Team:Colorado State/Safety

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'preview' : 'site'; var googleAnalytics = ""; </script>  <script type="text/javascript"> var adData = {"topLabel":"<span class=\"smallMusa\">(Wix-Logo) </span>Get a <span class=\"smallLogo\">Wix</span> site!","topContent":"Beautiful designs<br />No coding needed<br /><span class=\"emphasis spacer\">Get started now ></span>","footerLabel":"<div class=\"adFootBox\"><div class=\"siteBanner\"><div class=\"siteBanner\"><div class=\"wrapper\"><div class=\"bigMusa\">(Wix Logo)</div><div class=\"txt shd\">Create your stunning website with </div><div class=\"logoDot\">Wix.</div><div class=\"txt shd\"> It's super easy and free! <span class=\"emphasis\">Go></span></div></div></div></div>","adUrl":"http://www.wix.com?utm_campaign=vir_wixad_live&experiment_id=3073daeb-1138-41e7-aa9b-1adf82059d74"}; var usersDomain = "https://users.wix.com/wix-users" ; var wixData = {"document_data":{"image1z3f":{"type":"Image","id":"image1z3f","metaData":{"isPreset":true,"schemaVersion":"1.0","isHidden":false},"target":"","linkType":"FREE_LINK","href":"","title":"I'm a title","uri":"84770f_433691bbff25c6ae37b2ab6a93ad1296.jpg","description":"I'm a description. Click to edit me","width":1280,"height":960,"alt":""},"#Linkfty":{"type":"TextLink","id":"#Linkfty","metaData":{"isPreset":true,"schemaVersion":"1.0","isHidden":false},"target":"_self","linkType":"PAGE","href":"#!Contact/c1ttv"},"c1pep":{"type":"Page","id":"c1pep","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"title":"Protocols","hideTitle":true,"icon":"","descriptionSEO":"","metaKeywordsSEO":"","pageTitleSEO":"","pageUriSEO":"blank","hidePage":false,"underConstruction":false,"tpaApplicationId":0},"c4ci":{"type":"Page","id":"c4ci","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"title":"Safety","hideTitle":true,"icon":"","descriptionSEO":"","metaKeywordsSEO":"","pageTitleSEO":"","pageUriSEO":"safety","hidePage":false,"underConstruction":false,"tpaApplicationId":0},"c18ye":{"type":"RichText","id":"c18ye","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"text":"<p>\n\t<span class=\"font_2\">More Than A Great Beer</span></p>\n","defaultStyle":"font_2"},"#Link1m78":{"type":"TextLink","id":"#Link1m78","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"target":"_blank","linkType":"WEBSITE","href":"http://ginkgobioworks.com/support/"},"c1zwy":{"type":"Page","id":"c1zwy","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"title":"Modeling ","hideTitle":true,"icon":"","descriptionSEO":"","metaKeywordsSEO":"","pageTitleSEO":"","pageUriSEO":"modeling-","hidePage":false,"underConstruction":false,"tpaApplicationId":0},"c1ttv":{"type":"Page","id":"c1ttv","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"title":"Parts","hideTitle":true,"icon":"","descriptionSEO":"","metaKeywordsSEO":"","pageTitleSEO":"","pageUriSEO":"contact","hidePage":false,"underConstruction":false,"tpaApplicationId":0},"c23d0":{"type":"RichText","id":"c23d0","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"text":"<p>\n\t<span class=\"font_6\">Ethan Ho</span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">A young up-and-coming </span></span><span class=\"font_6\"><span class=\"color_2\">tech expert, Ethan has been writing code since you were in diapers. You can expect big things from this kid. Before you know it, he'll have us all saying Mark Zucker...WHO?</span></span></p>\n","defaultStyle":"font_6"},"image1vw":{"type":"Image","id":"image1vw","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"target":"","linkType":"FREE_LINK","href":"","title":"","uri":"5bd9eb_9ddd9ed386384c28f94f53261ba076db.jpg","description":"","width":1920,"height":1080,"alt":""},"c1ng2":{"type":"Page","id":"c1ng2","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"title":"Dave Packard","hideTitle":true,"icon":"","descriptionSEO":"","metaKeywordsSEO":"","pageTitleSEO":"","pageUriSEO":"dave-packard","hidePage":false,"underConstruction":false,"tpaApplicationId":0},"image212v":{"type":"Image","id":"image212v","metaData":{"isPreset":true,"schemaVersion":"1.0","isHidden":false},"target":"","linkType":"FREE_LINK","href":"","title":"I'm a title","uri":"84770f_7d8b729f23507fe45afa5bf60412cfc5.jpg","description":"I'm a description. Click to edit me","width":800,"height":982,"alt":""},"c1zf0":{"type":"Image","id":"c1zf0","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"target":"","linkType":"FREE_LINK","href":"","title":"","uri":"5bd9eb_7f9d6789b9321b56e3c60655841226cf.jpg","description":"","width":1440,"height":1920,"alt":""},"component_10457":{"type":"ImageList","id":"component_10457","metaData":{"isPreset":true,"schemaVersion":"1.0","isHidden":false},"items":["#component_items_0_27497","#component_items_1_78671","#component_items_2_78748"]},"ccd":{"type":"RichText","id":"ccd","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"text":"<p>\n\t<span class=\"font_8\"><span class=\"color_26\"></span></span><span class=\"font_8\"><span class=\"color_26\">We set out on a mission to design a yeast that secretes an enzyme to break down gluten. Come on, don't act like you're not impressed.</span></span></p>\n","defaultStyle":"font_6"},"c1m3p":{"type":"Page","id":"c1m3p","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"title":"Sponsors","hideTitle":true,"icon":"","descriptionSEO":"","metaKeywordsSEO":"","pageTitleSEO":"","pageUriSEO":"gallery","hidePage":false,"underConstruction":false,"tpaApplicationId":0},"c1d2p":{"type":"RichText","id":"c1d2p","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"text":"<p>\n\t<span class=\"font_6\">Guy Stewart</span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\"></span></span><span class=\"color_2\"></span><span class=\"font_6\"><span class=\"color_2\">He claims he's just a regular guy, but we don't believe him. Guy is the embodiment of natural biology and you can expect to see him on the cover of Home Brewer magazine any day now.</span></span></p>\n","defaultStyle":"font_6"},"c1x1l":{"type":"RichText","id":"c1x1l","metaData":{"isPreset":true,"schemaVersion":"1.0","isHidden":false},"text":"<p>\n\t<span class=\"font_2\">Latest Projects</span></p>\n","defaultStyle":"font_2"},"c13x":{"type":"Page","id":"c13x","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"title":"Notebook","hideTitle":true,"icon":"","descriptionSEO":"","metaKeywordsSEO":"","pageTitleSEO":"","pageUriSEO":"news","hidePage":false,"underConstruction":false,"tpaApplicationId":0},"c13v9":{"type":"RichText","id":"c13v9","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"text":"<p>\n\t<span class=\"font_5\"><span class=\"color_26\">RamStrong</span></span></p>\n","defaultStyle":"font_2"},"c1vq3":{"type":"RichText","id":"c1vq3","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"text":"<p>\n\t<span class=\"font_6\"><span class=\"color_2\">Yeast System<br>\n\tYeast Vectors 1<br>\n\tYeast Vectors 2<br>\n\tA System of Shuttle Vectors and Yeast Host Strains Designed for Efficient Manipulation of DNA in Saccharomyces ceratisiae</span></span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">Yeast Integrative Plasmid Vectors (YIp)<br>\n\tGAL1 and GAL10 (galactose catabolism) selection<br>\n\tTRP1 selection</span></span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">Prokaryotic System</span></span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">Promoters<br>\n\tBBa-J23100 (strong)<br>\n\tBBa-J23108 (medium)<br>\n\tBBa-J23109 (weak)<br>\n\tTerminators<br>\n\tBBa-B0015<br>\n\tRBS<br>\n\tConstitutive</span></span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">Secretion<br>\n\tHlyA Gene Sequence<br>\n\tA novel C-terminal signal sequence targets Escherichia coli haemolysin directly to the medium<br>\n\tAnalysis of the haemolysin transport process through the secretion from Escherichia coli of PCM, CAT or beta-galactosidase fused to the Hly C-terminal signal domain<br>\n\tAnalysis of the haemolysin secretion system by PhoA-HlyA fusion proteins</span></span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">Kumamolisin Info<br>\n\t57 kDa (beta-galactosidase: 540 kDa; source)<br>\n\t1701 bp<br>\n\t567 aa</span></span><span class=\"color_2\"></span></p>\n","defaultStyle":"font_6"},"c20l4":{"type":"RichText","id":"c20l4","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"text":"<p>\n\t<a dataquery=\"#Linknef\"><span class=\"font_6\"><span class=\"color_2\">Quick Yeast Transformation Protocol</span></span></a><span class=\"color_2\"></span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">Here is a link to the general biobricks assembly manual <a dataquery=\"#Link1m78\">[1]</a></span></span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">BioRad Protein Expression and Purification Series<a dataquery=\"#Link4z\">[2]</a></span></span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">Synthetic Biology Overview [3]</span></span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">Restriction Digest for 3A Assembly [4]</span></span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">QIAGEN® QIAprep® Spin Miniprep Kit File:QIAprep Miniprep Handbook.pdf</span></span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi?ID=2019 Yeast Genomic DNA Isolation David Amberg</span></span><span class=\"color_2\"></span></p>\n<p>\n\t<span class=\"color_2\"></span><br>\n\t<span class=\"font_6\"><span class=\"color_2\">Analysis of gluten in foods by MALDI-TOFMS.[5]</span></span></p>\n","defaultStyle":"font_6"},"imagev04":{"type":"Image","id":"imagev04","metaData":{"isPreset":true,"schemaVersion":"1.0","isHidden":false},"target":"","linkType":"FREE_LINK","href":"","title":"I'm a title","uri":"84770f_182f6254df3464b7d8a9e994bc8f5cf0.jpg","description":"I'm a description. Click to edit me","width":1280,"height":960,"alt":""},"c20rx":{"type":"RichText","id":"c20rx","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"text":"<p>\n\t<span class=\"font_5\"><span class=\"color_26\">Contact Us</span></span></p>\n","defaultStyle":"font_2"},"c15lo":{"type":"SiteButton","id":"c15lo","metaData":{"isPreset":true,"schemaVersion":"1.0","isHidden":false},"target":"","linkType":"FREE_LINK","href":"","label":"Read more"},"#Linknef":{"type":"TextLink","id":"#Linknef","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"target":"_blank","linkType":"WEBSITE","href":"http://home.cc.umanitoba.ca/~gietz/Quick.html"},"c1urk":{"type":"SiteButton","id":"c1urk","metaData":{"isPreset":true,"schemaVersion":"1.0","isHidden":false},"target":"","linkType":"ADMIN_LOGIN","href":"","label":"Webmaster Login"},"i11y26":{"type":"Image","id":"i11y26","metaData":{"isPreset":true,"schemaVersion":"1.0","isHidden":false},"target":"other","linkType":"WEBSITE","href":"http://www.twitter.com/wix","title":"Wix Twitter page","uri":"82cbd7e2e002355b856f7c0f638820de.wix_mp","width":32,"height":32,"alt":""},"c1apg":{"type":"RichText","id":"c1apg","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"text":"<p>\n\t<span class=\"font_2\"><span color=\"color_0\">Safety in the Workplace</span></span></p>\n","defaultStyle":"font_6"},"c19px":{"type":"SiteButton","id":"c19px","metaData":{"isPreset":true,"schemaVersion":"1.0","isHidden":false},"target":"_self","linkType":"PAGE","href":"#!Landscaping|c24f1","label":"READ MORE"},"cj0l":{"type":"RichText","id":"cj0l","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"text":"<p>\n\t<span class=\"font_6\">Brian Searcy</span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">You name it, he's lived there. Our coach </span></span><span class=\"font_6\"><span class=\"color_2\">and mentor is a Renaissance Man of the microbiology world. If you have a question, he has an answer, and boy did that come in handy.</span></span></p>\n","defaultStyle":"font_6"},"crff":{"type":"RichText","id":"crff","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"text":"<p>\n\t<span class=\"font_6\"><span class=\"color_2\">Each student team member also used an individual notebook page to post their research and update their progress over the course of the project.</span></span><span class=\"font_6\"> <span class=\"color_2\">The links to these notebook pages can be found by holding your cursor over the notebook icon and then clicking the individual team member's link.</span></span></p>\n","defaultStyle":"font_6"},"c1qrs":{"type":"RichText","id":"c1qrs","metaData":{"isPreset":true,"schemaVersion":"1.0","isHidden":false},"text":"<p>\n\t<span class=\"font_5\"><span class=\"color_11\"><span color=\"color_5\">I'm Another title</span></span></span></p>\n","defaultStyle":"font_6"},"cnfy":{"type":"Page","id":"cnfy","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"title":"Ryan Clouse","hideTitle":true,"icon":"","descriptionSEO":"","metaKeywordsSEO":"","pageTitleSEO":"","pageUriSEO":"notebook","hidePage":false,"underConstruction":false,"tpaApplicationId":0},"i01fa9":{"type":"Image","id":"i01fa9","metaData":{"isPreset":true,"schemaVersion":"1.0","isHidden":false},"target":"other","linkType":"WEBSITE","href":"http://www.facebook.com/wix","title":"Wix Facebook page","uri":"e7e2a8374fe89d6ac16b130302c5d978.wix_mp","width":32,"height":32,"alt":""},"component_items_1_78671":{"type":"Image","id":"component_items_1_78671","metaData":{"isPreset":true,"schemaVersion":"1.0","isHidden":false},"target":"_blank","linkType":"WEBSITE","href":"http://www.twitter.com/wix","title":"w-twitter","uri":"4941bced49cf4de3e8d7ea1582892fb8.wix_mp","description":"","width":205,"height":205,"alt":""},"c1aii":{"type":"RichText","id":"c1aii","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"text":"<p>\n\t<span class=\"font_6\"><span class=\"color_2\">Stable/Genomic encoding</span></span><span class=\"color_2\"></span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">http://partsregistry.org/wiki/index.php?title=Part:BBa_K590023</span></span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">I am currently viewing Yeast integrative plasmid,(YIp), whhich are yeast vectors that utilize integration into the yeast chromosome for survival and are used when studying a DNA gene. http://en.wikipedia.org/wiki/Yeast_artificial_chromosome http://www.annualreviews.org/doi/pdf/10.1146/annurev.mi.37.100183.001345 http://www.pnas.org/content/75/4/1929.full.pdf http://nar.oxfordjournals.org/content/29/12/e59.full http://www.blackwellpublishing.com/genecloning/pdfs/chapter7.pdf (7.1.3) https://static.igem.org/mediawiki/2008/4/4e/JHU_0708_paper_Shuttle_Vectors_with_Multiple_Unique_Restriction_Sites.pdf</span></span><span class=\"color_2\"></span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">Vectors derived from the 2mm plasmid are called yeast episomal plasmids (YEps). Some YEps contain the entire 2mm plasmid, others include just the 2mm origin of replication. An example of the latter type is YEp13 (Figure 7.3). YEp13 illustrates several general features of yeast cloning vectors. First, it is a shuttle vector. As well as the 2mm origin of replication and the selectable LEU2 gene, YEp13 also includes the entire pBR322 sequence, and can therefore replicate and be selected for in both yeast and E. coli. There are several lines of reasoning behind the use of shuttle vectors. One is that it may be difficult to recover the recombinant DNA molecule from a transformed yeast colony. This is not such a problem with YEps, which are present in yeast cells primarily as plasmids, but with other yeast vectors, which may integrate into one of the yeast chromosomes (p. 135), purification may be impossible.This is a disadvantage because in many cloning experiments purification of recombinant DNA is essential in order for the correct construct to be identified by, for example, DNA sequencing. The standard procedure when cloning in yeast is therefore to perform the initial cloning experiment with E. coli, and to select recombinants in this organism. Recombinant plasmids can then be purified, characterized, and the correct molecule introduced into yeast (Figure 7.4). (1)</span></span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">(1) Yeast integrative plasmids (YIps) are basically bacterial plasmids carrying a yeast gene. An example is YIp5, which is pBR322 with an inserted URA3 gene (Figure 7.6(a)). This gene codes for orotidine-5¢-phosphate decarboxylase (an enzyme that catalyses one of the steps in the biosynthesis pathway for pyrimidine nucleotides) and is used as a selectable marker in exactly the same way as LEU2. A YIp cannot replicate as a plasmid as it does not contain any parts of the 2mm plasmid, and instead depends for its survival on integration into yeast chromosomal DNA. Integration occurs just as described for a YEp (Figure 7.5). (2) Yeast replicative plasmids (YRps) are able to multiply as independent plasmids because they carry a chromosomal DNA sequence that includes an origin of replication. Replication origins are known to be located very close to several yeast genes, including one or two which can be used as selectable markers. YRp7 (Figure 7.6(b)) is an example of a replicative plasmid. It is made up of pBR322 plus the yeast gene TRP1. This gene, which is involved in tryptophan biosynthesis, is located adjacent to a chromosomal origin of replication.The yeast DNA fragment present in YRp7 contains both TRP1 and the origin. (1)</span></span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">1. \"Chapter 7 Cloning Vectors.\" Gene Cloning. N.p.: n.p., n.d. 135-37. Http://www.blackwellpublishing.com/genecloning/pdfs/chapter7.pdf. 22 Aug. 2005. Web.</span></span></p>\n","defaultStyle":"font_6"},"cv41":{"type":"RichText","id":"cv41","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"text":"<p>\n\t<span class=\"font_2\">Our Gluten-Free Mission</span></p>\n","defaultStyle":"font_2"},"c1z00":{"type":"RichText","id":"c1z00","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"text":"<p>\n\t<span class=\"font_6\">Steven Denham</span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">The Houdini of the lab, Steven can do things in the lab that will have you wondering where you are and what just happened. And as though that wasn't enough, he's a research genius. He makes Post-Docs look like High School Freshman</span></span><span class=\"font_6\"><span class=\"color_2\">.</span></span></p>\n","defaultStyle":"font_6"},"c1ocy":{"type":"RichText","id":"c1ocy","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"text":"<p>\n\t<span class=\"font_2\">Our Proud Sponsors</span></p>\n","defaultStyle":"font_6"},"image1ttr":{"type":"Image","id":"image1ttr","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"target":"","linkType":"FREE_LINK","href":"","title":"","uri":"5bd9eb_2258f1d27b99d86f410ccadd47efec26.jpg","description":"","width":1279,"height":652,"alt":""},"c11wu":{"type":"ImageList","id":"c11wu","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"items":["#image1ttr","#image1vw","#image11ey","#imagegv0","#imaget0y"]},"MAIN_MENU":{"type":"Menu","id":"MAIN_MENU","metaData":{"isPreset":true,"schemaVersion":"1.0","isHidden":false},"items":[{"refId":"#mainPage","items":[]},{"refId":"#c1uzr","items":[]},{"refId":"#c1ttv","items":[]},{"refId":"#c13x","items":[{"refId":"#cnfy","items":[]},{"refId":"#c1cnb","items":[]},{"refId":"#c2449","items":[]},{"refId":"#c1ye4","items":[]},{"refId":"#c1ng2","items":[]},{"refId":"#c1pep","items":[]}]},{"refId":"#c1zwy","items":[]},{"refId":"#c4ci","items":[]},{"refId":"#c1m3p","items":[]},{"refId":"#c1n3s","items":[]}]},"c2449":{"type":"Page","id":"c2449","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"title":"Guy Stewart","hideTitle":true,"icon":"","descriptionSEO":"","metaKeywordsSEO":"","pageTitleSEO":"","pageUriSEO":"guy-stewart","hidePage":false,"underConstruction":false,"tpaApplicationId":0},"cw5o":{"type":"RichText","id":"cw5o","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"text":"<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">I am currently researching how to create a screen test for gluten in a beer sample. More specifically Hordein, the gluten protein found in barley which is a key ingredient in beer.</span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\"></span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">According to various group research we suspect there is a repeating sequence in both wheat and barley gluten proteins that will allow Hordein, the Gluten protein of barley, to be broken down in a similar manner to gliadins, the Gluten protein of wheat. This could potentially be conducted using an enzyme to break down the gluten protein, in a method similar to that used by the University of Washington in 2011's iGEM competition.</span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">here is a link on beer and hordein quantification: </span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">http://pubs.acs.org/doi/pdf/10.1021/pr2008434 https://2011.igem.org/Team:Washington/Protocols/Purified_Enzyme_Assay</span></span></span></p>\n<p>\n\t </p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">I am currently searching for a purified polypeptide sequence of gluten that we can use as a control for experimental basis. The University of Washington used a PQPQLP sequence with a fluorophore and quencher attached using a custom sequencing option from a company called Anaspec (Anaspec provided a 20% discount for educational research). According to Anaspec's website the average delivery time for a custome peptide strand is between 2 and 3 weeks. This could be problematic and another solution may need to be found.</span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\"></span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">I found today that CSU has a Lab dealing with DNA sequencing, I sent an email asking if they also did custom peptide sequencing. Their product turnaround is faster than many commercial biotechnology companies. Also, I found several other companies that have a quicker turnaround than Anaspec for custom sequencing. It turns out that CSU's department does not do custom peptide sequencing, but a company called biomatik is looking promising.</span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\"></span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">Here is a patent for a gluten assay that goes incredibly in-depth into the foudation of Gluten, its components and sequencing, its relation to celiac patients, etc. Very informing. </span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">http://www.patentstorm.us/patents/7534426/description.html</span></span></span></p>\n<p>\n\t </p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">(6/11/12) Over the weekend I read the reading for the journal club, requested quotes from two more companies on the custom peptide sequence and updated the goodle document. The reading was very informative and confirmed my other research that the sequence PQPQLP was primarily responsible for the digestive difficulties of Celiac patients. I received a repsonse from Biomatik stating that my quote could not be completed because of the quencher modification on the C-terminus. I am still awaiting a reply from three companies as of 6/11/12. I updated some gluten information on the google doc as well as the temperatures for which ale and lager are brewed. This allows us to be certain that our concept would work under actual brewing temperatures.</span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\"></span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">Here is a link with great information on yeast expression and secretion vectors. http://www.patentstorm.us/patents/5024941/fulltext.html</span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">I have done a lot of research on the definition of \"gluten-free\" according to various organizations and standards and it appears as though <20 ppm seems to be classified as \"glute-free.\" As something like our idea has never been done before, from my understanding we would qualify as \"gluten-free\" assuming we could meet the <20 ppm requirement.</span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\"></span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">Here is a patent for a genetically engineered strain of yeast that produces a gluten-free wheat protein that is indistinguishable from the actual protein. </span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">http://www.freepatentsonline.com/4826765.html</span></span></span></p>\n<p>\n\t </p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">Today I received my quote back from Anaspec with a time frame of 4-5 weeks for delivery. I returned their email with a response explaining the iGEM competition through CSU and our educational research purpose. I mentioned that UW's iGEM team from 2011 received a 20% discount and I asked if we could receive a similar deal. I also mentioned our time constraint and asked if there was anyway to receive the sample in a more timely fashion. I also emailed New Belgium to potentially setup a meeting to discuss the brewing process as well as our project idea.</span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\"></span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">Today I received the quote back from Anaspec and received a 20% discount on the previous quote, but the delivery time frame was still 4-5 weeks. This time frame is not acceptable given the limited window of the project. Therefore I am researching other fluorophore and quencher combinations offered by different biosynthetic companies. Here is a helpful link on how to choose quenchers and fluorophores. </span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">http://www.biosyn.com/faq.aspx?qid=303</span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\"></span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">Two companies, Biomatik and Genscript, got back to me today with other options for quencher and fluorophore combinations. Each company provided two combinations, with one of the combinations being the same from each company. Biomatik recommended 5-TARMA/5-FAM and DABCYL/Glu(EDANS)-NH2, while Genscript recommended Abz,Tyr(3-NO2) and DABCYL/Glu(EDANS)-NH2. The DABCYL/Glu(EDANS)-NH2 combination requires the addition of a Glucose chain attachment, but that does not appear to be a problem. I asked what the key differences in those combinations were from each company and am currently waiting a repsonse. We are also considering using a different assay while waiting for our custom peptide sequence. This assay is a 2D Silverstain gel and would potentially allow us to see is our enzyme is properly cleaving the PQPQLP sequence. I sent two quotes for purified PQPQLP sequences to Biomatik and Genscript to see if we could get a quicker and less expensive solution to our problem. I am currently waiting for a repsonse on those also.</span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\"></span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">I am going to start researching the qualifications for meeting \"gluten-free\" standards according to U.S. requirements. I am also going to do research on invertase as a yeast secretion solution.</span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\"></span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">There are no gluten-free regulatory definitions in place according to the FDA, but actions are under way to better define gluten-free. http://www.fda.gov/Food/LabelingNutrition/FoodAllergensLabeling/GuidanceComplianceRegulatoryInformation/ucm111487.htm#q8</span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\"></span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">I contacted the advisor and main contact (Dr. Mills) for the 2011 University of Washington iGEM team to see if they had any leftovers from last year's gluten project. We are piggybacking off many of their concepts and experiments for original purpose and it would likely save time and money if they were able to offer assistance in any form.</span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\"></span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">After conducting some initial research on invertase, it appears that most experimentation has been done using two strains of Saccharomyces called 303-67 and FH4C (mutant strain). The strains act differently according to the amount of glucose present in the media, as well as other fundamental differences.</span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\"></span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">I received a quote from GenScript for a purified gluten peptide sequence, but one of our advisors is concerned that in a gel run the fragments may be too small to distinguish from individual amino acids. The only solution appears to be to order the PQPQLP sequence with a quencher and fluorophore attached.</span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\"></span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">(6/15/12) I am still waiting for a response from both Biomatik and Genscript on the differences between the recommended quencher and fluorophore combinations (other than price). Both companies claimed I would be hearing back today at some time. After doing more research on the DABCYL/Glu(EDANS)-NH2 quencher and fluorophore, I found many papers documenting the combination for various protease assays. From my understanding it would be a viable combination for use in our project. Here is a link about the combination's use in an experiment for various assays http://www.jimmunol.org/content/183/10/6708.full.pdf</span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\"></span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">Here is a link to various protocols needed to run the enzyme activity assay from the University of Washington. </span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">https://2011.igem.org/Team:Washington/Protocols</span></span></span></p>\n<p>\n\t </p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">Over the weekend I researched the possibility of using mass spec as an assay to measure enzymatic activity. After speaking with an expert at CSU, Don Dick, I was referred to Jessica Prenni who has experience in mass spec with beer. I emailed Dr. Prenni and am currently awaiting her response.</span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">I also received a quote back from Genscript and Biomatik, but it appears we are foregoing the route of the straight PQPQLP sequence for the quencher and fluorophore option. I suspect we may order a sample of gluten online if we decide to use mass spec as an option for monitoring enzymatic activity.</span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\"></span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">Here is a very informative link on the quantification of wheat gluten using mass spec. The publication also mentions that one of the best ways to characterize gluten proteins in wheat is using 2D gel electrophoresis. http://www.springerlink.com/content/11u4752uu71510v1/fulltext.pdf</span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">I have emailed two different companies about the possibility of a gluten ELISA assay sponsorship. On the quote I received back from one company, the difference in quencher and fluorophore combinations is the wavelength they emit. This however does not explain why some combinations would cost more than 3x as much others. I emailed GenScript asking what the differences were as well as asking them for the discounted rates.</span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\"></span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">(6/19/12) I called both Genscript and Biomatik today. With Biomatik I was only able to leave a message, so I will be waiting for them to call me back. I spoke with customer service at GenScript and was told I would receive a quote with the 15% discount applied, and that they would forward me the response on the differences between the quencher and fluorophores as described by their specialists. If the quote and description are adequate, we should be able to order the sequence today.</span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\"></span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">(6/20/12) We met with Chris Strickland yesterday and he tasked us with finding an appropriate set of equations to model collision rates within the reactions. Specifically, I am researching the gas collision theory to see if it can be applied to fluids. From what I understand it should be a valid set of equations to use on a liquid reactions, assuming some modifications are made. The following are links on collision theory: This link is the collision theory based primarily around gas reactions. http://en.wikipedia.org/wiki/Collision_theory This slide show reviews some of the derivation of the collision theory. http://www.powershow.com/view/51cd1-YTM4Z/Collision_Theory_flash_ppt_presentation This link shows some different variations of the collision theory equations. http://www.life.illinois.edu/crofts/bioph354/diffusion1.html</span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">I did some various derivations and modifications of collision theory equations to find a viable modeling system. If we start by assuming there is no activation energy (i.e. a reaction occurs with every collision) then my equations should prove sufficient. This would provide a starting ground to build an initial model of the reaction between the K (Kumamolisin) enzyme and 33-Mer sequence of Gluten. After having an initial model, we could then add more advanced components depending on the information available about our two components.</span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\"></span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">(6/20/12)We met with Dr. Prenni today about the possibility of using mass spec as an enzymatic assay. She said she thought there would be too many proteins to identify the one we wanted, and even more difficult to see if this specific sequence had been broken down. She instead recommended a Western Blot test. In order to perform this test we need an antibody that targets a specific binding site on the gliadin sequence.</span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\"></span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">(6/21/12) We have a meeting with Chris Strickland today to further discuss the modeling component of our project. We are first trying to model the collision rate between the enzyme Kumamolisin and the 33-mer. David is currently working on a Thermodynamic based approach to the prolem, while I am researching a collision theory approach. We may more forward with both theories in parallel in order to have both options to compare our experimental results with. We met with Chris today about modeling, and he thinks starting from scratch will be too difficult and greatly inaccurate. So we are instead going to use an existing modeling software called Smoldyn, which specializes in micro-scale chemical reactions. Chris has tasked those interested in the modeling component of the project with reading the user manual for Smoldyn and to find the parameters of our reaction necessary to run an initial model of our system. http://www.smoldyn.org/index.html Here is the link for the Smoldyn user manual. http://www.smoldyn.org/Smoldyn_doc1.pdf</span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\"></span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">(6/26/12) Today I spoke with Dr. Brian Geiss about the protocol for preparing the enzyme and protein with the quencher and fluorophore. He said he wanted to set up a meeting with us sometime this week to discuss the assay as well as how to prepare for it. I sent him an email telling him we are available all week in the afternoon and am currently awaiting his response. We also have a meeting with Chris Strickland today about furthering the modeling component of our project. So far I have read over the more relevant pieces of the user manual for Smoldyn and it appears to be a very in-depth detailed software. I am trying to understand the various components we will need in order to successfully run an initial attempt. The manual is clear on setting up parameters within Smoldyn, but not necessarily the features we will need for our molecules.</span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">This link gives measurement of gluten in various types of beer. http://www.ncbi.nlm.nih.gov/pubmed/17071509</span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">Here is a link about EnvZ and ompR use from the Missouri Miners 2011 iGEM project </span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">https://2011.igem.org/wiki/index.php?title=Team:Missouri_Miners/Project&oldid=202637</span></span></span></p>\n<p>\n\t </p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">(7/2/12) Today I am going to be working on further determining the relative amounts of Kumamolisin and gluten necessary to run an accurate simulation in Smoldyn. I will also be periodically learning some Python coding for Smoldyn programming. I am still researching the possibility of the CXCR3 receptor gluten assay. I will be bouncing ideas off of Steven who is also researching the topic. It appears as though many schools in the past have used EnvZ and ompR in their projects as a receptor/signaling pathway.</span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">Here is the link for the full publication about the levels of gluten in various beers: </span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">http://web.ebscohost.com/ehost/pdfviewer/pdfviewer?sid=acda06bc-f950-461d-9cd2-62e8f7404356%40sessionmgr12&vid=2&hid=8</span></span></span></p>\n<p>\n\t </p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">In order to calculate the number of molecules of gluten in a given sample of beer, I used the molecular weight of the 33-mer repeating gluten sequence as a base model. In one publication I found that the average concentration of gluten for a lager beer was roughly 4.175 mg/L. I then used the molecular weight of the 33-mer (g/mol) to calculate the mol/L of the gluten. Then, using a 5-gallon (18.927 L) carboy as my volume, I calculated the number of moles of gluten. Finally, I multiplied this result by Avagodro's Number to find the number of molecules of gluten. I repeated the same process for wheat beer (28.6 mg/L) to obtain the number of molecules of gluten in a 5-gallon carboy. I don't know if I mentioned this, but on Friday 6/22/12 we ordered our custom peptide sequence for the enzymatic assay. After much research, debate, and frustration, we ended up going with the 5-FAM/TAMRA combination, as recommended by Dr. Brian Geiss. We ordered from Biomatik and received an educational research discount. The delivery time frame is estimated to be 3-4 weeks. We can expect delivery between 7/13 - 7/20. I will be checking up on our order's progress in the coming weeks.</span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\"></span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">(7/3/12) Today we had our meeting with Dr. Brian Geiss and spoke about several different topics. The first topic was about our desire to test enzymatic activity of crude Kumamolisin produced from bacteria using constituative promoters (they are always on, i.e. not inducible) in the DH5-alphas. Dr. Geiss said we could run an SDS-Page Gel to verify that our bacteria system was producing our enzyme. The next topic we discussed was using inducible promoters in BL-21 E. coli to produce enzyme K. Dr. Geiss is providing the bacterial cells and we are in the process of designing and ordering the promoters. Once the promoters arrive we will be able to test this as an alternative system to the constituative promoter system (inducibles will also be run on a Page gel). Dr. Geiss recommended we test our quencher and fluorophore system once we received it using Protinase K, or something that will cleave within that sequence. This would allow us to see that our custom peptide system was working correctly, so we could proceed with our assay. We also discussed our end goal of having our yeast secrete enzyme K. He said we needed to purify enzyme K in order to cleanly test it against our custom peptide sequence. Once purified, we will be able to run in on his platereader to obtain our data. We then moved to discussion of our novel idea to use the GPCR CXCR3 to detect gluten in samples. He said that our main concern would be the successful fusion of CXCR3 and signal transduction of the binding of gliadin. We told him that we were speaking with Dr. Kevin Morey and that we would further pursue the concept with him. The final topic we covered was our modeling component. We mentioned that we were having trouble finding data needed to accurately model enzyme kinetics. He mentioned the idea of using a light polarization assay to calculate values related to enzyme K binding to the custom peptide sequence.</span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\"></span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">(7/5/12) Today I learned several different procedures for bench work we are doing on a regular basis. These procedures include making/pouring agarose gel, purifying gel bands, and running a nanodrop device. I also emailed Biomatik asking for an update on our custom peptide order. I am working on finding diffusion coefficients for Kumamolisin and Gluten 33-mer for the modeling component of our project. Tomorrow we have the New Belgium microbiology tour where we should learn more about the brewing process as well as discuss our project with them.</span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\"></span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">(7/9/12) Today I am further researching the idea of creating a Gluten-detecting assay. Our method would utilize a gliadin-binding receptor with a two stage signal transduction system. Several other iGEM teams as well as researchers have published work on the fusion of receptors to create a transmembrane receptor for various purposes. One specific iGEM team used this concept to create a vanillin receptor that produced a fluorescent protein that varied in color based the concentration of ompR produced. Although similar constructs (similar to what we are trying to develop) have been created, none of them used G-protein coupled receptors. This is where the potential problem arises. Fusion of the CXCR3 GPCR may be possible, but may not necessarily transduce the signal properly. Since I cannot find published work on this topic, we may have to take a shot in the dark and just make an attempt. I will discuss several ideas and topics with fellow group members and advisers to see what they think. New Belgium brewery also discussed the possibility of having us design primers for them. They need primers to help detect the presence of certain contaminants in the brewing tanks. Here is a paper that better characterizes the GPCR CXCR3 http://onlinelibrary.wiley.com/doi/10.1002/eji.200324235/pdf</span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\"></span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">(7/11/12) Today I am trying to learn about some of the basic functionality of python. I have limited previous experience with coding, but python appears to be quite user-friendly. I also have been doing bench work with Guy to further expand my practical lab knowledge. The first thing we did was a gel extraction, which I did once before. This procedure takes about 20 mins and follows a very intuitive protocol. After completing the gel extraction, we went to Lucas' lab to use the fluorometer to measure the concentration of our 189 and Kan DNA. The protocol for this procedure is slightly more involved, but wasn't overly complicated. Our results however proved disappointing, with very low concentrations of 189 and Kan. We are going to have to redo one of the original procedures and maybe use some combination of the previous measures or modify them in some manner.</span></span></span></p>\n<p>\n\t </p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">(7/12/12) Today we met with Chris Strickland to discuss our modeling progress as well as information about using the Cray Supercomputer. Dave showed Chris the rate limiting equation he had derived for the breakdown of gluten by Kumamolisin. The equation appears to be solid and should prove useful in determining the rates at which gluten breaks down. Chris told us our accounts had been added to the Cray and that we could now log-in from anywhere on campus. After Chris left, Dave tasked Ethan and I with writing Python code that would put multiple output files into one unified file with labels that described which file corresponded to which output.</span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\"></span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">(7/13/12) Today we have a meeting with Dr. Brian Geiss to discuss operation of the platereader for when our custom peptide arrives. We also have questions about the experiment involving the polarization of light to measure various qualities of our enzyme and gluten substrate. After meeting with Dr. Geiss we now know how to use the platereader for several tests. These tests include fluorescent, kinetics, and ELISA. Brian Searcy brought in both a barley and sorghum beers to test on the Gluten ELISA. Since sorghum is gluten-free, it shouldn't show up on the ELISA, whereas barley should. We are trying to work in with another lab member who is using the platereader, so I am not sure when we will actually test our gluten ELISA.</span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\"></span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">Here is a paper on two commercial types of gluten ELISA assays. </span></span></span></p>\n<p>\n\t<span class=\"font_8\"><span class=\"color_15\"><span color=\"color_0\">http://www.sciencedirect.com/science/article/pii/S0002822308014090</span></span></span></p>\n","defaultStyle":"font_6"},"c8km":{"type":"RichText","id":"c8km","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"text":"<p>\n\t<span class=\"font_7\">Safety is an important part of every project, and is a key focus of our iGEM 2012 efforts. The chief safety concerns for this summer revolve mostly around lab practices and alcohol related activities.</span></p>\n<p>\n\t<span class=\"font_7\"></span></p>\n<p>\n\t<span class=\"font_7\"></span><span class=\"font_7\">When working in the lab, it is paramount to follow safety procedures in order to minimize health risks of the participants directly and indirectly involved. Examples of common safety followings involve wearing gloves when dealing with hazardous chemicals, maintaining sanitized work equipment when dealing with objects that come into contact with people, and keeping chemicals and materials in their proper storage environments. It is also crucial to keep a very clean and organized lab environment in order to avoid potentially dangerous situations. </span></p>\n<p>\n\t<span class=\"font_7\"></span></p>\n<p>\n\t<span class=\"font_7\">The other component of our summer project is the alcohol-related activities. These activities included (for those who are 21 and older) consumption of alcohol for taste and educational standards as they related to particular strains of yeast. The brewing of alcohol for testing and analytical purposes was also conducted. This allowed for a lab comparison of a standard yeast brew, yeast brew with brewer's Clarex, and our modified yeast strain. The most important safety feature of this part of the project is that whenever alcohol (beer) was consumed by members of the team at least 21 years of age, it was always done in a controlled environment with no option to operate a vehicle post-consumption. Consumption was also minimized for taste, so full-size beverages were rarely (if at all) consumed by any group member. Consumption was also monitored to keep the focus project-related and professional.</span></p>\n","defaultStyle":"font_6"},"mainPage":{"type":"Page","id":"mainPage","metaData":{"isPreset":true,"schemaVersion":"1.0","isHidden":false},"title":"Home","hideTitle":true,"icon":"","descriptionSEO":"","metaKeywordsSEO":"","pageTitleSEO":"","pageUriSEO":"home","hidePage":false,"underConstruction":false,"tpaApplicationId":0},"come":{"type":"RichText","id":"come","metaData":{"isPreset":true,"schemaVersion":"1.0","isHidden":false},"text":"<p>\n\t<span class=\"font_8\">I'm a paragraph. Click here to add your own text and edit me. I’m a great place for you to tell a story and let your users know a little more about you.</span></p>\n<p>\n\t<span class=\"font_8\"></span></p>\n<p>\n\t<span class=\"font_8\"><span color=\"color_0\">This is a great space to write long text about your company and your services. You can use this space to go into a little more detail about your company.</span></span></p>\n","defaultStyle":"font_6"},"cy03":{"type":"Image","id":"cy03","metaData":{"isPreset":true,"schemaVersion":"1.0","isHidden":false},"target":"_self","linkType":"PAGE","href":"#!Home|mainPage","title":"","uri":"84770f_3789376cfc52ef9574c70089ac7a0089.png","description":"","width":36,"height":36,"alt":""},"cn19":{"type":"RichText","id":"cn19","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"text":"<p>\n\t<span class=\"font_6\">Project Protocols</span></p>\n<p>\n\t </p>\n<p>\n\t<span class=\"font_8\">Over the course of the project our team used various protocols covering everything from E. coli transformation to gel band purification. Click the picture or the \"Read More\" link below to see all of them.<span class=\"font_8\"><span class=\"font_8\"></span></span></span></p>\n","defaultStyle":"font_6"},"c1pa3":{"type":"RichText","id":"c1pa3","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"text":"<p>\n\t<span class=\"font_0\">Follow The Protocol</span></p>\n","defaultStyle":"font_0"},"c1l9r":{"type":"SiteButton","id":"c1l9r","metaData":{"isPreset":true,"schemaVersion":"1.0","isHidden":false},"target":"_self","linkType":"PAGE","href":"#!Protocols/c1pep","label":"Read more"},"cm7i":{"type":"RichText","id":"cm7i","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"text":"<p>\n\t<span class=\"font_6\">David Packard</span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">Math Wizard, Code Genius</span></span><span class=\"font_6\"><span class=\"color_2\">, Racquet Sports Pro... We call him a lot of things, but he prefers Dave. </span></span></p>\n","defaultStyle":"font_6"},"SITE_STRUCTURE":{"type":"Document","id":"SITE_STRUCTURE","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"siteName":"Template Base","mainPage":"#mainPage"},"cebb":{"type":"RichText","id":"cebb","metaData":{"isPreset":true,"schemaVersion":"1.0","isHidden":false},"text":"<p>\n\t<span class=\"font_2\"><span color=\"color_0\">I'm a title. Click here to edit me</span></span></p>\n","defaultStyle":"font_6"},"c1dot":{"type":"RichText","id":"c1dot","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"text":"<p>\n\t<span class=\"font_8\">A gluten-free beer isn't the only thing we are working on. We are also designing primers for New Belgium Brewery, developing a novel gluten-detecting assay, and creating a computer-modeled simulation for the breakdown of gluten in beer. All of these various components have us teaming up with some of the best in the business, so don't forget to visit our sponsor and attribution pages.</span><span class=\"font_6\"></span></p>\n","defaultStyle":"font_6"},"component_2780":{"type":"RichText","id":"component_2780","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"text":"<p>\n\t<a dataquery=\"#Linkfty\" href=\"#%21Home%7CmainPage\" target=\"_self\"><span class=\"font_0\">Colorado State University iGEM 2012</span></a></p>\n","defaultStyle":"font_6"},"component_items_2_78748":{"type":"Image","id":"component_items_2_78748","metaData":{"isPreset":true,"schemaVersion":"1.0","isHidden":false},"target":"_blank","linkType":"WEBSITE","href":"https://plus.google.com/117167403531518744294","title":"w-googleplus","uri":"608e7725939b0eda16493c462180552c.wix_mp","description":"","width":205,"height":205,"alt":""},"c1ojt":{"type":"Image","id":"c1ojt","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"target":"","linkType":"FREE_LINK","href":"","title":"","uri":"5bd9eb_299a61aed7aee9a8fe1c2e1ca33ea264.jpg","description":"","width":450,"height":338,"alt":""},"cl8c":{"type":"RichText","id":"cl8c","metaData":{"isPreset":true,"schemaVersion":"1.0","isHidden":false},"text":"<p>\n\t<span class=\"font_5\"><span class=\"color_11\"><span color=\"color_5\">I'm Another title</span></span></span></p>\n","defaultStyle":"font_6"},"c1n3s":{"type":"Page","id":"c1n3s","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"title":"Attributions","hideTitle":true,"icon":"","descriptionSEO":"","metaKeywordsSEO":"","pageTitleSEO":"","pageUriSEO":"attributions","hidePage":false,"underConstruction":false,"tpaApplicationId":0},"c1al5":{"type":"RichText","id":"c1al5","metaData":{"isPreset":true,"schemaVersion":"1.0","isHidden":false},"text":"<p>\n\t<span class=\"font_7\"><span color=\"color_0\">I'm a paragraph. Click here to add your own text and edit me. It’s easy. Just click “Edit Text” or double click me and you can start adding your own content and make changes to the font. Feel free to drag and drop me anywhere you like on your page. I’m a great place for you to tell a story and let your users know a little more about you.</span></span></p>\n","defaultStyle":"font_6"},"c11a4":{"type":"Image","id":"c11a4","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"target":"_blank","linkType":"WEBSITE","href":"http://www.equinoxbrewing.com/","title":"","uri":"5bd9eb_6ab83abbdc0e863d0ae1482c9627ecde.jpg","description":"","width":469,"height":459,"alt":""},"cb":{"type":"Image","id":"cb","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"target":"","linkType":"FREE_LINK","href":"","title":"","uri":"5bd9eb_824d36de9675768f73820cc4c7ce1caa.jpg","description":"","width":400,"height":308,"alt":""},"image1zwu":{"type":"Image","id":"image1zwu","metaData":{"isPreset":true,"schemaVersion":"1.0","isHidden":false},"target":"","linkType":"FREE_LINK","href":"","title":"I'm a title","uri":"84770f_6edda2fdf28ee065c70b4f7aa4ab4681.jpg","description":"I'm a description. Click to edit me","width":1280,"height":960,"alt":""},"ciaw":{"type":"RichText","id":"ciaw","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"text":"<p class=\"alignCenter\">\n\t<span class=\"font_5\"><span class=\"color_26\">GO RAMS!</span></span></p>\n","defaultStyle":"font_2"},"c9zf":{"type":"Image","id":"c9zf","metaData":{"isPreset":true,"schemaVersion":"1.0","isHidden":false},"target":"","linkType":"FREE_LINK","href":"","title":"","uri":"84770f_2e2993fdb76fd293b62e3eb1323b4535.png","description":"","width":195,"height":200,"alt":""},"c1y1":{"type":"Image","id":"c1y1","metaData":{"isPreset":true,"schemaVersion":"1.0","isHidden":false},"target":"","linkType":"FREE_LINK","href":"","title":"","uri":"84770f_7ce1ddb86000aefa86f1a05553079857.jpg","description":"","width":1280,"height":960,"alt":""},"cw8o":{"type":"ImageList","id":"cw8o","metaData":{"isPreset":true,"schemaVersion":"1.0","isHidden":false},"items":["#imagev04","#image1zwu","#image212v","#image1z3f"]},"imaget0y":{"type":"Image","id":"imaget0y","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"target":"","linkType":"FREE_LINK","href":"","title":"","uri":"5bd9eb_0da132b68968259736220617b3a74023.jpg","description":"","width":1600,"height":1200,"alt":""},"imagegv0":{"type":"Image","id":"imagegv0","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"target":"","linkType":"FREE_LINK","href":"","title":"","uri":"5bd9eb_dd1d446e8fdf6aab11b88895aa6386aa.jpg","description":"","width":1920,"height":1440,"alt":""},"czh4":{"type":"Image","id":"czh4","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"target":"","linkType":"FREE_LINK","href":"","title":"","uri":"5bd9eb_5fd8137354dd49f875f7f50df3424d77.jpg","description":"","width":1440,"height":1920,"alt":""},"c20ui":{"type":"RichText","id":"c20ui","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"text":"<p>\n\t<span class=\"font_6\"><span class=\"color_2\">Currently I'm looking into 3 aspects of the transition to yeast:</span></span><span class=\"color_2\"></span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">1. Subcloning into a yeast expression plasmid.</span></span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">Lucas Argueso claims that this is quite easy.</span></span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">2. Codon bias.</span></span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">The issue in Codon bias is that different organisms can produce different RNA's much easier. If the RNA used in a codon is uncommon, it is likely to die off as was shown in the following Stanford research paper: Codon bias. Using the codon bias tools below, there is a strong indication that the Bio-brick UW developed last year uses a lot of RNA uncommon in yeast. It is likely that this strain will die out in yeast very quickly. However, the Codons can be altered to use more common RNA, which would make the odds of replication greater. An optimized sequence is given by the final two links, and I am now investigating methods of synthesizing such a sequence.</span></span><span class=\"color_2\"></span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">This is a tool for analyzing the codon bias of a given protein strand in a specific organism.</span></span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">Another tool for analyzing the codon bias of a given protein strand in a specific organism.</span></span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">This gives optimized nucleic acid sequence for a given amino acid sequence.</span></span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">3. Gene synthesis. Chemical synthesis is done by chemically creating the desired sequence. There is a lab on campus which would do this for us if we desired so: Chemical Synthesis at CSU.</span></span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">4. Secreting enzyme into beer.</span></span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">Guy has done quite a bit of research on secretion/expression vectors. See his page for more information, but a useful background article is given below.</span></span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">https://static.igem.org/mediawiki/2008/2/27/JHU_0708_paper_ForeignGeneExpression.pdf</span></span><span class=\"color_2\"></span></p>\n","defaultStyle":"font_6"},"c1ex9":{"type":"Image","id":"c1ex9","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"target":"","linkType":"FREE_LINK","href":"","title":"","uri":"5bd9eb_7b0fb962b18765bb29e10fc210885ee5.jpg","description":"","width":480,"height":640,"alt":""},"cz0u":{"type":"Image","id":"cz0u","metaData":{"isPreset":true,"schemaVersion":"1.0","isHidden":false},"target":"","linkType":"FREE_LINK","href":"","title":"","uri":"97acfd_8960aa625477c2189d48ac9311f25ad3.jpg","description":"","width":1280,"height":1280,"alt":""},"#Link4z":{"type":"TextLink","id":"#Link4z","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"target":"_blank","linkType":"WEBSITE","href":"http://www.bio-rad.com/webroot/web/pdf/lse/literature/pepsi_hr_1665067.pdf"},"component_items_0_27497":{"type":"Image","id":"component_items_0_27497","metaData":{"isPreset":true,"schemaVersion":"1.0","isHidden":false},"target":"_blank","linkType":"WEBSITE","href":"http://www.facebook.com/wix","title":"w-facebook","uri":"da00086a27cc2c52ec7a11ec468c4d29.wix_mp","description":"","width":205,"height":205,"alt":""},"c225t":{"type":"RichText","id":"c225t","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"text":"<p>\n\t<span class=\"font_6\">Ryan Clouse</span></p>\n<p>\n\t<span class=\"font_6\"></span></p>\n<p>\n\t<span class=\"font_6\">Blah Blah Blah</span></p>\n","defaultStyle":"font_6"},"c1e6a":{"type":"Image","id":"c1e6a","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"target":"","linkType":"FREE_LINK","href":"","title":"","uri":"5bd9eb_7ef5fd654f20a317552cf2933f9f5671.jpg","description":"","width":640,"height":409,"alt":""},"c1v3t":{"type":"RichText","id":"c1v3t","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"text":"<p>\n\t<span class=\"font_4\"><span color=\"color_0\">The Research</span></span></p>\n","defaultStyle":"font_6"},"cv0p":{"type":"RichText","id":"cv0p","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"text":"<p>\n\t<span class=\"font_6\">Important Group Research</span></p>\n<p>\n\t<span class=\"font_10\"><span class=\"italic\"></span></span></p>\n<p>\n\t<span class=\"font_8\">Throughout the research process, many important publications were found that contributed to how our team approached the project. These articles and publications can be viewed by clicking the picture or the \"Read More\" link below.<span class=\"font_8\"><span class=\"font_8\"></span></span></span></p>\n","defaultStyle":"font_6"},"image11ey":{"type":"Image","id":"image11ey","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"target":"","linkType":"FREE_LINK","href":"","title":"","uri":"5bd9eb_6c48e1727a23eee94773e54df2564b80.jpg","description":"","width":1920,"height":1440,"alt":""},"c10bu":{"type":"RichText","id":"c10bu","metaData":{"isPreset":true,"schemaVersion":"1.0","isHidden":false},"text":"<p>\n\t<span class=\"font_2\"><span color=\"color_0\">Let's Talk</span></span></p>\n<p>\n\t </p>\n<p>\n\t<span class=\"font_6\"><span color=\"color_0\">Company name</span></span></p>\n<p>\n\t </p>\n<p>\n\t<span class=\"font_8\"><span color=\"color_0\">2601 Mission St.<br>\n\tSan Francisco, CA 94110<br>\n\tinfo@mysite.com</span></span></p>\n<p>\n\t<span class=\"font_8\"><span color=\"color_0\">Tel: 123-456-7890<br>\n\tFax: 123-456-7890</span></span></p>\n","defaultStyle":"font_6"},"c19":{"type":"RichText","id":"c19","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"text":"<p>\n\t<span class=\"font_0\">Welcome to Dave's Page!</span></p>\n","defaultStyle":"font_0"},"cdpm":{"type":"RichText","id":"cdpm","metaData":{"isPreset":true,"schemaVersion":"1.0","isHidden":false},"text":"<p>\n\t<span class=\"font_8\"><span color=\"color_0\">I'm a paragraph. Click here to add your own text and edit me. I’m a great place for you to tell a story and let your users know a little more about you.<br>\n\t<br>\n\tThis is a great space to write long text about your company and your services. You can use this space to go into a little more detail about your company.</span></span></p>\n","defaultStyle":"font_6"},"c8b8":{"type":"Image","id":"c8b8","metaData":{"isPreset":true,"schemaVersion":"1.0","isHidden":false},"target":"_self","linkType":"PAGE","href":"#!Protocols/c1pep","title":"","uri":"97acfd_57e001de11fd13ceafce3d70b6719f7b.jpg","description":"","width":1280,"height":1025,"alt":""},"cixm":{"type":"RichText","id":"cixm","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"text":"<p>\n\t<span class=\"font_0\">Computer Modeling Component</span></p>\n","defaultStyle":"font_0"},"cjxj":{"type":"Image","id":"cjxj","metaData":{"isPreset":true,"schemaVersion":"1.0","isHidden":false},"target":"","linkType":"FREE_LINK","href":"","title":"","uri":"84770f_6cbe7263add591a385d4bb70c432014c.jpg","description":"","width":946,"height":1222,"alt":""},"c19ou":{"type":"RichText","id":"c19ou","metaData":{"isPreset":false,"schemaVersion":"1.0","isHidden":false},"text":"<p>\n\t<span class=\"font_6\"><span class=\"color_2\">We are currently working on modeling the reaction between the enzyme Kumamolisin and the protein responsible for causing an immune response in Celiac patients. Our model is based on Michaelis-Menten kinetics which treat enzyme kinetics as a two step reaction. Our assay can return reaction rates for pure substrate and Kumamolisin, however in beer several proteins other than gluten which will act as inhibitors to gluten breakdown. Both the gluten and inhibitor are broken down in the same two step reaction in the Michaelis-Menten model. First, the enzyme must bind to the protein, which is a reversible reaction. Then, the bound enzyme breaking down the protein is treated as its own reaction.</span></span><span class=\"color_2\"></span></p>\n<p>\n\t<span class=\"color_2\"></span><br>\n\t<span class=\"font_6\"><span class=\"color_2\">Smoldyn is a biochemical reaction modeling software we've chosen to carry out this model in. Currently, we have working a simulation based on the Michealis-Menten model. Our model includes three reactions for gluten breakdown:</span></span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">1. Forward binding Kumamolisin + Gluten -> Bound Kumamolisin and Gluten</span></span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">2. Reverse binding Bound Kumamolisin and Gluten -> Kumamolisin + Gluten</span></span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">3. Protein breakdown Bound Kumamolisin and Gluten -> Kumamolisin + Broken-down gluten</span></span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">Reactions 4-6 account for inhibitors:</span></span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">4. Forward binding Kumamolisin + Inhibitor -> Bound Kumamolisin and Inhibitor</span></span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">5. Reverse binding Bound Kumamolisin and Inhibitor -> Kumamolisin + Inhibitor</span></span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">6. Protein breakdown Bound Kumamolisin and Gluten -> Kumamolisin + Broken-down Inhibitor</span></span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">Since Kumamolisin is breaking down the same amino acid sequence in each protein, reactions 1 and 4, 2 and 5 and 3 and 6 have the same reaction rate when Kumamolisin is saturated with substrate. Smoldyn will then adjust these reaction rates for the concentrations of enzyme and protein input.</span></span><span class=\"color_2\"></span></p>\n<br>\n<p>\n\t<span class=\"color_2\"></span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">This simulation can be run on the iGEM laptop by entering the Unix command python /home/local_admin/igem/pythonScripts/KumaModelSweeper.py. Currently, this simulation sweeps over different reaction rates for each of the 3 possibilities. These parameters can be edited by any member of the CSU iGEM team, by opening the python file with a text editor (i.e. gedit /home/local_admin/igem/pythonScripts/KumaModelSweeper.py).</span></span></p>\n<p>\n\t<span class=\"color_2\"></span><br>\n\t<span class=\"font_6\"><span class=\"color_2\">Our focus is on determining accurate parameters to use in the simulation. The following are parameters we are hoping to find via research:</span></span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">1. Volume</span></span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">2. Total number of molecules</span></span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">3. Gluten concentration in beer</span></span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">4. Total protein concentration in beer</span></span></p>\n<p>\n\t<span class=\"font_6\"><span class=\"color_2\">5. 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