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<h1 class="style1" style="font-size: 50pt"> PROJECT </h1>
 
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<h2 style="color: #E7BB21"> .DESCRIPTION</h2>
 
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<p>Our  project is to construct a biological device that can be used to detect the  existence of <em>Streptococcus Pyogenes (S.  Pyogenes)</em> in a cheaper and faster way in comparison to currently used  techniques. The construct can be used as a detecting device for bacteria or  virus secreting protease by modifying the cleavage sequence in the array (for  more information see parts). The name of the project comes from &lsquo;<strong>Dia</strong>gnosing <strong><em>S.</em></strong><em> Pyo<strong>genes</strong></em>&rsquo;.<br />
 
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  <em>Streptococcus Pyogenes</em>, also  known as group A streptococci (GAS), is a Gram-positive pathogen responsible  for a wider variety of human disease than any other bacterial species including  pharyngitis (streptococcal sore throat), scarlet fever, impetigo, erysipelas,  cellulitis, septicemia, toxic shock syndrome, necrotizing fasciitis  (flesh-eating disease) and the sequelae, rheumatic fever and acute  glomerulonephritis.1  The  complications of current GAS infections are severe; bacteremia associated with aggressive  soft tissue infection, shock, adult respiratory distress syndrome and renal  failure are common; 30% to 70% of patients die in spite of aggressive modern  treatments.2 Patients with symptomatic pharyngitis rarely develop  streptococcal toxic shock syndrome, though such cases have been reported,  especially in the last year. Numerous cases have developed within 24 to 72  hours of minor nonpenetrating trauma, resulting in hematoma, deep bruise to the  calf, or even muscle strain.3 <br />
 
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  There  are currently several test methods to detect the existence of <em>S. Pyogenes. </em>TheStrep A Rapid Test Device (SARTD) is considered to be fastest one  with detecting antigen in 5 minutes with the accuracy of 72%.4 However  SARTD is an expensive device that most health institutions have difficulty affording.  Therefore blood agar plate culture is prepared which requires a long time  interval (one to two days) to show the results.4 Testing on the same  day is important to reduce unnecessary antibiotic use and to prevent possible  complications caused by <em>S. Pyogenes</em>.  In our project we aimed to shorten the amount of time needed to detect <em>S. Pyogenes </em>while making the higher  speed test more affordable. </p>
 
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<p><img src="file:///C|/Users/ÖZTÜRK/Documents/Adlandırılmamış Site 2/project1.jpg" alt="3D computer-generated image of a Streptococcus Pyogenes. Content Provider(s): Center for Disease Control and Prevention/ Melissa Brower" width="1128" height="762" title="3D computer-generated image of a Streptococcus Pyogenes. Content Provider(s): Center for Disease Control and Prevention/ Melissa Browe"/></p>
 
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<pre style="font-family: 'Lucida Grande', 'Lucida Sans Unicode', 'Lucida Sans', 'DejaVu Sans', Verdana, sans-serif; font-size: 14px;">3D computer-generated image of a <em>Streptococcus Pyogenes. </em>Content Provider(s): Center for Disease  Control and Prevention/ Melissa Brower</pre>
 
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<h2 style="color: #E7BB21">&nbsp;</h2>
 
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<h2 style="color: #E7BB21">.PARTS</h2>
 
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<blockquote>
 
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  <h3> .OmpA-SpeB_Cleavage_Site</h3>
 
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<p>We  accomplish the task of detecting the existence of <em>S. Pyogenes</em> with the base part OmpA-SpeB_Cleavage_Site.Research was conducted to discover the  proteins secreted by <em>Streptococcus  Pyogenes</em>; results showed that SpeB is one of the main virulence factor of <em>S. Pyogenes, </em>which is a cysteine  proteinase functioning protein secreted by the bacteria.5 After  further investigation on the mechanism of SpeB, we discovered that the amino  acid sequence is cleaved by SpeB.6 If there is <em>S. Pyogenes </em>in the medium, SpeBs secreted by <em>S. Pyogenes</em> split the amino acid sequence including SpeB cleavage  site, into two. By using this for our benefit, construct consisting of a cell  wall protein, linker sequence and SpeB cleavage site is designed. This is the  base part designed for <em>S. Pyogenes</em> detecting devices. To the end of this part, the protein, which will be used to  show that the part is cleaved, should be added. After the SpeB cleavage site is  cleaved, the amino acid sequence placed after the cleavage site can become free  and start action. (see part <strong>OmpA-SpeB_Cleavage_Site-xylE</strong>). <br />
 
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  The  cell wall protein keeps the construct on the cell wall, which helps to prevent  unnecessary transmission between the protein coming after the SpeB cleavage site  and the protein&rsquo;s reactants, if designed. The cell wall protein OmpA is  preferred, due to its abundant usage.7 The linker segregates the  OmpA and SpeB cleavage site and thus creating space for the SpeB to access the  cleavage site; the sequence of the linker is taken from the Imperial College  2010 project.8 The SpeB cleavage site is broken down in the  existence of the SpeB and splits the amino acids, thus unchaining the substance  from the cell wall. </p>
 
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<p><img src="file:///C|/Users/ÖZTÜRK/Documents/Adlandırılmamış Site 2/3partspic.jpg" width="1178" height="603" alt=""/></p>
 
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<p>&nbsp;</p>
 
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<blockquote>
 
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  <h3>.OmpA-SpeB_Cleavage_Site-xyIE</h3>
 
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</blockquote>
 
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<p>This part is constructed upon the base part <strong>OmpA-SpeB_Cleavage_Site</strong> by adding  Catechol 2,3-dioxygenase to the end of the sequence. <em>xylE</em>&nbsp;is thegene encoding the enzyme catechol-2,3-dioxygenase, which converts  catechol, a cheap colorless substance, to the bright yellow product  2-hydroxy-cis,cis-muconic semialdehyde, if provided with oxygen. The sequence  of xylE is taken from the partsregistry.9 In the existence of <em>S. Pyogenes</em>,  SpeB secreted by the bacteria splits the amino acid sequence from the cleavage  site, monomers of catechol-2,3-dioxygenase become liberated. Free monomers come  together and form the tetramer form to start activation. By using this part,  detecting organisms will be a lot easier. </p>
 
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<p><img src="file:///C|/Users/ÖZTÜRK/Documents/Adlandırılmamış Site 2/5partspic.jpg" width="1129" height="600" alt=""/></p>
 
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<h2><span style="color: #E7BB21">.MATH MODELLING</span></h2>
 
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<p>This part is constructed upon the base part <strong>OmpA-SpeB_Cleavage_Site</strong> by adding  Catechol 2,3-dioxygenase to the end of the sequence. <em>xylE</em>&nbsp;is thegene encoding the enzyme catechol-2,3-dioxygenase, which converts  catechol, a cheap colorless substance, to the bright yellow product  2-hydroxy-cis,cis-muconic semialdehyde, if provided with oxygen. The sequence  of xylE is taken from the partsregistry.9 In the existence of <em>S. Pyogenes</em>,  SpeB secreted by the bacteria splits the amino acid sequence from the cleavage  site, monomers of catechol-2,3-dioxygenase become liberated. Free monomers come  together and form the tetramer form to start activation. By using this part,  detecting organisms will be a lot easier. </p>
 
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<p>&nbsp;</p>
 
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<p>&nbsp;</p>
 
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<h3><span style="color: #E7BB21">.VISUAL MODELLING</span></h3>
 
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<p>This part is constructed upon the base part <strong>OmpA-SpeB_Cleavage_Site</strong> by adding  Catechol 2,3-dioxygenase to the end of the sequence. <em>xylE</em>&nbsp;is thegene encoding the enzyme catechol-2,3-dioxygenase, which converts  catechol, a cheap colorless substance, to the bright yellow product  2-hydroxy-cis,cis-muconic semialdehyde, if provided with oxygen. The sequence  of xylE is taken from the partsregistry.9 In the existence of <em>S. Pyogenes</em>,  SpeB secreted by the bacteria splits the amino acid sequence from the cleavage  site, monomers of catechol-2,3-dioxygenase become liberated. Free monomers come  together and form the tetramer form to start activation. By using this part,  detecting organisms will be a lot easier. </p>
 
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<p>&nbsp;</p>
 
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<h1><span style="color: #E7BB21">.RESULTS</span></h1>
 
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<p>&nbsp;</p>
 
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<p>&nbsp;</p>
 
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<p></p>
 
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<p>&nbsp;</p>
 
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<h1><span style="color: #E7BB21">.CONCLUSION</span></h1>
 
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<p>In the  end, we hope to produce a working OmpA-SpeB_Cleavage_Site-xylE construct along  with our base part OmpA-SpeB_Cleavage_Site. If we manage to show that the SpeB  cleavage part is working, we can easily detect <em>S. Pyogenes. </em>It is our hope that our construct will be able to  reduce the cost of rapid detecting systems and allow fast detections. In the  future, researches can use the benefits of our system to develop more effective  and faster methods for detection, helping patients to have more comfortable  treatment. </p>
 
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<p>&nbsp;</p>
 
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<h1><span style="color: #E7BB21">.REFERENCES</span></h1>
 
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<p>1. Ferretti, Joseph J. &quot;Complete Genome Sequence of an M1 Strain  of Streptococcus Pyogenes.&quot;&nbsp;<em>PNAS</em>&nbsp;98.8 (2001): 4658-663. Print. <br />
 
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  2. Stevens DL, Tanner  MH, Winship J, Swarts R, Reis KM, Schlievert PM, et al. Reappearance of scarlet  fever toxin A among streptococci in the Rocky Mountain West: severe group A  streptococcal infections associated with a toxic shock-like syndrome. N Engl J  Med 1989; 321:1-7.<br />
 
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  3. Dennis L. Stevens, Ph.D, M.D.  &quot;Streptococcal Toxic-Shock Syndrome: Spectrum of Disease, Pathogenesis,  and New Concepts in Treatment.&quot; <em>Emerging Infectious Diseases </em>1.3  (1995): 69-78. Print. <br />
 
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  4. Forward, Kevin R., David Haldane,  Duncan Webster, Carolyn Mills, and Diane Aylward. &quot;A Comparison between  the Strep A Rapid Test Device and Conventional Culture for the Diagnosis of  Streptococcal Pharyngitis.&quot;&nbsp;<em>Can J Infect Dis Med Microbiol</em>&nbsp;17.4 (2006): 221-23. Print. <br />
 
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  5. Nelson, DC, J. Garbe, and M. Collin.  &quot;Cysteine Proteinase SpeB from Streptococcus Pyogenes - a Potent Modifier  of Immunologically Important Host and Bacterial Proteins.&quot; (2011): n. pag.&nbsp;<em>PubMed</em>. Web. 12 June 2014.  &lt;http://www.ncbi.nlm.nih.gov/pubmed/22050223&gt;. <br />
 
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  6. Ender, Miriam, Federica Andreoni,  Annelies Sophie Zinkernagel, and Reto Andreas Schuepbach. &quot;Streptococcal  SpeB Cleaved PAR-1 Suppresses ERK Phosphorylation and Blunts Thrombin-Induced  Platelet Aggregation.&quot; (2013): n. pag. <em>PLOS</em>.  Web. 12 June 2014. <br />
 
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  7. &quot;P0A910 (OMPA_ECOLI).&quot;  (2014): n. pag.&nbsp;<em>UniProt</em>. Web. 12 June 2014. <br />
 
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  8. Imperial College London.  &quot;Parasight.&quot;&nbsp;<em>IGEM</em>. N.p., 2010. Web. 12 June 2014.  &lt;https://2010.igem.org/Team:Imperial_College_London/Parts&gt;. </p>
 
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<p>&nbsp;</p>
 

Revision as of 09:27, 17 June 2014