Team:ATOMS-Turkiye/Results3

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<center><p style="font-size:1.3em;color: rgb(192,0,0) !important;  text-shadow: 0px 0px 1px #000000 !important;        };"><a href="#" onclick="ddaccordion.collapseall('technology'); return false" style="
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">[Expand all]</a></p></center>
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<div class="technology">Cloning</div>
 
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<div class="thelanguage">
 
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<li>Here is our results page, you can analyze our constructed vectors for coding proteins and the agarose gel electrophoresis result of inserts ligated with interest vectors. TetR-VP16 double plasmid system is used in our project as an empowering system of expressing. 
 
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</li>
 
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<li>For Aprotinin, GPx, SOD and tPA;
 
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</li>
 
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<img src="https://static.igem.org/mediawiki/2014/thumb/f/f3/ATOMS_results_1.png/800px-ATOMS_results_1.png" style="margin-left:70px">
 
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<li>For ODD;
 
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</li>
 
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<img src="https://static.igem.org/mediawiki/2014/thumb/3/3f/ATOMS_results_2.png/800px-ATOMS_results_2.png" style="margin-left:70px">
 
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<li>For HRE and KB regulator genes;
 
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</li>
 
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<img src="https://static.igem.org/mediawiki/2014/5/57/HRExx.png" style="margin-left:70px; width:600px">
 
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<img src="https://static.igem.org/mediawiki/2014/thumb/6/63/ATOMS_results_4.png/800px-ATOMS_results_4.png" style="margin-left:70px">
 
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<li>This system includes few understructured elements called TetR-VP16 complex and two different plasmids, pTet-off and pTRE vectors. In the pTet-off plasmid, PCMV constitutive promoter codes for TetR-VP16 protein complex in medium strength which can bind to its responding element present in the second plasmid, pTRE. Tetracycline respond element (TetRE) is a protein binding domain which enables the binding of TetR component of the protein complex. Whereas, VP16 component works as a transcription activator for the weak constitutive promoter (PminiCMV) which exists in the pTRE vector. TetR-VP16 protein complex can activate this weak promoter in pTRE vector.
 
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<h1>At A Glance</h1>
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<ul style="text-indent:20px"> This year, we have disseminated two global health problems: <b>heart attacks and strokes</b>, and their viable clinical solutions. Due to the intricate components of the present mechanisms and treatments of such clinical methods we have set forth a project which has an ample  perspective and approach, together with our policy & practice activities.</ul>  
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<h1>Sensing</h1>
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<ul style="text-indent:20px"> <a href="https://2014.igem.org/Team:ATOMS-Turkiye/Design" style="text-decoration:none">Here</a>, you can take a look at the main components of our project design.
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<div class="technology">1. HRE</div>
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<div class="thelanguage">
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<li>Hypoxia response elements (HREs) are 234 bp long small DNA sequences present in most of the body cells which work as binding domains for hypoxia inducible factor-1α (HIF-1α), a common transcription factor found in our body cells released during hypoxic conditions. (Semenza et al. 1992)</li>
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<ul><li>We aimed to demonstrate its functionality by inserting it into pTRE-Luc vector.</li>
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<li>We expect that HRE, as an enhancer, would activate the promoter existing on the downstream region of it, depending on the level of HIF1alfa in the media which is increased in hypoxic conditions. </li>
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</ul>
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<li>The design of our vector possessing our part is shown below.</li>
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<a href="https://2014.igem.org/Team:ATOMS-Turkiye/Background-Problem" style="text-decoration:none"><h2>The Problem </h2></a>
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<img src="https://static.igem.org/mediawiki/2014/d/df/ATOMS-HREsnap.png">
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<div class="withImage" style="width:420px;height:auto;float:left;margin-right:25px">
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<li>We started our experiments by cloning our parts. The forward primer is designed to produce HRE sequence inserted before the CMV promoter sequence presenting on pTRE vector. Reverse primer is synthesized to produce the cloning region of the vector. HRE-CMV sequence is cloned from pTRE vector via performing PCR. </li>
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<a href="https://static.igem.org/mediawiki/2014/d/d8/ATOMS_at_a_glance_1.jpg"><img src="https://static.igem.org/mediawiki/2014/d/d8/ATOMS_at_a_glance_1.jpg" style="width:400px"></a>
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<li>The PCR product is purified with phenol chloroform method and, afterwards, is cut with XhoI and BamHI restriction enzymes. The product is ligated with pTRE-Luc vector been cut with same enzymes and been treated with Antarctic phosphatase. The ligation product is inserted in DH5alfa strain and we performed colony PCR from the plate. </li>
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<p><b>Expected</b></p>
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<img src="https://static.igem.org/mediawiki/2014/8/89/ATOMS-Hre_2.png" style="width:150px;height:300px" />
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<p>To understand which colony our gene is inserted among the colonies that we transformated pHRE-luciferase vector, we expected the picture above when we perform PCR when we use pTRE-Luc forward and MCS reverse primers.
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  <li>Ischemia is the restriction of blood supply to tissues, generally caused by the blockage of a clot due to the reduction or inhibition of blood flow. This process results in the shortage of oxygen and nutrition vital for metabolism and survival of cells. Since oxygen is delivered to tissues only via the blood stream, incommensurate blood supply causes tissue cells to spurn. <b>Particularly for the heart and brain, irreversible damage is parlously supposable to occur in as little as 3–4 minutes at body temperature.</b></li>
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  <li>Ischemia is said to be the major factor in many of the medical problems of today. The famous heart attack, which is culpable for most of the deaths worldwide, is, as a matter of fact, an ischemic condition. In addition to this, the loss of a specific region of the brain due to inadequate blood perfusion is associated with ischemia and is widely known as stroke.<b>In fact, every year, about 1.5 million Americans have heart attacks resulting in 500,000 deaths.</b> A heart attack occurs every 20 seconds, which is equivalent to loss of a life approximately every single minute.</li>
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<div>
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<div class="withImage">
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<p><b>Experimented</b></p>
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<a href="https://static.igem.org/mediawiki/2014/5/50/Backround2.gif"><img src="https://static.igem.org/mediawiki/2014/5/50/Backround2.gif" style="width:300px"></a>
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<img src="https://static.igem.org/mediawiki/2014/d/da/ATOMS_hre_3.png" />
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<p>From the samples we perform colony PCR by using pTRE-Luc forward and MCS reverse primers, we obtained a band in 428 bp line. This image proves that our HRE sequnce is inserted into the vector, successfully.
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  <li>As it can be expected, one treatment of ischemia is the extermination of the blockage. Nonetheless, such a removal has the likeliness of making the situation more radical. In the current day, it is possible to dissolve the elements of the clot with specific medication such as tissue adaption. However, the tissue adapted to hypoxic media may be poisoned from oxygen. The absence of oxygen and nutrients from the blood during the ischemic period creates a state in which the rejuvenation of circulation to its original state results in inflammation and oxidative damage due to the induction of oxidative stress instead of the restoration of normal function. </li>
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<h4>Luciferase Assay</h4>
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  <li>The state of oxidative damage may cause some unwilling reactions which have the aptitude to produce toxic chemicals called reactive oxygen species (ROS). ROS are intermediate products of oxygenation which have an absence of one electron in their covalent bonds. This makes them ultimately unstable. With such instability, ROS can disrupt almost all cellular structures, including DNA, cell membranes and organelles.  <b>Due to such elements in the nature of ischemic conditions, the final solution should incorporate a solution for all negative outcomes caused by ischemia</b></li>
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<li>The vectors isolated from the colonies we identified correct are co-transfected  into HEK293 and HepG2 cell lines. Transfected cells are incubated in hypoxic and normoxic conditions and the luminescence levels are measured by performing luciferase assay. To conduct hypoxic condition, we used 100 uM CoCl2. Here, the graphics we obtained after the measurement of luminescence is shown below.</li>
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</ul>
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<img src="https://static.igem.org/mediawiki/2014/thumb/7/79/ATOMS_hre_4.png/800px-ATOMS_hre_4.png">
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<a href="https://2014.igem.org/Team:ATOMS-Turkiye/Project-Approach" style="text-decoration:none"> <h2>Our Approach </h2></a>
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<li>According to the results, in hypoxic conditions comparing with normoxia, HRE been inserted into pTRE-luciferase vector shows 7 times more activity by producing more luciferase in HepG2 cell line. In HEK293 cell line, this activity is measured in hypoxia 1,5 times more than normoxia. This results prove that HRE sequence improves the production rate of desired protein in hypoxia comparing with normal oxygen levels.</li>
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<ul>
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<img src="https://static.igem.org/mediawiki/2014/thumb/8/89/ATOMS_hre_5.png/800px-ATOMS_hre_5.png">
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  <li>Our project design aims to assess and resolve two distinct cases. If a more holistic approach is to be taken towards ischemia, the issue could be better combatted. For this reason, we intend to use our vessel cells as a route to act in time to prevent the consequences of ischemia and heal them. <b>To do this, we have to sense hypoxic conditions to prevent or remove clot formation and oxidative stress factors.</b></li>
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  <li>Regarding the need of an efficient, <b>cost effective and rapid treatment option</b>, the approach to the issue was dissected into modules.
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Detection devices and activist proteins.
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Hypoxia will be detected in two levels; with a promoter device for detection in genomic level and a protein domain for protein level regulation. Also, the oxidant factors in the media will be sensed to empower sensor devices. These devices will then produce the activist proteins, which are clot dissolving agents, antioxidant enzymes and proteins. We have the prospect of issuing a novel approach to the problem which will take effect in a short time interval, and affect strongly to prevent the recurrence of the problem.
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<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 1); return false">[Collapse]</a></p>
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</li>
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<a href="https://2014.igem.org/Team:ATOMS-Turkiye/Future-Plan" style="text-decoration:none"> <h2>Future Aspects </h2></a>
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<ul>
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<div class="withImage" style="width:270px;height:auto;float:left;margin-right:25px">
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<a href="https://static.igem.org/mediawiki/2014/a/a5/ATOMS_at_a_glance_3.jpg"><img src="https://static.igem.org/mediawiki/2014/a/a5/ATOMS_at_a_glance_3.jpg" style="width:210px"></a>
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  <li>Gene therapy and tissue engineering are two different, but promising therapies which relate to our project. We have deliberated the advantages, disadvantages and also the possible side effects, ethical or social issues of these therapies to determine which of these would be the most beneficial. </li>
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<div class="technology">2. KB</div>
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  <li>Furthermore, we have designed the real-life application of our project to address the needs of both the clinic and the patient in a multiperspective manner.</li>
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<div class="thelanguage">
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<p>NF-kappaB (NF-kB) proteins comprise a family of structurally-related eukaryotic transcription factors that are involved in the control of a large number of normal cellular and organismal processes, such as immune and inflammatory responses, developmental processes, cellular growth, and apoptosis. In some circumstances, NF-kB/IkB complex can be separated by external effects such as radiation, cellular stress, pathogens, inflammation etc. In this case, NF-kB can enter into nucleus and integrate with compatible kB-RE sites in order to initiate transcription.</p>
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<li>As a final remark, we aim to feature computer modelling data to predict the potential effects of our treatment in order to determine its applicability as preventive medication. <b>We envision that our system will detect hypoxia before it has fatal effects and interfere to prohibit the occurrence of more austere conditions without causing any dismay in the target patient.</b></li>
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<img src="https://static.igem.org/mediawiki/2014/5/5c/ATOMS_KB_results_1.png" style="margin-left:100px">
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<img src="https://static.igem.org/mediawiki/2014/thumb/1/1d/ATOMS-unnamed.jpg/800px-ATOMS-unnamed.jpg">
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<p>We cloned kB-RE and inserted it into the downstream region of CMV mini promoter as it’s shown above.</p>
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<p>NF-kappaB (NF-kB) was synthesized to GenScript™ company and it came in pUC57 plasmid. We digested it with BamHI & PstI and exposed it to Antarctic phosphatase.  </p>
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<p>Afterwards, we purified our part via phenol chloroform method. We performed the same procedures onto the pTRE-luciferase vector. Eventually, we ligated them. </p>
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<p>We inserted our plasmid (pTRE-luciferase kB-RE) into DH5α strain and performed colony PCR by using CMV forward and kB reverse primers. At the end of this experiment, we expected a band seen in the 20-30 bp line. </p>
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<img src="https://static.igem.org/mediawiki/2014/e/ef/ATOMS_KB_results_3.png" style="margin-left:150px">
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<p>  And we observed correct bands in the expected region.</p>
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<img src="https://static.igem.org/mediawiki/2014/2/21/ATOMS_KB_results_4.png" style="margin-left:150px">
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<img src="https://static.igem.org/mediawiki/2014/d/d4/ATOMS_KB_results_5.png">
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<li>At the end of the experiment,  we could not see an increase in the luciferase assay of cells exposed to H202. We assumed that cell death due to the direct exposal of H2O2 was what limited our luciferase activity expectations. Our assumptions were further supported after the microscope analysis we performed, which showed that our cells were unattached from the plates upon being exposed to H202.</li>
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<a href="https://2014.igem.org/Team:ATOMS-Turkiye/Achievements" style="text-decoration:none">
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<h3>Reference</h3>
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<h2>Achievements</h2></a>
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<li>1. Zhang Y., Yang X., Bian F., et al. TNF-α promotes early atherosclerosis by increasing transcytosis of LDL across endothelial cells: Crosstalk between NF-κB and PPAR-γ. Journal of Molecular and Cellular Cardiology 72 (2014) 85–94</li>
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<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 2); return false">[Collapse]</a></p>
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  <li><b>We successfully propose a promising, novel approach and system design to treat ischemia, a common and lethal public health issue.</b></li>
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</div>
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<li>We have created a system which aims to treat ischemia: the number one cause of deaths worldwide.</li>
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<li>We successfully worked all of our seven parts in our engineered eukaryotic cells.</li>
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<li>We have accomplished modeling our gene expression dynamics and have also come up with the unique idea of modelling our safety experiment which enables us to prove graphically that we are able to switch our system off in the presence of a simple antibiotic such as tetracycline.</li>
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<li>We collaborated with the METU iGEM team and exchanged thoughts on how we can improve our modelling.</li>
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<li>We will hold a special metaphor event called ‘Think By Heart’ during the Giant Jamboree where we discover the unknowns of our heart simply beginning from its etymology to the cardiac cycle and how it is bizarrely similar to us human beings in terms of its style of functioning and many more.Have created a system which aims to treat ischemia: the number one cause of deaths worldwide.</li>
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<div class="technology">3. ODD</div>
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</ul>
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<div class="thelanguage">
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<a href="https://2014.igem.org/Team:ATOMS-Turkiye/Practices-Approach" style="text-decoration:none"> <h2>Policy & Practices</h2></a>
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<ul>
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<img src="https://static.igem.org/mediawiki/2014/c/c7/ATOMS-ODD1.jpg" style="width:400px">
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<a href="https://2014.igem.org/Team:ATOMS-Turkiye/Practices-Approach"style="
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<p><strong>ODD SYNTHESİS FROM HEP2G VİA PCR </strong></p>
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<p><b>Expected</b></p>
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padding: 3px;">Click and go to P&P section.</b>
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  <li>Apart from our project design, we also wanted to engender a better approach on ischemic conditions and diseases by working on human practice and social issues. <b>This year, we preferred to conduct an inclusive perspective and activity to address every aspect of the issue as well as all the stakeholders who are connected with it.</b> Therefore, we separated our P&P design into four columns; all of which present a respective process and interactive strategy.</li>
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<li><b>First, we need to analyze how intense the problem</b> is and how common these ischemic diseases are among people, specifically in the iGEM community. This module is the <b>“Identification”</b> module; which intends to identify the parameters of the problem, in order approach the problem accurately.</li>
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<td>
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<li>AAfterwards, our aim is to undergo some tasks to raise awareness and warn people who are at high risk from ischemic diseases such as heart attacks. We plan to present our “identification” data inquired from the public in an influential way. The data includes information such as the total calories consumed by individuals. This comprises our second column, <b>“clarification”</b>.</li>
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<div>
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<li>Thirdly, we need to be accurate and effective in influencing the stakeholders and improving public health. To do this, we chose the <b>“specialization”</b> step to consult experts, doctors, specialists and related stakeholders of sectors and describe what had have been done so far and inquire what could further be conveyed.</li>
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<p><b>Experimented</b></p>
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<li>LLastly, all the acquired data and knowledge could be utilized to form a product. However, we must obstruct the possible consequences and side effects of this product. The public should also be informed to prevent any social reaction. Therefore, we have conducted extensive research and performed several brain-storming sessions to depict the applicability of the product, and this is named as the <b>“application”</b> step.
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<a href="https://2014.igem.org/Team:ATOMS-Turkiye/Collaboration" style="text-decoration:none"> <h2>Collaboration</h2></a>
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<p>ODD (Oxygen Dependent Degredation) domain of HIF-1α was synthesized through liver cDNA using PCR with Sall enzyme restriction cites placed at the starting and ending points of the domain.</p>
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<ul>
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<p>The PCR product was purified using the Phenol Chloroform method. Following the isolation, the ODD and pTet-Off vector were cut using the Sall restriction enzyme and then ligated. Thus, the ODD insert was placed in between the tetR(DNA binding domain) and VP16(Transactivating domain) of the pTet-Off vector.</p>
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<a href="https://static.igem.org/mediawiki/2014/5/53/ATOMS_at_a_glance_5.jpg"><img src="https://static.igem.org/mediawiki/2014/5/53/ATOMS_at_a_glance_5.jpg"style="width:200px"></a>
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<p><strong>COLONY PCR</strong></p>
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<p><b>Expected</b></p>
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<img src="https://static.igem.org/mediawiki/2014/4/4c/ATOMS-ODD4.png" />
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  <li>For collaboration, we invited Paris Bettencourt iGEM team to the International Turgut Ozal Medical Congress, which our university hosts. The congress has the highest number of attendants in between medical congress’ across the nation. During the congress, together with the Paris Bettencourt team, a workshop on synthetic biology was organized. The unlimited potential that synthetic biology bears was delved into. </li>
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<li><b>Secondly, we invited METU Turkey iGEM team to our university for dinner. We presented our projects respectively and discussed some ideas regarding our projects. We also debated on how we can help each other and what we can do for collaboration purposes or P&P.</b> METU Turkey iGEM team requested to use some of the parts present in our iGEM kit plates for their project, which we gladly accepted.</li>
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<div>
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<p><b>Experimented</b></p>
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<img src="https://static.igem.org/mediawiki/2014/e/ec/ATOMS-ODD51.jpg" />
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<p>The DH-5α E.coli strains were transformated and, using CMV forward and SV40 polyA reverse primers, colony PCR was conducted and the vectors, in which the inserts were placed, were elected.</p>
 
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<p><strong>CUT-CHECK</strong></p>
 
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<p><b>Expected</b></p>
 
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<img src="https://static.igem.org/mediawiki/2014/b/b3/ATOMS-ODD6.png" />
 
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</td>
 
-
 
-
<td>
 
-
<div>
 
-
<p><b>Experimented</b></p>
 
-
<img src="https://static.igem.org/mediawiki/2014/7/7c/ATOMS-ODD7.jpg" />
 
-
</div>
 
-
</td>
 
-
</tr>
 
-
 
-
</table>
 
-
<p>Since the ODD insert’s contained the same restriction cite on both ends, the colonies that entered the sequence from the right end were cut-checked using EcoRI and BamHI restriction enzymes and the colony containing the desired vector was selected.</p>
 
-
 
-
<h4>ODD Results</h4>
 
-
 
-
<p>ODD (oxygen dependent degradation) domain, which is present in the HIF-1α (hypoxia inducible factor) protein that it activates in lower oxygen levels and breaks down in intermediate oxygen levels, is a protein domain that plays a key regulatory role in the transcription of the HIF-1α factor. In intermediate oxygen levels, the ODD domain of the HIF-1α protein is hydroxilized by the hydroxilase enzyme and the hydroxilized HIF-1α enzyme breaks down through ubiquitin attachment. Thus, the ODD causes the regulation of a transcription factor, which is active in hypoxic conditions and inactive in normoxic conditions. The Tet-Off is a strong system composed of two strong plasmids. Of the two plasmids that form this system, the TetR-VP16 fusion protein produced by the first plasmid acts as a transcriptionary factor regulating the Tet operator sequence of the second plasmid(pTRE). (TetR: DNA Binding Domain, DBD; VP16: Transactivating domain, TAD).</p>
 
-
<p>The Tet-Off system can be inhibited using tetracyclane.</p>
 
-
<p>In this study, through placing the ODD region of the HIF-1α in between the synthetic TetR - VP16 transcription factors (which are not present in mammallian cells and have been used in molecular biology experiments for a long time), the transcription factor was designed to gain sensitivity to oxygen. The therapeutic genes in the pTRE genes can be synthesized as sensitive to the hypoxic  conditions, controlled by the TetR-ODD-VP16 transcription factor.
 
-
</p>
 
-
<img src="https://static.igem.org/mediawiki/2014/8/8b/ATOMS-ODD8.png" style="width:700px;margin-left:80px">
 
-
 
-
<p><strong>Luciferase Assay</strong></p>
 
-
 
-
<p>To control the functionality of the TetR-ODD-VP16 system, the HEK 293T and Hep G2 cell lines were cotransfected using the pTET Off-ODD and pTRE-Luc vectors.</p>
 
-
 
-
<p>100 µM of CoCl2 was added to the cell medium to establish 1% O2 in the medium. The cells were collected 9 hours later. Luminometric measurement under Thermo Varioscan for 613 nm was done and the data of the following graph were acquired:</p>
 
-
 
-
<img src="https://static.igem.org/mediawiki/2014/f/fc/ATOMS-ODD9.png" style="width:500px">
 
-
 
-
<p> The observation of the operational level of the TetR-ODD-VP16 did not yield fruitful results as the cells reached out of the dish when 100 µM of CoCl2 were added to HEK 293T medium.</p>
 
-
 
-
<p> When the Tet Off-ODD and pTRE-Luc vectors were cotranfected to Hep G2 cells in hypoxic medium, there was a 4 times increase in Luciferase concentration in respect to normoxic medium.</p>
 
-
<img src="https://static.igem.org/mediawiki/2014/2/23/ATOMS-ODD10.png" style="width:500px">
 
-
 
-
<p> In the light of these results, it is possible to say that the Tet-ODD-VP16 system was successfully synthesized and a novel hypoxia inducible system was introduced for future use in studies indulged into examining hypoxic conditions.</p>
 
-
 
-
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 3); return false">[Collapse]</a></p>
 
-
</div>
 
-
 
-
 
-
<h1>Therapy</h1>
 
-
 
-
 
-
 
-
<div class="technology">4. tPA</div>
 
-
<div class="thelanguage">
 
-
 
-
<p><b>Tissue plasminogen activator</b> (abbreviated <b>tPA</b> or <b>PLAT</b>) is a protein involved in the breakdown of blood clots. It is a serine protease (EC 3.4.21.68) found in endothelial cells that can be secreted into the plasma as well as the cells that line the blood vessels. As an enzyme, it catalyzes the conversion of plasminogen to plasmin, the major enzyme responsible for clot breakdown. Due to its ability of working on the clotting system, tPA is used in clinical medicine to treat embolic or thrombotic stroke. However, use is contraindicated in hemorrhagic stroke and head trauma.
 
-
 
-
</p>
 
-
<img src="https://static.igem.org/mediawiki/2014/thumb/a/a2/ATOMS-tpa_results1.png/565px-ATOMS-tpa_results1.png" style="margin-left:110px">
 
-
<p>We questioned the best possible was of measuring the tPA enzyme activity and sought the answer to this question by examining the yield of a tPA catalyzed reaction. We searched the sector and discovered the Human tPA Activity Kit of the company ASSAYPRO which we then ordered to use. In the reaction which catalyzes tPA, our aim was to show that tPA was active when plasminogen was transformed to plasmine.
 
-
 
-
</p>
 
-
<p>To acquire the tPA gene, the tPA forward and tPA revers primers were synthesized from the cDNA’s we were in possession of. Using these primers, we acquired the tPA genes by performing the PCR of the cDNA. The head and neck cancer cell line was used as the source for cDNA.
 
-
 
-
</p>
 
-
<h2>GENE SYNTHESIS FROM cDNA OF 64A CELL LINE
 
-
</h2>
 
-
<table class="blueborder">
 
-
<tr>
 
-
<td>
 
-
<div>
 
-
<p><b>Expected</b></p>
 
-
<img src="https://static.igem.org/mediawiki/2014/9/97/ATOMS-tpa_results2.png" />
 
-
<p>The base length of tPA is 1762 bp’dir.
 
-
The electrophoresis of the PCR was expected to show a base length of around 1700 bp. The image below shows the expected result of the electrophoresis.
 
-
</p>
 
-
</div>
 
-
</td>
 
-
 
-
<td>
 
-
<div>
 
-
<p><b>Experimented</b></p>
 
-
<img src="https://static.igem.org/mediawiki/2014/f/f4/ATOMS-tpa_results3.png" />
 
-
<p>Through the primers that we ordered and the cDNA, we acquired tPA inserts. The insert was, as expected, portraying that the base length was around 1700 bp.
 
-
</p>
 
-
</div>
 
-
</td>
 
-
</tr>
 
-
 
-
</table>
 
-
 
-
<h2>CLONING CONTROL-1
 
-
</h2>
 
-
<table class="blueborder">
 
-
<tr>
 
-
<td>
 
-
<div>
 
-
<p><b>Expected</b></p>
 
-
<img src="https://static.igem.org/mediawiki/2014/5/5c/ATOMS-tpa_results4.png" />
 
-
<p>We used EcoRI and BamHI enzymes to cut the inserts and pTRE vectors which was followed by the ligation of our insert and vector using the ligation process. We then transformed our plasmids to
 
-
The E. coli’nin DH5-α strains. Colony PCR was then conducted to verify the accuracy of our ligation.
 
-
 
-
</p>
 
-
</div>
 
-
</td>
 
-
 
-
<td>
 
-
<div>
 
-
<p><b>Experimented</b></p>
 
-
<img src="https://static.igem.org/mediawiki/2014/0/0c/ATOMS-tpa_results5.png" />
 
-
<p>The specified primers were put into colony PCR. As it can be seen in the results above, a right insert was not achieved. This process was repeated several times but no result was achieved. Seeing that ligation did not provide a solution to the problem, synthetically produced inserts were ordered.
 
-
 
-
</p>
 
-
</div>
 
-
</td>
 
-
</tr>
 
-
 
-
</table>
 
-
</h2>
 
-
 
-
<p>The tPA gene which was ligated with the synthetically synthesized Puc57 vector was cut using the EcorI and BamHI restriction enzymes and then ligated with pTRE vector to be transformed into the DH5-α strain. We again used Colony PCR to control the accuracy of our ligation.
 
-
 
-
</p>
 
-
<h2>CLONING CONTROL-2</h2>
 
-
<table class="blueborder">
 
-
<tr>
 
-
<td>
 
-
<div>
 
-
<p><b>Expected</b></p>
 
-
<img src="https://static.igem.org/mediawiki/2014/b/be/ATOMS-tpa_results6.png" />
 
-
<p>The received genes were ligated with the pTRE vector, and then transformated to E.coli strands DH5-α. To test the validity of the transformation, we again applied colony PCR through CMV forward and SV40 reverse primers. The expected base length was 1984 bp.</p>
 
-
</div>
 
-
</td>
 
-
 
-
<td>
 
-
<div>
 
-
<p><b>Experimented</b></p>
 
-
<img src="https://static.igem.org/mediawiki/2014/0/09/ATOMS-tpa_results7.png" />
 
-
<p>Colony PCR was applied to the inserts acquired from transformation and ligation. As it can be seen in the figure above, the second colony contains the appropriate base length in respect to the ladder.</p>
 
-
</div>
 
-
</td>
 
-
</tr>
 
-
 
-
</table>
 
-
 
-
 
-
 
-
<h2>WESTERN BLOTTİNG
 
-
</h2>
 
-
<table class="blueborder">
 
-
<tr>
 
-
<td>
 
-
<div>
 
-
<p><b>Expected</b></p>
 
-
<img src="https://static.igem.org/mediawiki/2014/f/f6/ATOMS-tpa_results8.png" />
 
-
<p>The confirmed genes were transfected into HEK293 cells. The lysates acquired from the cells were run through Western Blot. tPA, that is known to have 63 kD, was expected to have the image on the left.
 
-
</p><p>http://www.emdmillipore.com/TR/en/product/Anti-tPA-%28Tissue-Plasminogen-Activator%29-Antibody%2C-clone-GMA-043,MM_NF-05-883
 
-
</p>
 
-
</div>
 
-
</td>
 
-
 
-
<td>
 
-
<div>
 
-
<p><b>Experimented</b></p>
 
-
<img src="https://static.igem.org/mediawiki/2014/1/18/ATOMS-tpa_results9.png" />
 
-
<p>The bands in the Western Blot were accurate. The transfection of the inserts were verified and assays of tPA were prepared from the lysates.
 
-
</p>
 
-
</div>
 
-
</td>
 
-
</tr>
 
-
 
-
</table>
 
-
 
-
 
-
 
-
<p>After having proven the presence of tPA expression in the cells transfected with Western Blotting, the concentration of the amount of tPA in the cell and the amount of secreted tPA were measured using the ‘Human tPA Activity Kit’ to show that the expressed proteins are functionally active.
 
-
 
-
</p>
 
-
<p>The real parameter of the measurement in the Assay was the product of the reaction of the plasmin enzyme. Since tPA shifts the inactive plasminogen to active plasmine, the measured value also presents tPA activity. The yield gives absorbance at 405 nm.
 
-
 
-
</p>
 
-
 
-
 
-
 
-
 
-
 
-
<h2>tPA ASSAY</h2>
 
-
<h3>Experimented</h3>
 
-
<img src="https://static.igem.org/mediawiki/2014/0/05/ATOMS-tpa_results10.png">
 
-
 
-
<p>According to the tPA assay results we obtained, we were able to prove that the tPA enzyme could be produced and secreted from the cell successfully hence also showing that tPA is  functionally active. 
 
-
 
-
</p>
 
-
 
-
 
-
<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 4); return false">[Collapse]</a></p>
 
-
</div>
 
-
 
-
<div class="technology">5. Aprotinin</div>
 
-
<div class="thelanguage">
 
-
<p>Aprotinin(Bovine Pancrease Tripsine Inhibitor) is a serine protease which inhibits 80% of the formation of superoxide by blocking the transformation of xanhtine dehidrogenase into xanthine oksidase.</p>
 
-
<img src="https://static.igem.org/mediawiki/2014/5/5b/ATOMS_Aprotinin_Results1.png">
 
-
<p>Aprotinin and pTRE vector was digested using EcoRI ve BamHI enzymes following by their ligation which cloned aprotinin into the pTRE vector. The isolated plasmid from the bacteria was then cut-checked using  EcoRI ve BamHI  enzymes and the following results were achieved:</p>
 
-
<img src="https://static.igem.org/mediawiki/2014/thumb/a/aa/ATOMS_Aprotinin_Results2.png/796px-ATOMS_Aprotinin_Results2.png">
 
-
<h3>Aprotinin Assay</h3>
 
-
<p> As Aprotinin is a serine protease inhibitor aprotinin activity can be measured using the correlation of serine protease inhibition the aprotinin inhibits.</p>
 
-
<img src="https://static.igem.org/mediawiki/2014/5/54/ATOMS_Aprotinin_Results3.png">
 
-
<p>HEK 293T cell was transfected with 1 µg pTRE-Aprotinin and cells were obtained after 36 hours which continued with the isolation of proteins. Afterwards, the isolated protein was placed into a tube containing the serine protease and its activity was measured. </p>
 
-
<p>The graphic shows that serine protease inhibition was not observed in the isolated proteins of the cells transfected with the pTRE vectors and our negative controls only.</p>
 
-
<p>The inhibition values obtained by adding 6 mg/dl Bovine Aprotinin(sigmaA1153) samples obtained by cattles and the cotransfected Tet off – pTRE Aprotinin, show that the transfected HEK 293 T cells perform a lower amount of inhibition compared to our standard sample however that is a very minimal difference.  As a result, our transfected aprotinin has accomplished more inhibition than our negative control and has proved that it can pe produced functionally. Afterwards the HEK 293 T cells cotransfected with Tet off – pTRE Aprotinin have been transfected in different amounts to characterise and measure its serine protease activity. </p>
 
-
<h3>Characterisation</h3>
 
-
<img src="https://static.igem.org/mediawiki/2014/thumb/7/71/ATOMS_Aprotinin_Results4.png/703px-ATOMS_Aprotinin_Results4.png">
 
-
<p>HEK 293T cells  have been cotransfected with 1 µg, 2 µg, 3µg ve 4 µg Tet off – pTRE Aprotinin. The cells collected and protein isolation was performed to measure the serine protease inhibition. According to the positive control test, these results Show dose-dependent reduction and the production of aprotinin has increased according to the amount of cells successfully transfected.</p>
 
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<p style="text-align:right;font-size:1.3em;"><a href="" class="collapseLink" onClick="ddaccordion.collapseone('technology', 5); return false">[Collapse]</a></p>
 
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Revision as of 03:40, 18 October 2014

At A Glance

    This year, we have disseminated two global health problems: heart attacks and strokes, and their viable clinical solutions. Due to the intricate components of the present mechanisms and treatments of such clinical methods we have set forth a project which has an ample perspective and approach, together with our policy & practice activities.
    Here, you can take a look at the main components of our project design.

The Problem

  • Ischemia is the restriction of blood supply to tissues, generally caused by the blockage of a clot due to the reduction or inhibition of blood flow. This process results in the shortage of oxygen and nutrition vital for metabolism and survival of cells. Since oxygen is delivered to tissues only via the blood stream, incommensurate blood supply causes tissue cells to spurn. Particularly for the heart and brain, irreversible damage is parlously supposable to occur in as little as 3–4 minutes at body temperature.
  • Ischemia is said to be the major factor in many of the medical problems of today. The famous heart attack, which is culpable for most of the deaths worldwide, is, as a matter of fact, an ischemic condition. In addition to this, the loss of a specific region of the brain due to inadequate blood perfusion is associated with ischemia and is widely known as stroke.In fact, every year, about 1.5 million Americans have heart attacks resulting in 500,000 deaths. A heart attack occurs every 20 seconds, which is equivalent to loss of a life approximately every single minute.
  • As it can be expected, one treatment of ischemia is the extermination of the blockage. Nonetheless, such a removal has the likeliness of making the situation more radical. In the current day, it is possible to dissolve the elements of the clot with specific medication such as tissue adaption. However, the tissue adapted to hypoxic media may be poisoned from oxygen. The absence of oxygen and nutrients from the blood during the ischemic period creates a state in which the rejuvenation of circulation to its original state results in inflammation and oxidative damage due to the induction of oxidative stress instead of the restoration of normal function.
  • The state of oxidative damage may cause some unwilling reactions which have the aptitude to produce toxic chemicals called reactive oxygen species (ROS). ROS are intermediate products of oxygenation which have an absence of one electron in their covalent bonds. This makes them ultimately unstable. With such instability, ROS can disrupt almost all cellular structures, including DNA, cell membranes and organelles. Due to such elements in the nature of ischemic conditions, the final solution should incorporate a solution for all negative outcomes caused by ischemia

Our Approach

  • Our project design aims to assess and resolve two distinct cases. If a more holistic approach is to be taken towards ischemia, the issue could be better combatted. For this reason, we intend to use our vessel cells as a route to act in time to prevent the consequences of ischemia and heal them. To do this, we have to sense hypoxic conditions to prevent or remove clot formation and oxidative stress factors.
  • Regarding the need of an efficient, cost effective and rapid treatment option, the approach to the issue was dissected into modules. Detection devices and activist proteins. Hypoxia will be detected in two levels; with a promoter device for detection in genomic level and a protein domain for protein level regulation. Also, the oxidant factors in the media will be sensed to empower sensor devices. These devices will then produce the activist proteins, which are clot dissolving agents, antioxidant enzymes and proteins. We have the prospect of issuing a novel approach to the problem which will take effect in a short time interval, and affect strongly to prevent the recurrence of the problem.

Future Aspects

  • Gene therapy and tissue engineering are two different, but promising therapies which relate to our project. We have deliberated the advantages, disadvantages and also the possible side effects, ethical or social issues of these therapies to determine which of these would be the most beneficial.
  • Furthermore, we have designed the real-life application of our project to address the needs of both the clinic and the patient in a multiperspective manner.
  • As a final remark, we aim to feature computer modelling data to predict the potential effects of our treatment in order to determine its applicability as preventive medication. We envision that our system will detect hypoxia before it has fatal effects and interfere to prohibit the occurrence of more austere conditions without causing any dismay in the target patient.

Achievements

  • We successfully propose a promising, novel approach and system design to treat ischemia, a common and lethal public health issue.
  • We have created a system which aims to treat ischemia: the number one cause of deaths worldwide.
  • We successfully worked all of our seven parts in our engineered eukaryotic cells.
  • We have accomplished modeling our gene expression dynamics and have also come up with the unique idea of modelling our safety experiment which enables us to prove graphically that we are able to switch our system off in the presence of a simple antibiotic such as tetracycline.
  • We collaborated with the METU iGEM team and exchanged thoughts on how we can improve our modelling.
  • We will hold a special metaphor event called ‘Think By Heart’ during the Giant Jamboree where we discover the unknowns of our heart simply beginning from its etymology to the cardiac cycle and how it is bizarrely similar to us human beings in terms of its style of functioning and many more.Have created a system which aims to treat ischemia: the number one cause of deaths worldwide.

Policy & Practices

  • Apart from our project design, we also wanted to engender a better approach on ischemic conditions and diseases by working on human practice and social issues. This year, we preferred to conduct an inclusive perspective and activity to address every aspect of the issue as well as all the stakeholders who are connected with it. Therefore, we separated our P&P design into four columns; all of which present a respective process and interactive strategy.
  • First, we need to analyze how intense the problem is and how common these ischemic diseases are among people, specifically in the iGEM community. This module is the “Identification” module; which intends to identify the parameters of the problem, in order approach the problem accurately.
  • AAfterwards, our aim is to undergo some tasks to raise awareness and warn people who are at high risk from ischemic diseases such as heart attacks. We plan to present our “identification” data inquired from the public in an influential way. The data includes information such as the total calories consumed by individuals. This comprises our second column, “clarification”.
  • Thirdly, we need to be accurate and effective in influencing the stakeholders and improving public health. To do this, we chose the “specialization” step to consult experts, doctors, specialists and related stakeholders of sectors and describe what had have been done so far and inquire what could further be conveyed.
  • LLastly, all the acquired data and knowledge could be utilized to form a product. However, we must obstruct the possible consequences and side effects of this product. The public should also be informed to prevent any social reaction. Therefore, we have conducted extensive research and performed several brain-storming sessions to depict the applicability of the product, and this is named as the “application” step.

Collaboration

  • For collaboration, we invited Paris Bettencourt iGEM team to the International Turgut Ozal Medical Congress, which our university hosts. The congress has the highest number of attendants in between medical congress’ across the nation. During the congress, together with the Paris Bettencourt team, a workshop on synthetic biology was organized. The unlimited potential that synthetic biology bears was delved into.
  • Secondly, we invited METU Turkey iGEM team to our university for dinner. We presented our projects respectively and discussed some ideas regarding our projects. We also debated on how we can help each other and what we can do for collaboration purposes or P&P. METU Turkey iGEM team requested to use some of the parts present in our iGEM kit plates for their project, which we gladly accepted.