Look at results from the hydrogen peroxide experiment</h3>
<p>Participants: Caitlin, (Mads)</p>
<ul>
<li>The plates were removed from the incubator and we took pictures before they were disposed of safely</li>
</ul>
</div>
</section>
<section id="section4">
<div class="desc">
<h3>23/09/24 <br>Goal: Transformation of E. Coli</h3>
<p>Participants: Caitlin, Mads, Viktor <br> E-Coli transformation and Oxalate Oxidase production: <br>We successfully transformed the E. coli and grew it on petri dishes.</p>
<ul>
<li>The CaCl*6H2O was supposed to be in a solid form, whereas the water had escaped from the powdered crystal formation, so a rather unknown amount CaCl had been added. The added CaCl consisted of around 0,2-0,3 ml of the solution of the separated crystals. We further added sterile water until the volume was 10 ml to dilute the solution. The original solution with the fully crystalized CaCl is unknown.</li>
<li>The laboratory did not have any supply of gas left for use with a bunsen-burner, so we resorted to burning ethanol instead to keep an open flame above the petri dishes to minimize risk of contamination.</li>
</ul>
</div>
</section>
<section id="section5">
<div class="desc">
<h3>27/09/24 <br>Goal: Look at results from transformation and transfer to LB-medium</h3>
<p>Participants: Caitlin, Marie, Viktor</p>
<ul>
<li>The transformation went perfectly.</li>
<li>All positive controls had growth and all negative controls had none.</li>
</ul>
</div>
</section>
<section id="section6">
<div class="desc">
<h3>30/09/24 <br>Goal: Cell lysis to predate the enzyme tests</h3>
<p>Participants: Viktor, Mads</p>
<ul>
<li>We successfully isolated the bacteria from the lb medium and used 2 kinds of lysis solution to lyse the cells, so we can later isolate the enzyme.</li>
<ul>
<li>6 of the Eppendorf tubes with cells were lysed with a bio-rad lysis solution that expired in 2017.</li>
<li>One out of the 6 tubes lysed with 10% Tween 20 solution were lost to the overflow beaker.</li>
</ul>
</ul>
</div>
</section>
<section id="section7">
<div class="desc">
<h3>Title 2</h3>
<p>Content of title...</p>
</div>
<section id="subsection1_1">
<h4>Subsection 1.1</h4>
<p>Content of subsection 1.1...</p>
</section>
</section>
</section>
</div>
</div>
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