Difference between revisions of "Team:Georgia State/Design"

 
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<h1 style="color:#1F618D; text-align: center; font-size: 36px; line-height: 40px;">Background</h1>
 
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<h1 style="color:#ffffff; background-color:#1F618D;; -moz-border-radius: 15px; -webkit-border-radius: 15px; padding:15px; text-align: center; font-family: Trebuchet MS">What is Lipopolysaccharide?</h1>  <br>
 
Endotoxin is a Lipopolysaccharide (LPS) it consists of a core oligosaccharide, O-antigen a glycan polymer and the lipid A. The lipid A is a phosphorylated glucosamine disaccharide with multiple fatty acids and is the cause for endotoxin toxicity.  LPS are bacterial poisons and can impact numerous biological activities. When gram-negative bacteria enter the body a complement immune response is initiated. Once the cell wall and/or bacteria are destroyed a significant of endotoxins are released which can lead to endotoxemia the symptoms of which are vomiting, nausea, diarrhea, fever, disseminated intravascular coagulation, vascular collapse, organ failure and possibly death. Antibiotics will not inactivate the endotoxins, therefore detection of the endotoxin before they enter the body is prudent.<br>
 
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<h1 style="color:#ffffff; background-color:#1F618D;; -moz-border-radius: 15px; -webkit-border-radius: 15px; padding:15px; text-align: center; font-family: Trebuchet MS">What do horseshoe crabs have to do with endotoxin?</h1> <br>
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                    <h1><i><font style="text-transform: none;">"As soon as your brain starts telling you that you can't have a tree that is blue then you stop being able to paint trees."</font></i></h2>
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                    <h2>Steven Johnson</h4>
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<img src="https://static.igem.org/mediawiki/2017/c/cf/T--Georgia_State--horseshoebranfromaquarium.jpg" alt="Horseshoe Crab in Aquarium" width="250" height="250" style="float:right;">  
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<b> Limulus polyphemus (Atlantic horseshoe crab)</b> is protected against infection by their immune system and blood coagulation system. They use hemocyanin to carry oxygen instead of hemoglobin. The blue color of their blood is due to the presence of copper in hemocyanin. The amebocytes (blood cells) are similar to white blood cells. Inside the amebocytes are proteins that are released in response to unwanted organisms like gram-negative bacteria. These proteins bind to and inactivate endotoxin. Assistance in wound control is moderated by components of their blood which prevent bleeding and form a physical barrier against additional infection. <br>
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In the presence of endotoxin, a clotting cascade is invoked to activate the proclotting enzyme which is used to transform coagulogen into coagulin. The zymogen Factor C is a glycoprotein that is 123kD, and the only enzyme that is endotoxin sensitive. It consists of an H chain (80kD) and L chain (43kD). Factor C activates in the presence of LPS and undergoes autocatalysis, the phenylalanine- isoleucine bond on the L chain is cleaved resulting in a B chain (34kD) and an A chain (8.5 kD). Factor C then activates Factor B which activates the proclotting enzyme. An activated proclotting enzyme is called the clotting enzyme which converts coagulogen into coagulin. <br>
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<h1 style="color:#ffffff; background-color:#1F618D;; -moz-border-radius: 15px; -webkit-border-radius: 15px; padding:15px; text-align: center; font-family: Trebuchet MS">Limulus Factor C cDNA Design</h1>  <br>
<img src="https://static.igem.org/mediawiki/2017/9/9a/T--Georgia_State--FactorCcascade.jpg"  alt="Cotting Cascade" width="300" height="300" style="left;">
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<img src="https://static.igem.org/mediawiki/2017/1/1d/T--Georgia_State--factorcdiagram.jpg"  alt="Factor C diagram" width="250" height="250" style="float:right;">  
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<b>Limulus Factor C </b>is a relatively large protein (1020 amino acids), so the synthesis of its coding sequence presented a challenge. We determined that instead of trying to have the entire construct synthesized as one piece it would be more cost effective to build it in pieces. The coding sequence for Limulus Clotting Factor C (LFC) was designed as four contiguous blocks to be joined together via overlap extension PCR. Three of the four blocks were 800 bp long, the fourth block was 660 bp long. We designed primers for overlap extension PCR to join the g-blocks together into the final Factor C cDNA sequence. Primers were designed in both forward and reverse complement to the top and bottom strands of the blocks. For an example, the forward primer of Block 2 was created with a few base pairs from Block 1- 5’ top strand end. These base pairs were added to the beginning of the 5’ top strand to start amplification of Block 2- 5’ top strand. The reverse complement primer for Block 1 was created by taking a few base pairs from the end of the 3’ bottom strand of Block 1 and adding it to the 5’ bottom strand of Block 2. The goal of creating these primers is to create overhangs from the previous block to bind to the next block. During amplification the blocks would then be joined and extend the 5’ top strand and 3’ bottom strand of each block. Once all 4 blocks and a total of 8 primers were created, we began PCR amplification of Blocks 1 and 2 and then amplified Blocks 3 and 4. The fusion of the coupled blocks would then be fused together to make the final product, LFC Blocks 1-4 via overlap extension PCR. (LFC Blocks 1-4 fusion illustrated in the image below to the right).
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<center><img src="https://static.igem.org/mediawiki/2017/2/25/T--Georgia_State--7.20OverlapFACTORCMAP.jpg"  alt="Horseshoe Crab in Aquarium" width="800" height="250" style="float:right;"></center>
  
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<center><img src= "https://static.igem.org/mediawiki/2017/d/d1/T--Georgia_State--7.20OverlapDIAGRAM.jpg" alt="Clotting Cascade" width="300" height="100" style="left;"></center>
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<b> PCR Overlap Extension</b>
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Due to the expense of purchasing the entire Factor C sequence as a new part and the opportunity to attempt a method that has never been done in the Georgia State University iGEM lab Factor C is planned to be synthesized via overlap extension PCR. A modified protocol written by Ichiro Matsumura was used. This method uses PCR to recombine DNA sequences instead of using restriction sites. By using a 3'-end primer that matches each template block and a 5'-end primer that matches a part of the block sequence and a part of the new block sequence. The two blocks can be recombined using DNA polymerase. The basic mechanism for overlap begins with a PCR to generate the two fragments (AB and CD)  that have ends modified by mispriming so that they have homologous regions. Through mixture, denaturation, and annealing the AB 3'-end will anneal onto the 3'-end of the bottom strand of the CD. Extension of the overlap product is used to form the recombinant product (the overlapping ends of the fragments are created by primers b and c while a and d match the individual fragments). The method can be repeated to add together more than two DNA fragments.
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The image above and to the left is a figure to illustrate the mechanism described above.
  
<h1 style="color:#ffffff; background-color:#1F618D;; -moz-border-radius: 15px; -webkit-border-radius: 15px; padding:15px; text-align: center; font-family: Trebuchet MS"> What is LAL assay? </h1>   
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<h1 style="color:#ffffff; background-color:#1F618D;; -moz-border-radius: 15px; -webkit-border-radius: 15px; padding:15px; text-align: center; font-family: Trebuchet MS">Human Chorionic Gonadotropin</h1>  <br><br>
<img src= "https://static.igem.org/mediawiki/2017/2/24/T--Georgia_State--LALcascadeandBLUEblood.jpg" alt="Clotting Cascade" width="300" height="300" style="float:right;">
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Limulus Amebocyte Lysate test is an extract of the Limulus polyphemus (Atlantic horseshoe crab) blood cells (amoebocytes) it detects small concentrations of endotoxin.  Horseshoe crabs are bleed through the pericardium. A third of their blood is taken and they are released back into the water. Through centrifugation, their blood cells are separated from the serum. In order to release the chemicals from inside the blood cells, they are placed in distilled water where they burst and form the lysate. Once the lysate is made the test becomes simple. A sample is mixed with lysate and water. If endotoxin is present if coagulation transpires.
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An injectable healthcare product like a vaccine and any other healthcare product like implantables that come in contact with a patients blood or cerebrospinal fluid must be sterile. However, the process to kill bacteria results in the release of endotoxin into the product because the cell wall can withstand steam sterilization. The products must be tested for endotoxin before use.
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<img src= "https://static.igem.org/mediawiki/2017/5/52/T--Georgia_State--horseshoecrabbleeding.jpg" alt="horseshoe crab bleeding" width="350" height="450" style="float:right;">
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<center><img src="https://static.igem.org/mediawiki/2017/f/f5/T--Georgia_State--recombiantHCGPCRsufixpreffix_.jpg"  alt="Horseshoe Crab in Aquarium" width="800" height="250" style="float:center;"></center><br><br>
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When designing this part we used the recombinant<b> human chorionic gonadotropin </b> cDNA sequence, sequenced by The National Institutes of Health Mammalian Gene Collection (MGC) Program . While the same amino acids are present, the cDNA sequence is altered. To detect the activation of Factor C autocatalysis, a hCG-beta subunit sequence component was a necessary addition to our final Factor C cDNA sequence. Similar to the primers designed for Factor C, we designed a forward and a reverse complement primer with iGEM prefix and suffix to join Block 4 of Factor C and Block 5 of hCG-beta subunit together. For our idea of a detection system, we plan to use a pregnancy test to detect the recombinant hCG-beta subunit once Factor C is activated.
 
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<h1 style="color:#ffffff; background-color:#1F618D;; -moz-border-radius: 15px; -webkit-border-radius: 15px; padding:15px; text-align: center; font-family: Trebuchet MS">Factor C-hCG Fusion </h1> <br>
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<center><img src="https://static.igem.org/mediawiki/2017/c/c4/T--Georgia_State--FactorCfusion.jpg"  alt="Horseshoe Crab in Aquarium" width="800" height="250" style="float:right;"></center>
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The basic mechanism for overlap begins with a PCR to generate two fragments (Block 1 and Block 2) that have ends modified by mispriming so that they have homologous regions. Through mixture, denaturation, and annealing of Block 1 5'-end of the top strand will anneal onto the 3'-end of the bottom strand of Block 2. Extension of the overlap product is used to form the recombinant product (the overlapping ends of the fragments are created by the forward primers and reverse complement primers to match the individual fragments). The method can be repeated to add together more than two DNA fragments (Block 3 and Block 4 + hCG-beta subunit). Together, all 5 g-blocks and a total of 10 primers would be joined via overlap extension PCR. If this joining of LFC blocks 1-4 and hCG-beta subunit block 5 is successful, the final product of <b>Limulus Clotting Factor C + hCG-beta subunit sequence</b> would be used as our complete detection system.
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<h1 style="color:#ffffff; background-color:#1F618D;; -moz-border-radius: 15px; -webkit-border-radius: 15px; padding:15px; text-align: center; font-family: Trebuchet MS">So where does GSU come in to play? </h1> 
 
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Using horseshoe crab blood is an unsustainable practice due to the impact on the crabs and the expense of the LAL assay. A third of the blood is taken from the horseshoe crab before they are realsed back into the water. The theory is that the crabs eventually heal however up to 30% of bled crabs die. The total population is decreasing rapidly while producers are forced to increase harvests to keep up with global demand. The LAL test is expensive to make; a quart of blood is sold for $15,000. Part of affordable healthcare means that the production of the products used also need to be affordable. <br>
 
Creating an alternative form of testing is unavoidable. Instead of creating a recombinant version of the entire clotting cascade it is more efficient to create a recombinant version of factor c and include a detection mechanism to detect the autocatalysis in the presence of LPS.  Human chorionic gonadotropin (hCG) beta subunit is detected through a human pregnancy test. Using the subunit in tandem with the pregnancy test allows for an efficient and inexpensive autocatalysis detection method. The goal of our project is to create a fusion protein of Factor c with hCG and immobilize it in a solution. When the LPS is exposed to the factor c it will undergo autocatalysis and release hCG into the solution where it is then detected by the pregnancy test. <br>
 
  
<img src= "https://static.igem.org/mediawiki/2017/4/4b/T--Georgia_State--factorcdiagrampart1.jpg" alt="factorc1" width="350" height="450" style="float:left;">
 
<img src= "https://static.igem.org/mediawiki/2017/a/a3/T--Georgia_State--factorc2.jpg" alt="factorc1" width="350" height="450" style="float:right;">
 
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<b>References</b><br>
 
<b>References</b><br>
- - -B. Akbar John, K.C.A. Jalal, Y.B. Kamaruzzaman and K. Zaleha, 2010.<i>Mechanism in the Clot Formation of Horseshoe Crab Blood during Bacterial Endotoxin Invasion (July 10, 2010). Journal of Applied Sciences, 10: 1930-1936. Retrieved June 11, 2017, from</i><a http://scialert.net/fulltext/?doi=jas.2010.1930.1936">http://scialert.net/fulltext/?doi=jas.2010.1930.1936</a> <br>
 
  
- - - <i>Elizabeth Cox. (2017,September, 21). Why do we harvest horseshoe crab blood?  [Video file]. </i> <a href="https://www.youtube.com/watch?v=VgEbcQxFUu8/"> https://www.youtube.com/watch?v=VgEbcQxFUu8/</a> <br>
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- - -Horton, R. M., Cai, Z., Ho, S. N., Pease, L. R., & Mayo Clinic. (2013). <i>Gene Splicing by Overlap Extension: Tailor-Made Genes Using the Polymerase Chain Reaction. BioTechniques, 54(3), 129-130 . doi:10.2144/000114017</i><a https://www.biotechniques.com/multimedia/archive/00190/BTN_A_000114017_O_190204a.pdf ">https://www.biotechniques.com/multimedia/archive/00190/BTN_A_000114017_O_190204a.pdf </a> <br>
  
- - - <i>Ecological Research & Development Group. (2013). <br> Horseshoe Crabs and Endotoxin Testing . . Retrieved October 31, 2017, from </i> <a href="http://www.horseshoecrab.org/med/sustainable.html"> http://www.horseshoecrab.org/med/sustainable.html</a><br>
 
  
- - - <i>Ecological Research & Development Group. (2013). [Copper Based "Blue blood" and Cascade of Enzymes and Proteins ]. </i> JRetrieved October 31, 2017, from<br> <a href="http://www.horseshoecrab.org/med/testing.html">http://www.horseshoecrab.org/med/testing.html</a><br>
 
  
- - - <i>PBS. (2014, February 26). A still for the PBS Nature documentary Crash [Digital image]. Retrieved October 31, 2017, from <br><a href="https://www.theatlantic.com/technology/archive/2014/02/the-blood-harvest/284078/">https://www.theatlantic.com/technology/archive/2014/02/the-blood-harvest/284078/</a><br>
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- - - <i>CGB protein [Homo sapiens] - Protein - NCBI. (2003, October 7).<br> Retrieved July 7 2017, from </i> <a href="https://www.ncbi.nlm.nih.gov/protein/26996824?report=genbank&log%24=protalign&blast_rank=1&RID=PU2MZS8Z014"> https://www.ncbi.nlm.nih.gov/protein/26996824?report=genbank&log%24=protalign&blast_rank=1&RID=PU2MZS8Z014</a><br>
  
 
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- - - <i>P28175 (LFC_TACTR) Tachypleus tridentatus (Japanese horseshoe crab). (n.d.). Retrieved June 21, 2017, from
 
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</i> <br> <a href=" https://swissmodel.expasy.org/repository/uniprot/P28175?csm=5BC2864C6715289B"> https://swissmodel.expasy.org/repository/uniprot/P28175?csm=5BC2864C6715289B</a><br>
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Latest revision as of 03:37, 2 November 2017