http://2009.igem.org/wiki/index.php?title=Special:Contributions/ParthivA&feed=atom&limit=50&target=ParthivA&year=&month=2009.igem.org - User contributions [en]2024-03-29T09:30:40ZFrom 2009.igem.orgMediaWiki 1.16.5http://2009.igem.org/Team:Queens/TeamTeam:Queens/Team2009-10-21T21:09:58Z<p>ParthivA: </p>
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<td align="top"><img src="https://static.igem.org/mediawiki/2009/6/6b/QueensParthivAmin.png" alt="Image not Found" /></td><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Parthiv Amin</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Nickname: francium phosphide (Fr<sub>3</sub>P) / fatal attrACTION</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Program: B.Sc. BioChemical Engineering 2011</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">QGEM Position: Student Volunteer/Webmaster</P><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
About: I am currently entering my third year in BioChemical Engineering at Queen's University. I garnered an interest in iGEM due to my fascination of the field of biofuels, especially the use of engineered algal strains for biodiesel production. As a volunteer member of the QGEM, my focus is mostly on the technological aspects of iGEM: maintaining the Wiki, compiling data, and animations. Outside of QGEM I'll be working on the Queen's BAJA SAE car, as well as getting some of the paperwork filed for a new chemical engineering design team a few of my friends and I are developing. In my free time I'll be playing my guitar & Halo 2, surfing the net, and of course doing a metric tonne of homework.</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Michael Freeman</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Nickname: The Naked Sasquatch / Microbicurious</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Program: B.Sc. BioChemical Engineering 2010</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">QGEM Position: Student Employee</P><br />
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About: I am in my fourth year at Queen's after transferring degree programs from chemistry to biochemical engineering. iGEM is a competition that grabbed my interest immediately because of the unbelievable possibilities synthetic biology makes available. Solving problems in general is incredibly interesting to me, and solving them in unconventional or obscure ways is even more exciting. I came into iGEM with a few project ideas like including engineered microbes in specific waste water treatment processes, creating a more conclusive indicator test for aggressive prostate cancers, and creating some microbial suspension which could be ingested and used to break down cellulose in the human stomach, a hugely abundant source of food and energy. For iGEM this year I was a 'wet labber' responsible for creating, engineering and testing our Heme/Heme-Oxygenase system which would be used as one of our plaque degrading effectors. In order to keep from going nuts from talking with bacteria all day I bar tend at night, I compete in triathlons and varsity rowing for Queen's, I am deeply in love with traveling and hosting travelers, and I have just entered the wonderful (and expensive) world of skydiving! Ever touched a cloud? This guy has.</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Jonas Elliott Gerson</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Nickname: Sex Pili</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Program:</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">QGEM Position:</P><br />
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About: More to come later</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">James MacLeod</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Nickname: Bond</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Program: B.Sc.H. Biology</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">QGEM Position: Co-Founder</P><br />
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About: I am currently in my third year at Queen's University pursuing a Bachelor's of Science (BScH). I was born in Britain, lived in the Middle East for a period, and now reside in Canada. I love the skiing that Canada has to offer, and I'm very excited that Queen's will be participating in the 2009 Jamboree and am looking forward to meeting teams from across the world! </p><br />
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<td align="top"><img src="https://static.igem.org/mediawiki/2009/9/94/QueensBogdanMomciu.png" alt="Image not Found" /></td><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Bogdan Momciu</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Nickname: The Count </p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Program: B.Sc.H. Life Sciences SSP 2010</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">QGEM Position: Student Employee</P><br />
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About: I am 4th year student in the Queen's University Life Sciences program with a focus towards research and biomedical applications. Once I heard about iGEM I was greatly enthusiastic about the idea due to the sheer number of innovations this kind of cooperative initiative could end up producing. During this past summer I worked on the design and synthesis of our SAA and Endothelial Cell Binding Constructs as well as testing and troubleshooting.<br />
In my spare time I like to play a variety of sports including hockey, soccer, and volleyball. </p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Chris Palmer</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Nickname: FRECsecutioner</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Program: B.Sc. Engineering Chemistry 2012</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">QGEM Position: Student Volunteer</P><br />
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About: I am a second year student in the Engineering Chemistry program at Queen's university, but I have always had a strong interest in microbiology and biochemistry as well. As a result, I was intrigued by the opportunities presented by the iGEM competition. As soon as I heard that Queen's was forming an iGEM team, I knew that I wanted to be a part of it and help get it off the ground in its first year. I have thoroughly enjoyed my time this summer working with everyone else on the team, and I look forward to meeting people and hearing about everyone's exciting projects at the jamboree!</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Bryant Shum</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Nickname: Blossom</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Program: Life Science SSP 2011</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">QGEM Position: Student Employee</P><br />
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About: I am currently a third year undergraduate student at Queen's, with a concentration in Life Science. Given the nature of my studies, I have always been fascinated by the sheer variety of phenotypes that can arise from a simple genetic code; therefore, the chance to play "Lego" with these complex molecules of life was an irresistible opportunity for me. Specifically, my role on the team this year was to help construct the genetic components required for plaque binding, as well as to conduct background research on the pathogenesis of atherosclerosis. And, so far, this has proven to be a challenging but unforgettable project.</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Kate Turner</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Nickname: Chicken Joe / The Octagon</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Program: B.Sc. BioChemical Engineering 2010</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">QGEM Position: Student Employee</P><br />
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About: I am in my fourth year of a chemical engineering degree at Queen’s, focusing on the biomedical stream. I spent last summer working as a field engineer in the Alberta oil patch, but this year I wanted to broaden my horizons and focus on biotechnology or biomedical research. I was especially interested in iGEM due to the wide-ranging applications of synthetic biology – from smog cleanup to combating tumours to degrading plastics – and now I hope to continue in this field after graduation. During the summer I worked in one of the labs testing the heme/HO-1 effector system and researching the amelioration of atherosclerotic plaques by CO, HO-1, and biliverdin. Outside of QGEM, I spent my summer camping, BBQing, and reading like a fiend.</p><br />
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<td align="top"><img src="https://static.igem.org/mediawiki/2009/9/9a/QueensChrisYan.png" alt="Image not Found" /></td><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Christopher Yan</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Nickname: YanTASTIC</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Program: B.Sc.H. Life Sciences 2012</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">QGEM Position: Student Employee</P><br />
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About: I am a second year Life Sciences student at Queen's University. This is my first time participating in an event of such grand scale and am very much looking forward to the Jamboree.</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Harry Zhou</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Nickname: Bubbles</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Program: B.Sc.H. Biochemistry 2011</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">QGEM Position: Co-Founder</P><br />
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About: I am a third-year Biochemistry student at Queen’s University. Starting in high school, I had a strong interest in biotechnology that has the potential of benefiting health care and environment. When I was in grade 12, a professor at University of Calgary introduced me to synthetic biology and the iGEM competition and I volunteered for the UCalgary team in 2007. I thoroughly enjoyed the experience and was fascinated by the iGEM program, which provides a platform for students to tackle important problems concerning the environment, health care, and new technology. This was why I decided to put together a multidisciplinary team of undergraduate students at Queen’s to participate in this year’s iGEM competition. I had tons of fun this summer planning and carrying out experiments for the project and working with students from other discipline. In my spare time, I like to play sports, guitar, and read. </p><br />
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<td align="left"><p style="font-size:175%;font-family:corbel;color:#172C4E;font-weight:bold"><i>The Faculty Advisors</i></p></td><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Ian D. Chin-Sang, Ph.D.</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Faculty: Arts & Science</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Department: Biology</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Position: Associate Professor and CCS/NCIC Research Scientist</P><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Research: Molecular Genetics of <i>C. elegans</i> Development</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Peter A. Greer, Ph.D.</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Faculty: Arts & Science</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Department: BioChemistry</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Position: Professor of Biochemistry and Pathology & Molecular Medicine</P><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Research: Cancer Signal Transduction</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">David P. Lebrun, MD</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Faculty: Medicine</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Department: Pathology and Molecular Medicine</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Position: Associate Professor of Pathology & Molecular Medicine</P><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Research: Molecular Mechanisms of Leukemia & Clinicopathological Correlations in Malignant Lymphoma</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Nancy Martin, Ph.D.</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Faculty: Arts & Science</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Department: Microbiology and Immunology</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Position: Associate Professor</P><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Research: Sensing and Adaptation to Environmental Changes in <i>Salmonella typhimurium</i></p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Ronald J. Neufeld, Ph.D.</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Faculty: Applied Science</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Department: Chemical Engineering</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Position: Professor</P><br />
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Research: Bioencapsulation & Bioactives Processing and Controlled Release</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Waheed Sangrar, Ph.D.</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Faculty: Medicine</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Department: Pathology and Molecular Medicine</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Position: Assistant Professor</P><br />
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Research: Breast Cancer</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Virginia K. Walker, Ph.D.</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Faculty: Arts & Science</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Department: Biology</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Position: Professor</P><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Research: Genetics and Molecular Biology of Resistance</p><br />
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<center><br />
<p style="font-size:110%; font-family:corbel;color:#172C4E"><br />
Last Updated: October 21, 2009 by Fr<subs>3</subs>P</p></center><br />
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</html></div>ParthivAhttp://2009.igem.org/Team:Queens/TeamTeam:Queens/Team2009-10-21T21:09:26Z<p>ParthivA: </p>
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<a href="https://2009.igem.org/Team:Queens"><img src="https://static.igem.org/mediawiki/2009/4/4f/QueensBackground.png"></a><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Parthiv Amin</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Nickname: francium phosphide (Fr<sub>3</sub>P) / fatal attrACTION</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Program: B.Sc. BioChemical Engineering 2011</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">QGEM Position: Student Volunteer/Webmaster</P><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
About: I am currently entering my third year in BioChemical Engineering at Queen's University. I garnered an interest in iGEM due to my fascination of the field of biofuels, especially the use of engineered algal strains for biodiesel production. As a volunteer member of the QGEM, my focus is mostly on the technological aspects of iGEM: maintaining the Wiki, compiling data, and animations. Outside of QGEM I'll be working on the Queen's BAJA SAE car, as well as getting some of the paperwork filed for a new chemical engineering design team a few of my friends and I are developing. In my free time I'll be playing my guitar & Halo 2, surfing the net, and of course doing a metric tonne of homework.</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Michael Freeman</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Nickname: The Naked Sasquatch / Microbicurious</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Program: B.Sc. BioChemical Engineering 2010</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">QGEM Position: Student Employee</P><br />
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About: I am in my fourth year at Queen's after transferring degree programs from chemistry to biochemical engineering. iGEM is a competition that grabbed my interest immediately because of the unbelievable possibilities synthetic biology makes available. Solving problems in general is incredibly interesting to me, and solving them in unconventional or obscure ways is even more exciting. I came into iGEM with a few project ideas like including engineered microbes in specific waste water treatment processes, creating a more conclusive indicator test for aggressive prostate cancers, and creating some microbial suspension which could be ingested and used to break down cellulose in the human stomach, a hugely abundant source of food and energy. For iGEM this year I was a 'wet labber' responsible for creating, engineering and testing our Heme/Heme-Oxygenase system which would be used as one of our plaque degrading effectors. In order to keep from going nuts from talking with bacteria all day I bar tend at night, I compete in triathlons and varsity rowing for Queen's, I am deeply in love with traveling and hosting travelers, and I have just entered the wonderful (and expensive) world of skydiving! Ever touched a cloud? This guy has.</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Jonas Elliott Gerson</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Nickname: Sex Pili</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Program:</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">QGEM Position:</P><br />
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About: More to come later</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">James MacLeod</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Nickname: Bond</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Program: B.Sc.H. Biology</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">QGEM Position: Co-Founder</P><br />
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About: I am currently in my third year at Queen's University pursuing a Bachelor's of Science (BScH). I was born in Britain, lived in the Middle East for a period, and now reside in Canada. I love the skiing that Canada has to offer, and I'm very excited that Queen's will be participating in the 2009 Jamboree and am looking forward to meeting teams from across the world! </p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Bogdan Momciu</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Nickname: The Count </p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Program: B.Sc.H. Life Sciences SSP 2010</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">QGEM Position: Student Employee</P><br />
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About: I am 4th year student in the Queen's University Life Sciences program with a focus towards research and biomedical applications. Once I heard about iGEM I was greatly enthusiastic about the idea due to the sheer number of innovations this kind of cooperative initiative could end up producing. During this past summer I worked on the design and synthesis of our SAA and Endothelial Cell Binding Constructs as well as testing and troubleshooting.<br />
In my spare time I like to play a variety of sports including hockey, soccer, and volleyball. </p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Chris Palmer</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Nickname: FRECsecutioner</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Program: B.Sc. Engineering Chemistry 2012</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">QGEM Position: Student Volunteer</P><br />
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About: I am a second year student in the Engineering Chemistry program at Queen's university, but I have always had a strong interest in microbiology and biochemistry as well. As a result, I was intrigued by the opportunities presented by the iGEM competition. As soon as I heard that Queen's was forming an iGEM team, I knew that I wanted to be a part of it and help get it off the ground in its first year. I have thoroughly enjoyed my time this summer working with everyone else on the team, and I look forward to meeting people and hearing about everyone's exciting projects at the jamboree!</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Bryant Shum</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Nickname: Blossom</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Program: Life Science SSP 2011</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">QGEM Position: Student Employee</P><br />
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About: I am currently a third year undergraduate student at Queen's, with a concentration in Life Science. Given the nature of my studies, I have always been fascinated by the sheer variety of phenotypes that can arise from a simple genetic code; therefore, the chance to play "Lego" with these complex molecules of life was an irresistible opportunity for me. Specifically, my role on the team this year was to help construct the genetic components required for plaque binding, as well as to conduct background research on the pathogenesis of atherosclerosis. And, so far, this has proven to be a challenging but unforgettable project.</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Kate Turner</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Nickname: Chicken Joe / The Octagon</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Program: B.Sc. BioChemical Engineering 2010</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">QGEM Position: Student Employee</P><br />
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About: I am in my fourth year of a chemical engineering degree at Queen’s, focusing on the biomedical stream. I spent last summer working as a field engineer in the Alberta oil patch, but this year I wanted to broaden my horizons and focus on biotechnology or biomedical research. I was especially interested in iGEM due to the wide-ranging applications of synthetic biology – from smog cleanup to combating tumours to degrading plastics – and now I hope to continue in this field after graduation. During the summer I worked in one of the labs testing the heme/HO-1 effector system and researching the amelioration of atherosclerotic plaques by CO, HO-1, and biliverdin. Outside of QGEM, I spent my summer camping, BBQing, and reading like a fiend.</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Christopher Yan</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Nickname: YanTASTIC</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Program: B.Sc.H. Life Sciences 2012</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">QGEM Position: Student Employee</P><br />
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About: I am a second year Life Sciences student at Queen's University. This is my first time participating in an event of such grand scale and am very much looking forward to the Jamboree.</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Harry Zhou</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Nickname: Bubbles</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Program: B.Sc.H. Biochemistry 2011</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">QGEM Position: Co-Founder</P><br />
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About: I am a third-year Biochemistry student at Queen’s University. Starting in high school, I had a strong interest in biotechnology that has the potential of benefiting health care and environment. When I was in grade 12, a professor at University of Calgary introduced me to synthetic biology and the iGEM competition and I volunteered for the UCalgary team in 2007. I thoroughly enjoyed the experience and was fascinated by the iGEM program, which provides a platform for students to tackle important problems concerning the environment, health care, and new technology. This was why I decided to put together a multidisciplinary team of undergraduate students at Queen’s to participate in this year’s iGEM competition. I had tons of fun this summer planning and carrying out experiments for the project and working with students from other discipline. In my spare time, I like to play sports, guitar, and read. </p><br />
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<td align="left"><p style="font-size:175%;font-family:corbel;color:#172C4E;font-weight:bold"><i>The Faculty Advisors</i></p></td><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Ian D. Chin-Sang, Ph.D.</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Faculty: Arts & Science</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Department: Biology</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Position: Associate Professor and CCS/NCIC Research Scientist</P><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Research: Molecular Genetics of <i>C. elegans</i> Development</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Peter A. Greer, Ph.D.</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Faculty: Arts & Science</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Department: BioChemistry</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Position: Professor of Biochemistry and Pathology & Molecular Medicine</P><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Research: Cancer Signal Transduction</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">David P. Lebrun, MD</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Faculty: Medicine</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Department: Pathology and Molecular Medicine</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Position: Associate Professor of Pathology & Molecular Medicine</P><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Research: Molecular Mechanisms of Leukemia & Clinicopathological Correlations in Malignant Lymphoma</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Nancy Martin, Ph.D.</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Faculty: Arts & Science</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Department: Microbiology and Immunology</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Position: Associate Professor</P><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Research: Sensing and Adaptation to Environmental Changes in <i>Salmonella typhimurium</i></p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Ronald J. Neufeld, Ph.D.</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Faculty: Applied Science</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Department: Chemical Engineering</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Position: Professor</P><br />
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Research: Bioencapsulation & Bioactives Processing and Controlled Release</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Waheed Sangrar, Ph.D.</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Faculty: Medicine</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Department: Pathology and Molecular Medicine</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Position: Assistant Professor</P><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Research: Breast Cancer</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Virginia K. Walker, Ph.D.</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Faculty: Arts & Science</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Department: Biology</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Position: Professor</P><br />
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Research: Genetics and Molecular Biology of Resistance</p><br />
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<p style="font-size:110%; font-family:corbel;color:#172C4E"><br />
Last Updated: October 20, 2009 by Fr<subs>3</subs>P</p></center><br />
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</html></div>ParthivAhttp://2009.igem.org/File:QueensChrisPalmer.pngFile:QueensChrisPalmer.png2009-10-21T21:09:09Z<p>ParthivA: </p>
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<div></div>ParthivAhttp://2009.igem.org/Team:Queens/ResultsTeam:Queens/Results2009-10-21T13:04:20Z<p>ParthivA: </p>
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<table style="background-color:#922334; position:relative; overflow:auto; left:200px; top:-215px; width:750px"><br />
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<td align="left"><p style="font-size:175%;font-family:corbel;color:#172C4E;font-weight:bold"><i>Results</i><br />
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<p style="font-size=120%;font-family:palatino linotype;color:#ECB528"><br />
The following summarizes the results of the various aspects of this year's QGEM Project. <br />
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<a href="#Part1"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part One: SAA<br />
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<a href="#Part2"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Two: Binding Construct<br />
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<a href="#Part3"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Three: Heme and HO-1<br />
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<a href="#Part4"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Four: Future Directions<br />
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<a href="#Part5"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Safety Considerations<br />
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<a name="Part1"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part One: SAA</p></a><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Serum amyloid A (SAA) featured prominently on our list of possible effectors to release at the site of atherosclerotic plaques due to research done at Queen’s University by Tam et al. (2005). This research showed that treatment of SAA caused macrophages to reverse and prevent esterification of cholesterol, thereby allowing it to be exported out of plaques by high-density lipoprotein (HDL). In order to get the SAA to be picked up by the macrophages we needed to produce large quantities of the molecule, and then secrete it extracellularly. In order to secrete the protein we decided on using the twin-arginine translocase (TAT) system, which can transport fully folded proteins outside the cell (Sargent et al., 2006) and is found in the majority of prokaryotes. We then fused the TAT signal sequence to the front of our SAA sequence in order to produce a construct that should be able produce and secrete the protein, allowing it to be taken up by macrophages.<br />
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<center><img src="https://static.igem.org/mediawiki/2009/c/cb/QueensFigure1.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 1</i> SAA expression and secretion by <i>E. coli</i> cells. Cultures of <i>E. coli</i> cells containing either the SAA construct or control plasmid were spun down at 0, 4, and 12 hours after seeding. The culture medium and whole cell lysates were analyzed by SDS-PAGE and Western blot analysis with a polyclonal antibody recognizing whole SAA protein. <br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Our <i>E. coli</i> cells transformed with SAA construct did not appear to express or secrete SAA into the growth medium even after growing for 12 hours in optimum condition. This might be due to defective ligation of the Ptet-RBS fragment to the SAA-encoding gene. We plan to sequence the construct in the future. <br />
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References:<br />
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Benditt, E.P., Hoffman, J.S. & Eriksen, N. (1982). SAA, an apoprotein of HDL: its structure and function. <i> New York Academy of Sciences. </i> 82:183-189.<br />
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Ancsin, J.B. & Kisilevsky, R. (1999). The heparin/heparin sulfate-binding site on apo-serum amyloid A. <i>Journal of Biological Chemistry.</i> Vol. 274, 11:7172-7181.<br />
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<a name="Part2"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Two: Binding Construct</p></a><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In our original design of the binding construct, constitutive promoter BBa_J23119 was used to control the expression of the chimeric protein receptor consisted of the Lpp-OmpA fusion, TEV protease cut sites and ITGA4 fragment. We ordered our construct to be synthesized by Mr. GENE in early June. However, Mr. GENE notified us in August that our construct appeared to be unstable in and toxic to <i>E. coli</i> cells. The reason might be that high expression level of Lpp-OmpA might interfere with bacterial physiology, causing severe growth inhibition and reduced viability. (Daugherty et al, 1999) Mr. Gene was able to give us a sequence-confirmed PCR product of the synthesized construct. Thus, we decided to replace P¬const with P¬tet (BBa_R0040) by PCR. Below is a schematic diagram of the construction process. <br />
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<center><img src="https://static.igem.org/mediawiki/2009/0/08/QueensFigure2.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 2</i> Flowchart of PCR stitching for constructing the binding construct. <br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Due to time constraint, we were only able to confirm the stitching of Ptet-RBS fragment to Lpp-OmpA-Linker-TEVx2-Linker fragment (PCR round 2) using Agarose gel electrophoresis. The third round of PCR stitching did not yield expected band. Construction of the binding construct is ongoing. <br />
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<center><img src="https://static.igem.org/mediawiki/2009/1/14/QueensFigure3.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 3</i> <i>In silico</i> model of the binding construct. <br />
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References:<br />
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Daugherty, P.S., Olsen, M.J., Iverson, B.L. & Georgiou, G. (1999). Development of an optimized expression system for the screening of antibody libraries displayed on the <i>Escherichia coli</i> surface. <i>Protein Engineering.</i> Vol. 12, 7:613-621.<br />
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Yang, Z., Lui, Q., Wang, Q. & Zhang, Y. (2008). Novel bacterial surface display systems based on outer membrane anchoring elements from the marine bacterium <i>Vibrio anguillarum</i>. <i> American Society for Microbiology.</i> Vol. 74, 14: 4359-4365.<br />
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<a name="Part3"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Three: Heme and HO-1</p></a><br />
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Before presenting our results some useful parts have been outlined briefly.<br />
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BioBrick and Construct Legend:<br />
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BioBrick I716390: Plasmid containing a composite of Heme A, B C & D with T7-promoter and RBS. Amp Resistance.<br />
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BioBrick K098010: HO and phycocaynobilin:ferredoxin oxidoreductase. KAN Resistance.<br />
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BioBrick I15009: HO-1 from <i>Synechocystis</i>. Amp Resistance.<br />
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NLM 350: Rough <i> E. coli</i> strain containing T7-polymerase. Queen's University Research<br />
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In order to determine whether or not the BioBrick I716390 submitted by the Berkeley iGEM team in 2007 was producing heme, NLM350. Overnight cultures containing 100 μg/mL ampicillin were set up and inoculated with NLM350 containing I716390. The culture was analyzed daily by wavelength scan in a spectrometer. We ran the wavelength scan twice, once with a blank of LB and once with a blank of LB culture containing NLM350 without the BioBrick. Both of the scan results were identical, which indicates that the peaks present are due to the BioBrick plasmid and not resulting from compounds produced by NLM350. The results from day three with a blank of LB are shown in Figure 4.<br />
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<center><img src="https://static.igem.org/mediawiki/2009/c/ce/QueensFigure4.png"></center><br />
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<i>Fig. 4</i> Results of a wavelength scan between 350 and 550nm of the NLM350 + I716390 culture three days after inoculation. Strongest peaks at 412nm. <br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In order to check for the presence of the heme/HO-1 complex, NLM350 + I716390 cells were made electrocompetent and two sets of electroporations were performed. The BioBricks K098010 and I15008 were taken up by NLM350 + I716390 and the resulting colonies were used to inoculate cultures containing 40 μg/mL kanamycin and 100 μg/mL ampicillin, respectively. Triplicate testing of NLM350 + 176390 + KO98010 as well as NLM350 + 176390 + I15008 was conducted over five days. Both tests produced similar strong peaks for heme/HO-1 complexes, ranging from 407-409 nm. Again, we ran the scans twice with blanks of LB and LB inoculated with NLM350 and found that results were identical. The results from day five with blanks of LB are shown in Figures 5 and 6.<br />
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<center><img src="https://static.igem.org/mediawiki/2009/2/2b/QueensFigure5.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 5</i> Wavelength scan between 350 and 550 nm of the NLM350 + I716390 + K098010 culture 5 days after inoculation. Strong peak of 407.5 nm. <br />
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<center><img src="https://static.igem.org/mediawiki/2009/7/74/QueensFigure6.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 6</i> Wavelength scan between 350 and 550 nm of the NLM350 + I716390 + I15008 culture 5 days after inoculation. Strong peak of 407.5 nm. <br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
As can be seen from our results above, the peaks in the cultures containing heme and HO-1 producing BioBricks become clearer and occur at a lower absorbance, around 408nm, than the peaks from cultures containing only heme-producing BioBricks. Cultures containing only the heme-producing BioBrick has the strongest peaks at 412nm, which corresponds to the peak found by Moffet et al. in 2003 <sup>1</sup>. Since HO-1 does not have an absorbance peak of its own <sup>2,3,4</sup>, the presence of HO-1 can only be inferred by identifying the heme/HO-1 complexes via spectroscopy. According to several authors <sup>2,4</sup>, Soret bands for the oxy-bound form of the heme/HO-1 complex occur at 410nm. The peaks seen in Figures 5 and 6 are slightly blue-shifted compared to the literature values, which may be the result of different biochemical subunits present in the heme protein<sup>5</sup>. However, the difference between the spectra of Figures 4, 5, and 6 indicates that HO-1 is being produced and is binding with heme to form a complex which peaks at a different wavelength than heme alone.<br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
It was expected to see a peak for biliverdin as well, which would have occurred between 440 – 460nm according to the literature <sup>6,7</sup>, though we did not. This might have been due to the fact that the cultures containing heme and HO-1 producing BioBricks had some exposure to light. Researchers have noted in their methods that they had avoided light when handling biliverdin <sup>8,9</sup>. If biliverdin is photosensitive, this might be why these peaks did not appear in our wavelength scans.<br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In order to continue on this project track, the genetic effector system would have to be streamlined and optimized. More thorough kinetics testing for the production of heme as well as its metabolism by HO-1 would be required to maximize effector quality and concentration. Though many of the products of heme metabolism were likely produced, bilirubin was almost certainly not. <br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
As mentioned previously<sup>2</sup>, there is a lack of biliverdin reductases available for cloning into bacterial systems, especially mammalian. Recently the entire genome of <i>Synechocystis sp.</i> PP6803 was sequenced, along with a cyanobacterial biliverdin reductase homologue. This enzyme is used in the production of light-harvesting pigments called phycobilins which is a product in the breakdown of heme<sup>10</sup>. With more research into extraction of this gene from blue-green algae, a very useful biliverdin reductase BioBrick could be contributed the registry, allowing full degradation of heme to bilirubin. This would enable the microbe as well as the system to which it is contributing, to reap the strong antioxidant properties of this molecule.<br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
References:<br />
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1. Moffet, D.A., Foley J. & Hecht M.H. (2003). Midpoint reduction potentials and heme binding stoichiometries of de novo proteins from designed combinatorial libraries. <i>Biophysical Chemistry</i>. 105:231-239.<br />
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2. Frankenberg-Dinkel, N. (2004). Forum review: bacterial Heme Oxygenases. Antioxidants and Redox Signalling. 6:825 - 834.<br />
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3. Huber W. & Backes W. (2008). Quantitation of heme oxygenase 1: Heme titration increases yield of purified protein. <i>Analytical Biochemistry</i>. 373:167-169.<br />
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4. Migita C.T., Zhang X. & Yoshida T. (2003). Expression and characterization of cyanobacterium heme oxygenase, a key enzyme in the phycobilin synthesis. <i>European Journal of Biochemistry</i>. 270:687 – 698.<br />
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5. Wang N., Zhao X. & Lu Y. (2005). Role of Heme Types in Heme-Copper Oxidases: Effects of Replacing a Heme b with a Heme to Mimic in an Engineered Heme-Copper Center in Myoglobin. <i>Journal of American Chemical Society</i>. 127:16541 – 16547.<br />
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6. Yoshida T. & Kikuchi G. (1979). Purification and Properties of Heme Oxygenase from Rat Liver Microsomes. The <i>Journal of Biological Chemistry</i>. 254:4487 – 4491.<br />
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7. Ding Z.K. & Xu Y.Q. (2002). Purification and characterization of biliverdin IXalpha from Atlantic salmon (Salmo salar) bile. <i>Biochemistry</i>. 67:927 – 932. <br />
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8. Ollinger R., Yamashita K., Bilban M., Erat A., Kogler P., Thomas M., Csizmadia E., Usheva A., Margreiter R. & Bach F. H. (2007). Bilirubin and Biliverdin Treatment of Atherosclerotic Diseases. <i>Cell Cycle</i>. 6:39 – 43. <br />
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9. Ishikawa K., Navab M., Leitinger N., Fogelman A.M. & Lusis A. J. (1997). Induction of heme oxygenase-1 inhibits the monocyte transmigration induced by mildly oxidized LDL. <i>Journal of Clinical Investigation</i>. 100:1209-1216.<br />
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10. Cornejo J., Willows R. D. & Beale S. I. (1998). Phytobilin biosynthesis: cloning and expression of a gene encoding ferredoxin-dependent heme oxygenase from Synechocystis sp. PCC 6803. <i>The Plant Journal</i>. 15:99 – 107.<br />
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<a name="Part4"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Four: Future Directions</p></a><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Due to the delay of the synthesis of our binding construct, we were unable to test the presentation of the ITGA-4 chain of the VLA-4 antigen on <i>E. coli</i> outer membrane and the binding of the <i>E. coli</i> to endothelial cells expressing VCAM-1. In the future, we are planning to pursue this project further in the following areas.<br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"> <br />
<u>Construction</u><br />
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<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
1. Complete round 3 of PCR stitching and insert the binding construct into BioBrick backbone.<br />
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2. Sequence the binding construct and the SAA construct.<br />
</pre><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<u>Endothelial Adhesion Assay</u><br />
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<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
1. Transform the binding construct and constitutive GFP expression construct into E. coli cells. <br />
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2. Plate C166 murine endothelial cells on a 6-well plate. Incubate overnight. <br />
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3. Prepare serial dilutions of the binding E. coli and control cells (containing plasmid backbone). <br />
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4. Incubate C166 cells with E. coli for two hours and then wash wells 3X with PBS. <br />
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5. Quantify GFP emission using fluorimeter. <br />
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6. Alternative test: ELISA binding assay with recombinant VCAM-1 protein. <br />
</pre><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<u>Atrial Natriuretic Peptide</u><br />
</p><br />
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
1. Transform <i>E. coli</i> cells with ANP construct (P<sub>tet</sub>-RBS-Lpp-OmpA-Linker-TEVx2-Linker-ANP)<br />
and constitutive GFP expression construct. <br />
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2. Use the same protocol as endothelial adhesion assay to test the activation of membrane-bound <br />
guanylate cyclase (GCA) on endothelial cells by ANP. <br />
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3. Harvest the endothelial cells and prepare whole cell lysates. <br />
<br />
4. Run SDS-PAGE and Western blotting analysis with antibodies recognizing whole and <br />
phosphorylated Vasodilator Stimulated Phosphoprotein (VASP). VASP is phosphorylated <br />
by cGMP dependent kinase, which is activated by ANP. <br />
</pre><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<u>Inducible Effector System</u><br />
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In order to make our <i>E. coli</i> cells releasing the effectors at the site of plaque, we plan to put the SAA and HO-1 gene under the control of the P<sub>lux</sub> promoter, which will be activated by a threshold concentration of AHL at the site of plaque.<br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/2/27/QueensFigure7.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 7</i> Planned inducible effector construct.<br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
We plan to transform <i>E. coli</i> cells with the effector construct and the binding construct, and conduct the endothelial adhesion assay. We will then analyze the growth medium of the endothelial culture by SDS-PAGE and Western blot analysis to determine the concentration of AHL, SAA, and biliverdin in the medium. We will also determine the expression level of TEV protease, which is located downstream of a double terminator (BBa_B0015) and RBS (BBa_B0034) on the effector construct. Expression level of TEV protease directly contributes to the efficiency of detachment from the plaque and termination by the kill switch.<br />
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<a name="Part5"><br />
<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Safety Considerations</p></a><br />
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<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
We currently have no reason to believe that there are any safety concerns with our project.<br />
The iGEM safety questionnaire is below. <br />
<br />
1. Would any of your project ideas raise safety issues in terms of:<br />
* researcher safety,<br />
* public safety, or<br />
* environmental safety? <br />
<br />
At the current stage the project poses no safety concerns of any kind.<br />
<br />
2. Is there a local biosafety group, committee, or review board at your institution?<br />
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In short, yes there is an ethics and safety board that we must pass new ideas through.<br />
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3. What does your local biosafety group think about your project?<br />
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The project is considered within the approved guidelines.<br />
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4. Do any of the new BioBrick parts that you made this year raise any safety issues?<br />
* If yes, did you document these issues in the Registry? <br />
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The only issue is the toxicity of BBa_K214001 to host bacteria if over-expressed.<br />
This issue is mentioned in the registry.<br />
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Please note that Results are also available in PDF Format. <br />
Please click on the document you wish to view.<br />
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<a href="https://static.igem.org/mediawiki/2009/f/fc/QueensResultsJamesBogdanBryantHarry.pdf"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">Laboratory One: Harry, Bogdan, James, Bryant</p></a><br />
<a href="https://static.igem.org/mediawiki/2009/2/26/QueensResultsKateMike.pdf"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">Laboratory Two: Kate, Mike</p></a><br />
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<td align="left"><p style="font-size:175%;font-family:corbel;color:#172C4E;font-weight:bold"><i>Results</i><br />
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<p style="font-size=120%;font-family:palatino linotype;color:#ECB528"><br />
The following summarizes the results of the various aspects of this year's QGEM Project. <br />
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<a href="#Part1"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part One: SAA<br />
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<a href="#Part2"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Two: Binding Construct<br />
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<a href="#Part3"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Three: Heme and HO-1<br />
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<a href="#Part4"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Four: Future Directions<br />
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<a href="#Part5"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Safety Considerations<br />
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<a name="Part1"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part One: SAA</p></a><br />
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Serum amyloid A (SAA) featured prominently on our list of possible effectors to release at the site of atherosclerotic plaques due to research done at Queen’s University by Tam et al. (2005). This research showed that treatment of SAA caused macrophages to reverse and prevent esterification of cholesterol, thereby allowing it to be exported out of plaques by high-density lipoprotein (HDL). In order to get the SAA to be picked up by the macrophages we needed to produce large quantities of the molecule, and then secrete it extracellularly. In order to secrete the protein we decided on using the twin-arginine translocase (TAT) system, which can transport fully folded proteins outside the cell (Sargent et al., 2006) and is found in the majority of prokaryotes. We then fused the TAT signal sequence to the front of our SAA sequence in order to produce a construct that should be able produce and secrete the protein, allowing it to be taken up by macrophages.<br />
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<i>Fig. 1</i> SAA expression and secretion by <i>E. coli</i> cells. Cultures of <i>E. coli</i> cells containing either the SAA construct or control plasmid were spun down at 0, 4, and 12 hours after seeding. The culture medium and whole cell lysates were analyzed by SDS-PAGE and Western blot analysis with a polyclonal antibody recognizing whole SAA protein. <br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Our <i>E. coli</i> cells transformed with SAA construct did not appear to express or secrete SAA into the growth medium even after growing for 12 hours in optimum condition. This might be due to defective ligation of the Ptet-RBS fragment to the SAA-encoding gene. We plan to sequence the construct in the future. <br />
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References:<br />
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Benditt, E.P., Hoffman, J.S. & Eriksen, N. (1982). SAA, an apoprotein of HDL: its structure and function. <i> New York Academy of Sciences. </i> 82:183-189.<br />
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<br/><br />
Ancsin, J.B. & Kisilevsky, R. (1999). The heparin/heparin sulfate-binding site on apo-serum amyloid A. <i>Journal of Biological Chemistry.</i> Vol. 274, 11:7172-7181.<br />
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<a name="Part2"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Two: Binding Construct</p></a><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In our original design of the binding construct, constitutive promoter BBa_J23119 was used to control the expression of the chimeric protein receptor consisted of the Lpp-OmpA fusion, TEV protease cut sites and ITGA4 fragment. We ordered our construct to be synthesized by Mr. GENE in early June. However, Mr. GENE notified us in August that our construct appeared to be unstable in and toxic to <i>E. coli</i> cells. The reason might be that high expression level of Lpp-OmpA might interfere with bacterial physiology, causing severe growth inhibition and reduced viability. (Daugherty et al, 1999) Mr. Gene was able to give us a sequence-confirmed PCR product of the synthesized construct. Thus, we decided to replace P¬const with P¬tet (BBa_R0040) by PCR. Below is a schematic diagram of the construction process. <br />
</p><br />
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<i>Fig. 2</i> Flowchart of PCR stitching for constructing the binding construct. <br />
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Due to time constraint, we were only able to confirm the stitching of Ptet-RBS fragment to Lpp-OmpA-Linker-TEVx2-Linker fragment (PCR round 2) using Agarose gel electrophoresis. The third round of PCR stitching did not yield expected band. Construction of the binding construct is ongoing. <br />
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<i>Fig. 3</i> <i>In silico</i> model of the binding construct. <br />
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References:<br />
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Daugherty, P.S., Olsen, M.J., Iverson, B.L. & Georgiou, G. (1999). Development of an optimized expression system for the screening of antibody libraries displayed on the <i>Escherichia coli</i> surface. <i>Protein Engineering.</i> Vol. 12, 7:613-621.<br />
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Yang, Z., Lui, Q., Wang, Q. & Zhang, Y. (2008). Novel bacterial surface display systems based on outer membrane anchoring elements from the marine bacterium <i>Vibrio anguillarum</i>. <i> American Society for Microbiology.</i> Vol. 74, 14: 4359-4365.<br />
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<a name="Part3"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Three: Heme and HO-1</p></a><br />
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Before presenting our results some useful parts have been outlined briefly.<br />
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BioBrick and Construct Legend:<br />
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BioBrick I716390: Plasmid containing a composite of Heme A, B C & D with T7-promoter and RBS. Amp Resistance.<br />
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BioBrick K098010: HO and phycocaynobilin:ferredoxin oxidoreductase. KAN Resistance.<br />
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BioBrick I15009: HO-1 from <i>Synechocystis</i>. Amp Resistance.<br />
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NLM 350: Rough <i> E. coli</i> strain containing T7-polymerase. Queen's University Research<br />
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In order to determine whether or not the BioBrick I716390 submitted by the Berkeley iGEM team in 2007 was producing heme, NLM350. Overnight cultures containing 100 μg/mL ampicillin were set up and inoculated with NLM350 containing I716390. The culture was analyzed daily by wavelength scan in a spectrometer. We ran the wavelength scan twice, once with a blank of LB and once with a blank of LB culture containing NLM350 without the BioBrick. Both of the scan results were identical, which indicates that the peaks present are due to the BioBrick plasmid and not resulting from compounds produced by NLM350. The results from day three with a blank of LB are shown in Figure 4.<br />
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<i>Fig. 4</i> Results of a wavelength scan between 350 and 550nm of the NLM350 + I716390 culture three days after inoculation. Strongest peaks at 412nm. <br />
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In order to check for the presence of the heme/HO-1 complex, NLM350 + I716390 cells were made electrocompetent and two sets of electroporations were performed. The BioBricks K098010 and I15008 were taken up by NLM350 + I716390 and the resulting colonies were used to inoculate cultures containing 40 μg/mL kanamycin and 100 μg/mL ampicillin, respectively. Triplicate testing of NLM350 + 176390 + KO98010 as well as NLM350 + 176390 + I15008 was conducted over five days. Both tests produced similar strong peaks for heme/HO-1 complexes, ranging from 407-409 nm. Again, we ran the scans twice with blanks of LB and LB inoculated with NLM350 and found that results were identical. The results from day five with blanks of LB are shown in Figures 5 and 6.<br />
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<i>Fig. 5</i> Wavelength scan between 350 and 550 nm of the NLM350 + I716390 + K098010 culture 5 days after inoculation. Strong peak of 407.5 nm. <br />
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<i>Fig. 6</i> Wavelength scan between 350 and 550 nm of the NLM350 + I716390 + I15008 culture 5 days after inoculation. Strong peak of 407.5 nm. <br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
As can be seen from our results above, the peaks in the cultures containing heme and HO-1 producing BioBricks become clearer and occur at a lower absorbance, around 408nm, than the peaks from cultures containing only heme-producing BioBricks. Cultures containing only the heme-producing BioBrick has the strongest peaks at 412nm, which corresponds to the peak found by Moffet et al. in 2003 <sup>1</sup>. Since HO-1 does not have an absorbance peak of its own <sup>2,3,4</sup>, the presence of HO-1 can only be inferred by identifying the heme/HO-1 complexes via spectroscopy. According to several authors <sup>2,4</sup>, Soret bands for the oxy-bound form of the heme/HO-1 complex occur at 410nm. The peaks seen in Figures 5 and 6 are slightly blue-shifted compared to the literature values, which may be the result of different biochemical subunits present in the heme protein<sup>5</sup>. However, the difference between the spectra of Figures 4, 5, and 6 indicates that HO-1 is being produced and is binding with heme to form a complex which peaks at a different wavelength than heme alone.<br />
</p><br />
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It was expected to see a peak for biliverdin as well, which would have occurred between 440 – 460nm according to the literature <sup>6,7</sup>, though we did not. This might have been due to the fact that the cultures containing heme and HO-1 producing BioBricks had some exposure to light. Researchers have noted in their methods that they had avoided light when handling biliverdin <sup>8,9</sup>. If biliverdin is photosensitive, this might be why these peaks did not appear in our wavelength scans.<br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In order to continue on this project track, the genetic effector system would have to be streamlined and optimized. More thorough kinetics testing for the production of heme as well as its metabolism by HO-1 would be required to maximize effector quality and concentration. Though many of the products of heme metabolism were likely produced, bilirubin was almost certainly not. <br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
As mentioned previously<sup>2</sup>, there is a lack of biliverdin reductases available for cloning into bacterial systems, especially mammalian. Recently the entire genome of <i>Synechocystis sp.</i> PP6803 was sequenced, along with a cyanobacterial biliverdin reductase homologue. This enzyme is used in the production of light-harvesting pigments called phycobilins which is a product in the breakdown of heme<sup>10</sup>. With more research into extraction of this gene from blue-green algae, a very useful biliverdin reductase BioBrick could be contributed the registry, allowing full degradation of heme to bilirubin. This would enable the microbe as well as the system to which it is contributing, to reap the strong antioxidant properties of this molecule.<br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
References:<br />
<br/><br />
<br/><br />
1. Moffet, D.A., Foley J. & Hecht M.H. (2003). Midpoint reduction potentials and heme binding stoichiometries of de novo proteins from designed combinatorial libraries. <i>Biophysical Chemistry</i>. 105:231-239.<br />
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2. Frankenberg-Dinkel, N. (2004). Forum review: bacterial Heme Oxygenases. Antioxidants and Redox Signalling. 6:825 - 834.<br />
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<br/><br />
3. Huber W. & Backes W. (2008). Quantitation of heme oxygenase 1: Heme titration increases yield of purified protein. <i>Analytical Biochemistry</i>. 373:167-169.<br />
<br/><br />
<br/><br />
4. Migita C.T., Zhang X. & Yoshida T. (2003). Expression and characterization of cyanobacterium heme oxygenase, a key enzyme in the phycobilin synthesis. <i>European Journal of Biochemistry</i>. 270:687 – 698.<br />
<br/><br />
<br/><br />
5. Wang N., Zhao X. & Lu Y. (2005). Role of Heme Types in Heme-Copper Oxidases: Effects of Replacing a Heme b with a Heme to Mimic in an Engineered Heme-Copper Center in Myoglobin. <i>Journal of American Chemical Society</i>. 127:16541 – 16547.<br />
<br/><br />
<br/><br />
6. Yoshida T. & Kikuchi G. (1979). Purification and Properties of Heme Oxygenase from Rat Liver Microsomes. The <i>Journal of Biological Chemistry</i>. 254:4487 – 4491.<br />
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<br/><br />
7. Ding Z.K. & Xu Y.Q. (2002). Purification and characterization of biliverdin IXalpha from Atlantic salmon (Salmo salar) bile. <i>Biochemistry</i>. 67:927 – 932. <br />
<br/><br />
<br/><br />
8. Ollinger R., Yamashita K., Bilban M., Erat A., Kogler P., Thomas M., Csizmadia E., Usheva A., Margreiter R. & Bach F. H. (2007). Bilirubin and Biliverdin Treatment of Atherosclerotic Diseases. <i>Cell Cycle</i>. 6:39 – 43. <br />
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<br/><br />
9. Ishikawa K., Navab M., Leitinger N., Fogelman A.M. & Lusis A. J. (1997). Induction of heme oxygenase-1 inhibits the monocyte transmigration induced by mildly oxidized LDL. <i>Journal of Clinical Investigation</i>. 100:1209-1216.<br />
<br/><br />
<br/><br />
10. Cornejo J., Willows R. D. & Beale S. I. (1998). Phytobilin biosynthesis: cloning and expression of a gene encoding ferredoxin-dependent heme oxygenase from Synechocystis sp. PCC 6803. <i>The Plant Journal</i>. 15:99 – 107.<br />
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<a name="Part4"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Four: Future Directions</p></a><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Due to the delay of the synthesis of our binding construct, we were unable to test the presentation of the ITGA-4 chain of the VLA-4 antigen on <i>E. coli</i> outer membrane and the binding of the <i>E. coli</i> to endothelial cells expressing VCAM-1. In the future, we are planning to pursue this project further in the following areas.<br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"> <br />
<u>Construction</u><br />
</p><br />
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
1. Complete round 3 of PCR stitching and insert the binding construct into BioBrick backbone.<br />
<br />
2. Sequence the binding construct and the SAA construct.<br />
</pre><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<u>Endothelial Adhesion Assay</u><br />
</p><br />
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
1. Transform the binding construct and constitutive GFP expression construct into E. coli cells. <br />
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2. Plate C166 murine endothelial cells on a 6-well plate. Incubate overnight. <br />
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3. Prepare serial dilutions of the binding E. coli and control cells (containing plasmid backbone). <br />
<br />
4. Incubate C166 cells with E. coli for two hours and then wash wells 3X with PBS. <br />
<br />
5. Quantify GFP emission using fluorimeter. <br />
<br />
6. Alternative test: ELISA binding assay with recombinant VCAM-1 protein. <br />
</pre><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<u>Atrial Natriuretic Peptide</u><br />
</p><br />
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
1. Transform <i>E. coli</i> cells with ANP construct (P<sub>tet</sub>-RBS-Lpp-OmpA-Linker-TEVx2-Linker-ANP)<br />
and constitutive GFP expression construct. <br />
<br />
2. Use the same protocol as endothelial adhesion assay to test the activation of membrane-bound <br />
guanylate cyclase (GCA) on endothelial cells by ANP. <br />
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3. Harvest the endothelial cells and prepare whole cell lysates. <br />
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4. Run SDS-PAGE and Western blotting analysis with antibodies recognizing whole and <br />
phosphorylated Vasodilator Stimulated Phosphoprotein (VASP). VASP is phosphorylated <br />
by cGMP dependent kinase, which is activated by ANP. <br />
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<u>Inducible Effector System</u><br />
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In order to make our <i>E. coli</i> cells releasing the effectors at the site of plaque, we plan to put the SAA and HO-1 gene under the control of the P<sub>lux</sub> promoter, which will be activated by a threshold concentration of AHL at the site of plaque.<br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 7</i> Planned inducible effector construct.<br />
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We plan to transform <i>E. coli</i> cells with the effector construct and the binding construct, and conduct the endothelial adhesion assay. We will then analyze the growth medium of the endothelial culture by SDS-PAGE and Western blot analysis to determine the concentration of AHL, SAA, and biliverdin in the medium. We will also determine the expression level of TEV protease, which is located downstream of a double terminator (BBa_B0015) and RBS (BBa_B0034) on the effector construct. Expression level of TEV protease directly contributes to the efficiency of detachment from the plaque and termination by the kill switch.<br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Safety Considerations</p></a><br />
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We currently have no reason to believe that there are any safety concerns with our project.<br />
The iGEM safety questionnaire is below. <br />
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1. Would any of your project ideas raise safety issues in terms of:<br />
* researcher safety,<br />
* public safety, or<br />
* environmental safety? <br />
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At the current stage the project poses no safety concerns of any kind.<br />
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2. Is there a local biosafety group, committee, or review board at your institution?<br />
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In short, yes there is an ethics and safety board that we must pass new ideas through.<br />
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3. What does your local biosafety group think about your project?<br />
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The project is considered within the approved guidelines.<br />
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4. Do any of the new BioBrick parts that you made this year raise any safety issues?<br />
* If yes, did you document these issues in the Registry? <br />
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The only issue is the toxicity of BBa_K214001 to host bacteria if over-expressed.<br />
This issue is mentioned in the registry.<br />
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Please note that Results are also available in PDF Format. <br />
Please click on the document you wish to view.<br />
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<a href="https://static.igem.org/mediawiki/2009/f/fc/QueensResultsJamesBogdanBryantHarry.pdf"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">Laboratory One: Harry, Bogdan, James, Bryant</p></a><br />
<a href="https://static.igem.org/mediawiki/2009/2/26/QueensResultsKateMike.pdf"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">Laboratory Two: Kate, Mike</p></a><br />
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<td align="left"><p style="font-size:175%;font-family:corbel;color:#172C4E;font-weight:bold"><i>Results</i><br />
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The following summarizes the results of the various aspects of this year's QGEM Project. <br />
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Part One: SAA<br />
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Part Two: Binding Construct<br />
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Part Three: Heme and HO-1<br />
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<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Four: Future Directions<br />
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<a href="#Part5"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Safety Considerations<br />
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<a name="Part1"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part One: SAA</p></a><br />
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Serum amyloid A (SAA) featured prominently on our list of possible effectors to release at the site of atherosclerotic plaques due to research done at Queen’s University by Tam et al. (2005). This research showed that treatment of SAA caused macrophages to reverse and prevent esterification of cholesterol, thereby allowing it to be exported out of plaques by high-density lipoprotein (HDL). In order to get the SAA to be picked up by the macrophages we needed to produce large quantities of the molecule, and then secrete it extracellularly. In order to secrete the protein we decided on using the twin-arginine translocase (TAT) system, which can transport fully folded proteins outside the cell (Sargent et al., 2006) and is found in the majority of prokaryotes. We then fused the TAT signal sequence to the front of our SAA sequence in order to produce a construct that should be able produce and secrete the protein, allowing it to be taken up by macrophages.<br />
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<i>Fig. 1</i> SAA expression and secretion by <i>E. coli</i> cells. Cultures of <i>E. coli</i> cells containing either the SAA construct or control plasmid were spun down at 0, 4, and 12 hours after seeding. The culture medium and whole cell lysates were analyzed by SDS-PAGE and Western blot analysis with a polyclonal antibody recognizing whole SAA protein. <br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Our <i>E. coli</i> cells transformed with SAA construct did not appear to express or secrete SAA into the growth medium even after growing for 12 hours in optimum condition. This might be due to defective ligation of the Ptet-RBS fragment to the SAA-encoding gene. We plan to sequence the construct in the future. <br />
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References:<br />
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Benditt, E.P., Hoffman, J.S. & Eriksen, N. (1982). SAA, an apoprotein of HDL: its structure and function. <i> New York Academy of Sciences. </i> 82:183-189.<br />
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<br/><br />
Ancsin, J.B. & Kisilevsky, R. (1999). The heparin/heparin sulfate-binding site on apo-serum amyloid A. <i>Journal of Biological Chemistry.</i> Vol. 274, 11:7172-7181.<br />
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<a name="Part2"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Two: Binding Construct</p></a><br />
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In our original design of the binding construct, constitutive promoter BBa_J23119 was used to control the expression of the chimeric protein receptor consisted of the Lpp-OmpA fusion, TEV protease cut sites and ITGA4 fragment. We ordered our construct to be synthesized by Mr. GENE in early June. However, Mr. GENE notified us in August that our construct appeared to be unstable in and toxic to <i>E. coli</i> cells. The reason might be that high expression level of Lpp-OmpA might interfere with bacterial physiology, causing severe growth inhibition and reduced viability. (Daugherty et al, 1999) Mr. Gene was able to give us a sequence-confirmed PCR product of the synthesized construct. Thus, we decided to replace P¬const with P¬tet (BBa_R0040) by PCR. Below is a schematic diagram of the construction process. <br />
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<i>Fig. 2</i> Flowchart of PCR stitching for constructing the binding construct. <br />
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Due to time constraint, we were only able to confirm the stitching of Ptet-RBS fragment to Lpp-OmpA-Linker-TEVx2-Linker fragment (PCR round 2) using Agarose gel electrophoresis. The third round of PCR stitching did not yield expected band. Construction of the binding construct is ongoing. <br />
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<i>Fig. 3</i> <i>In silico</i> model of the binding construct. <br />
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References:<br />
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Daugherty, P.S., Olsen, M.J., Iverson, B.L. & Georgiou, G. (1999). Development of an optimized expression system for the screening of antibody libraries displayed on the <i>Escherichia coli</i> surface. <i>Protein Engineering.</i> Vol. 12, 7:613-621.<br />
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Yang, Z., Lui, Q., Wang, Q. & Zhang, Y. (2008). Novel bacterial surface display systems based on outer membrane anchoring elements from the marine bacterium <i>Vibrio anguillarum</i>. <i> American Society for Microbiology.</i> Vol. 74, 14: 4359-4365.<br />
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<a name="Part3"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Three: Heme and HO-1</p></a><br />
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Before presenting our results some useful parts have been outlined briefly.<br />
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BioBrick and Construct Legend:<br />
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BioBrick I716390: Plasmid containing a composite of Heme A, B C & D with T7-promoter and RBS. Amp Resistance.<br />
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BioBrick K098010: HO and phycocaynobilin:ferredoxin oxidoreductase. KAN Resistance.<br />
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BioBrick I15009: HO-1 from <i>Synechocystis</i>. Amp Resistance.<br />
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NLM 350: Rough <i> E. coli</i> strain containing T7-polymerase. Queen's University Research<br />
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In order to determine whether or not the BioBrick I716390 submitted by the Berkeley iGEM team in 2007 was producing heme, NLM350. Overnight cultures containing 100 μg/mL ampicillin were set up and inoculated with NLM350 containing I716390. The culture was analyzed daily by wavelength scan in a spectrometer. We ran the wavelength scan twice, once with a blank of LB and once with a blank of LB culture containing NLM350 without the BioBrick. Both of the scan results were identical, which indicates that the peaks present are due to the BioBrick plasmid and not resulting from compounds produced by NLM350. The results from day three with a blank of LB are shown in Figure 4.<br />
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<i>Fig. 4</i> Results of a wavelength scan between 350 and 550nm of the NLM350 + I716390 culture three days after inoculation. Strongest peaks at 412nm. <br />
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In order to check for the presence of the heme/HO-1 complex, NLM350 + I716390 cells were made electrocompetent and two sets of electroporations were performed. The BioBricks K098010 and I15008 were taken up by NLM350 + I716390 and the resulting colonies were used to inoculate cultures containing 40 μg/mL kanamycin and 100 μg/mL ampicillin, respectively. Triplicate testing of NLM350 + 176390 + KO98010 as well as NLM350 + 176390 + I15008 was conducted over five days. Both tests produced similar strong peaks for heme/HO-1 complexes, ranging from 407-409 nm. Again, we ran the scans twice with blanks of LB and LB inoculated with NLM350 and found that results were identical. The results from day five with blanks of LB are shown in Figures 5 and 6.<br />
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<i>Fig. 5</i> Wavelength scan between 350 and 550 nm of the NLM350 + I716390 + K098010 culture 5 days after inoculation. Strong peak of 407.5 nm. <br />
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<i>Fig. 6</i> Wavelength scan between 350 and 550 nm of the NLM350 + I716390 + I15008 culture 5 days after inoculation. Strong peak of 407.5 nm. <br />
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As can be seen from our results above, the peaks in the cultures containing heme and HO-1 producing BioBricks become clearer and occur at a lower absorbance, around 408nm, than the peaks from cultures containing only heme-producing BioBricks. Cultures containing only the heme-producing BioBrick has the strongest peaks at 412nm, which corresponds to the peak found by Moffet et al. in 2003 <sup>1</sup>. Since HO-1 does not have an absorbance peak of its own <sup>2,3,4</sup>, the presence of HO-1 can only be inferred by identifying the heme/HO-1 complexes via spectroscopy. According to several authors <sup>2,4</sup>, Soret bands for the oxy-bound form of the heme/HO-1 complex occur at 410nm. The peaks seen in Figures 5 and 6 are slightly blue-shifted compared to the literature values, which may be the result of different biochemical subunits present in the heme protein<sup>5</sup>. However, the difference between the spectra of Figures 4, 5, and 6 indicates that HO-1 is being produced and is binding with heme to form a complex which peaks at a different wavelength than heme alone.<br />
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It was expected to see a peak for biliverdin as well, which would have occurred between 440 – 460nm according to the literature <sup>6,7</sup>, though we did not. This might have been due to the fact that the cultures containing heme and HO-1 producing BioBricks had some exposure to light. Researchers have noted in their methods that they had avoided light when handling biliverdin <sup>8,9</sup>. If biliverdin is photosensitive, this might be why these peaks did not appear in our wavelength scans.<br />
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In order to continue on this project track, the genetic effector system would have to be streamlined and optimized. More thorough kinetics testing for the production of heme as well as its metabolism by HO-1 would be required to maximize effector quality and concentration. Though many of the products of heme metabolism were likely produced, bilirubin was almost certainly not. <br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
As mentioned previously<sup>2</sup>, there is a lack of biliverdin reductases available for cloning into bacterial systems, especially mammalian. Recently the entire genome of <i>Synechocystis sp.</i> PP6803 was sequenced, along with a cyanobacterial biliverdin reductase homologue. This enzyme is used in the production of light-harvesting pigments called phycobilins which is a product in the breakdown of heme<sup>10</sup>. With more research into extraction of this gene from blue-green algae, a very useful biliverdin reductase BioBrick could be contributed the registry, allowing full degradation of heme to bilirubin. This would enable the microbe as well as the system to which it is contributing, to reap the strong antioxidant properties of this molecule.<br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
References:<br />
<br/><br />
<br/><br />
1. Moffet, D.A., Foley J. & Hecht M.H. (2003). Midpoint reduction potentials and heme binding stoichiometries of de novo proteins from designed combinatorial libraries. <i>Biophysical Chemistry</i>. 105:231-239.<br />
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2. Frankenberg-Dinkel, N. (2004). Forum review: bacterial Heme Oxygenases. Antioxidants and Redox Signalling. 6:825 - 834.<br />
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3. Huber W. & Backes W. (2008). Quantitation of heme oxygenase 1: Heme titration increases yield of purified protein. <i>Analytical Biochemistry</i>. 373:167-169.<br />
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<br/><br />
4. Migita C.T., Zhang X. & Yoshida T. (2003). Expression and characterization of cyanobacterium heme oxygenase, a key enzyme in the phycobilin synthesis. <i>European Journal of Biochemistry</i>. 270:687 – 698.<br />
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<br/><br />
5. Wang N., Zhao X. & Lu Y. (2005). Role of Heme Types in Heme-Copper Oxidases: Effects of Replacing a Heme b with a Heme to Mimic in an Engineered Heme-Copper Center in Myoglobin. <i>Journal of American Chemical Society</i>. 127:16541 – 16547.<br />
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6. Yoshida T. & Kikuchi G. (1979). Purification and Properties of Heme Oxygenase from Rat Liver Microsomes. The <i>Journal of Biological Chemistry</i>. 254:4487 – 4491.<br />
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<br/><br />
7. Ding Z.K. & Xu Y.Q. (2002). Purification and characterization of biliverdin IXalpha from Atlantic salmon (Salmo salar) bile. <i>Biochemistry</i>. 67:927 – 932. <br />
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8. Ollinger R., Yamashita K., Bilban M., Erat A., Kogler P., Thomas M., Csizmadia E., Usheva A., Margreiter R. & Bach F. H. (2007). Bilirubin and Biliverdin Treatment of Atherosclerotic Diseases. <i>Cell Cycle</i>. 6:39 – 43. <br />
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9. Ishikawa K., Navab M., Leitinger N., Fogelman A.M. & Lusis A. J. (1997). Induction of heme oxygenase-1 inhibits the monocyte transmigration induced by mildly oxidized LDL. <i>Journal of Clinical Investigation</i>. 100:1209-1216.<br />
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10. Cornejo J., Willows R. D. & Beale S. I. (1998). Phytobilin biosynthesis: cloning and expression of a gene encoding ferredoxin-dependent heme oxygenase from Synechocystis sp. PCC 6803. <i>The Plant Journal</i>. 15:99 – 107.<br />
</p><br />
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<a name="Part4"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Four: Future Directions</p></a><br />
<br/><br />
<br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Due to the delay of the synthesis of our binding construct, we were unable to test the presentation of the ITGA-4 chain of the VLA-4 antigen on <i>E. coli</i> outer membrane and the binding of the <i>E. coli</i> to endothelial cells expressing VCAM-1. In the future, we are planning to pursue this project further in the following areas.<br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"> <br />
<u>Construction</u><br />
</p><br />
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
1. Complete round 3 of PCR stitching and insert the binding construct into BioBrick backbone.<br />
<br />
2. Sequence the binding construct and the SAA construct.<br />
</pre><br />
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<u>Endothelial Adhesion Assay</u><br />
</p><br />
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
1. Transform the binding construct and constitutive GFP expression construct into E. coli cells. <br />
<br />
2. Plate C166 murine endothelial cells on a 6-well plate. Incubate overnight. <br />
<br />
3. Prepare serial dilutions of the binding E. coli and control cells (containing plasmid backbone). <br />
<br />
4. Incubate C166 cells with E. coli for two hours and then wash wells 3X with PBS. <br />
<br />
5. Quantify GFP emission using fluorimeter. <br />
<br />
6. Alternative test: ELISA binding assay with recombinant VCAM-1 protein. <br />
</pre><br />
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<u>Atrial Natriuretic Peptide</u><br />
</p><br />
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
1. Transform <i>E. coli</i> cells with ANP construct (P<sub>tet</sub>-RBS-Lpp-OmpA-Linker-TEVx2-Linker-ANP)<br />
and constitutive GFP expression construct. <br />
<br />
2. Use the same protocol as endothelial adhesion assay to test the activation of membrane-bound <br />
guanylate cyclase (GCA) on endothelial cells by ANP. <br />
<br />
3. Harvest the endothelial cells and prepare whole cell lysates. <br />
<br />
4. Run SDS-PAGE and Western blotting analysis with antibodies recognizing whole and <br />
phosphorylated Vasodilator Stimulated Phosphoprotein (VASP). VASP is phosphorylated <br />
by cGMP dependent kinase, which is activated by ANP. <br />
</pre><br />
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<u>Inducible Effector System</u><br />
<br/><br />
<br/><br />
In order to make our <i>E. coli</i> cells releasing the effectors at the site of plaque, we plan to put the SAA and HO-1 gene under the control of the P<sub>lux</sub> promoter, which will be activated by a threshold concentration of AHL at the site of plaque.<br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/2/27/QueensFigure7.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 7</i> Planned inducible effector construct.<br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
We plan to transform <i>E. coli</i> cells with the effector construct and the binding construct, and conduct the endothelial adhesion assay. We will then analyze the growth medium of the endothelial culture by SDS-PAGE and Western blot analysis to determine the concentration of AHL, SAA, and biliverdin in the medium. We will also determine the expression level of TEV protease, which is located downstream of a double terminator (BBa_B0015) and RBS (BBa_B0034) on the effector construct. Expression level of TEV protease directly contributes to the efficiency of detachment from the plaque and termination by the kill switch.<br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Safety Considerations</p></a><br />
<br />
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We currently have no reason to believe that there are any safety concerns with our project.<br />
The iGEM safety questionnaire is below. <br />
<br />
1. Would any of your project ideas raise safety issues in terms of:<br />
* researcher safety,<br />
* public safety, or<br />
* environmental safety? <br />
<br />
At the current stage the project poses no safety concerns of any kind.<br />
<br />
2. Is there a local biosafety group, committee, or review board at your institution?<br />
<br />
In short, yes there is an ethics and safety board that we must pass new ideas through.<br />
<br />
3. What does your local biosafety group think about your project?<br />
<br />
The project is considered within the approved guidelines.<br />
<br />
4. Do any of the new BioBrick parts that you made this year raise any safety issues?<br />
* If yes, did you document these issues in the Registry? <br />
<br />
The only issue is the toxicity of BBa_K214001 to host bacteria if over-expressed.<br />
This issue is mentioned in the registry.<br />
</pre><br />
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Please note that Results are also available in PDF Format. <br />
Please click on the document you wish to view.<br />
</pre><br />
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<a href="https://static.igem.org/mediawiki/2009/2/26/QueensResultsKateMike.pdf"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">Laboratory Two: Kate, Mike</p></a><br />
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<br />
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All Part Documentation has been entered on the appropriate BioBrick Registry Part Page.<br />
The QGEM Team has submitted three parts this year. <br />
Please click on the part you wish to view more information for.<br />
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<a href="http://partsregistry.org/Part:BBa_K214001"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">BBa K214001 - ANP Outer Membrane Expression Construct</p></a><br />
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<a href="http://partsregistry.org/Part:BBa_K214002"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">BBa K214002 - Mature Atrial Natriuretic Peptide (ANP)</p></a><br />
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<a href="http://partsregistry.org/Part:BBa_K214003"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">BBa K214003 - Integrin Alpha 4 (ITGA4) Beta Epitope</p></a><br />
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</html></div>ParthivAhttp://2009.igem.org/Team:Queens/ResultsTeam:Queens/Results2009-10-21T02:03:26Z<p>ParthivA: </p>
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<td align="left"><p style="font-size:175%;font-family:corbel;color:#172C4E;font-weight:bold"><i>Results</i><br />
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<p style="font-size=120%;font-family:palatino linotype;color:#ECB528"><br />
The following summarizes the results of the various aspects of this year's QGEM Project. <br />
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<a href="#Part1"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part One: SAA<br />
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<a href="#Part2"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Two: Binding Construct<br />
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<a href="#Part3"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Three: Heme and HO-1<br />
</p></a><br />
<a href="#Part4"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Four: Future Directions<br />
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<a name="Part1"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part One: SAA</p></a><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Serum amyloid A (SAA) featured prominently on our list of possible effectors to release at the site of atherosclerotic plaques due to research done at Queen’s University by Tam et al. (2005). This research showed that treatment of SAA caused macrophages to reverse and prevent esterification of cholesterol, thereby allowing it to be exported out of plaques by high-density lipoprotein (HDL). In order to get the SAA to be picked up by the macrophages we needed to produce large quantities of the molecule, and then secrete it extracellularly. In order to secrete the protein we decided on using the twin-arginine translocase (TAT) system, which can transport fully folded proteins outside the cell (Sargent et al., 2006) and is found in the majority of prokaryotes. We then fused the TAT signal sequence to the front of our SAA sequence in order to produce a construct that should be able produce and secrete the protein, allowing it to be taken up by macrophages.<br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/c/cb/QueensFigure1.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 1</i> SAA expression and secretion by <i>E. coli</i> cells. Cultures of <i>E. coli</i> cells containing either the SAA construct or control plasmid were spun down at 0, 4, and 12 hours after seeding. The culture medium and whole cell lysates were analyzed by SDS-PAGE and Western blot analysis with a polyclonal antibody recognizing whole SAA protein. <br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Our <i>E. coli</i> cells transformed with SAA construct did not appear to express or secrete SAA into the growth medium even after growing for 12 hours in optimum condition. This might be due to defective ligation of the Ptet-RBS fragment to the SAA-encoding gene. We plan to sequence the construct in the future. <br />
</p><br />
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References:<br />
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Benditt, E.P., Hoffman, J.S. & Eriksen, N. (1982). SAA, an apoprotein of HDL: its structure and function. <i> New York Academy of Sciences. </i> 82:183-189.<br />
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<br/><br />
Ancsin, J.B. & Kisilevsky, R. (1999). The heparin/heparin sulfate-binding site on apo-serum amyloid A. <i>Journal of Biological Chemistry.</i> Vol. 274, 11:7172-7181.<br />
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<a name="Part2"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Two: Binding Construct</p></a><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In our original design of the binding construct, constitutive promoter BBa_J23119 was used to control the expression of the chimeric protein receptor consisted of the Lpp-OmpA fusion, TEV protease cut sites and ITGA4 fragment. We ordered our construct to be synthesized by Mr. GENE in early June. However, Mr. GENE notified us in August that our construct appeared to be unstable in and toxic to <i>E. coli</i> cells. The reason might be that high expression level of Lpp-OmpA might interfere with bacterial physiology, causing severe growth inhibition and reduced viability. (Daugherty et al, 1999) Mr. Gene was able to give us a sequence-confirmed PCR product of the synthesized construct. Thus, we decided to replace P¬const with P¬tet (BBa_R0040) by PCR. Below is a schematic diagram of the construction process. <br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/0/08/QueensFigure2.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 2</i> Flowchart of PCR stitching for constructing the binding construct. <br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Due to time constraint, we were only able to confirm the stitching of Ptet-RBS fragment to Lpp-OmpA-Linker-TEVx2-Linker fragment (PCR round 2) using Agarose gel electrophoresis. The third round of PCR stitching did not yield expected band. Construction of the binding construct is ongoing. <br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/1/14/QueensFigure3.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 3</i> <i>In silico</i> model of the binding construct. <br />
</p><br />
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References:<br />
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Daugherty, P.S., Olsen, M.J., Iverson, B.L. & Georgiou, G. (1999). Development of an optimized expression system for the screening of antibody libraries displayed on the <i>Escherichia coli</i> surface. <i>Protein Engineering.</i> Vol. 12, 7:613-621.<br />
<br/><br />
<br/><br />
Yang, Z., Lui, Q., Wang, Q. & Zhang, Y. (2008). Novel bacterial surface display systems based on outer membrane anchoring elements from the marine bacterium <i>Vibrio anguillarum</i>. <i> American Society for Microbiology.</i> Vol. 74, 14: 4359-4365.<br />
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<a name="Part3"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Three: Heme and HO-1</p></a><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Before presenting our results some useful parts have been outlined briefly.<br />
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BioBrick and Construct Legend:<br />
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BioBrick I716390: Plasmid containing a composite of Heme A, B C & D with T7-promoter and RBS. Amp Resistance.<br />
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BioBrick K098010: HO and phycocaynobilin:ferredoxin oxidoreductase. KAN Resistance.<br />
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BioBrick I15009: HO-1 from <i>Synechocystis</i>. Amp Resistance.<br />
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NLM 350: Rough <i> E. coli</i> strain containing T7-polymerase. Queen's University Research<br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In order to determine whether or not the BioBrick I716390 submitted by the Berkeley iGEM team in 2007 was producing heme, NLM350. Overnight cultures containing 100 μg/mL ampicillin were set up and inoculated with NLM350 containing I716390. The culture was analyzed daily by wavelength scan in a spectrometer. We ran the wavelength scan twice, once with a blank of LB and once with a blank of LB culture containing NLM350 without the BioBrick. Both of the scan results were identical, which indicates that the peaks present are due to the BioBrick plasmid and not resulting from compounds produced by NLM350. The results from day three with a blank of LB are shown in Figure 4.<br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/c/ce/QueensFigure4.png"></center><br />
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<i>Fig. 4</i> Results of a wavelength scan between 350 and 550nm of the NLM350 + I716390 culture three days after inoculation. Strongest peaks at 412nm. <br />
</p><br />
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In order to check for the presence of the heme/HO-1 complex, NLM350 + I716390 cells were made electrocompetent and two sets of electroporations were performed. The BioBricks K098010 and I15008 were taken up by NLM350 + I716390 and the resulting colonies were used to inoculate cultures containing 40 μg/mL kanamycin and 100 μg/mL ampicillin, respectively. Triplicate testing of NLM350 + 176390 + KO98010 as well as NLM350 + 176390 + I15008 was conducted over five days. Both tests produced similar strong peaks for heme/HO-1 complexes, ranging from 407-409 nm. Again, we ran the scans twice with blanks of LB and LB inoculated with NLM350 and found that results were identical. The results from day five with blanks of LB are shown in Figures 5 and 6.<br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/2/2b/QueensFigure5.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 5</i> Wavelength scan between 350 and 550 nm of the NLM350 + I716390 + K098010 culture 5 days after inoculation. Strong peak of 407.5 nm. <br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/7/74/QueensFigure6.png"></center><br />
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<i>Fig. 6</i> Wavelength scan between 350 and 550 nm of the NLM350 + I716390 + I15008 culture 5 days after inoculation. Strong peak of 407.5 nm. <br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
As can be seen from our results above, the peaks in the cultures containing heme and HO-1 producing BioBricks become clearer and occur at a lower absorbance, around 408nm, than the peaks from cultures containing only heme-producing BioBricks. Cultures containing only the heme-producing BioBrick has the strongest peaks at 412nm, which corresponds to the peak found by Moffet et al. in 2003 <sup>1</sup>. Since HO-1 does not have an absorbance peak of its own <sup>2,3,4</sup>, the presence of HO-1 can only be inferred by identifying the heme/HO-1 complexes via spectroscopy. According to several authors <sup>2,4</sup>, Soret bands for the oxy-bound form of the heme/HO-1 complex occur at 410nm. The peaks seen in Figures 5 and 6 are slightly blue-shifted compared to the literature values, which may be the result of different biochemical subunits present in the heme protein<sup>5</sup>. However, the difference between the spectra of Figures 4, 5, and 6 indicates that HO-1 is being produced and is binding with heme to form a complex which peaks at a different wavelength than heme alone.<br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
It was expected to see a peak for biliverdin as well, which would have occurred between 440 – 460nm according to the literature <sup>6,7</sup>, though we did not. This might have been due to the fact that the cultures containing heme and HO-1 producing BioBricks had some exposure to light. Researchers have noted in their methods that they had avoided light when handling biliverdin <sup>8,9</sup>. If biliverdin is photosensitive, this might be why these peaks did not appear in our wavelength scans.<br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In order to continue on this project track, the genetic effector system would have to be streamlined and optimized. More thorough kinetics testing for the production of heme as well as its metabolism by HO-1 would be required to maximize effector quality and concentration. Though many of the products of heme metabolism were likely produced, bilirubin was almost certainly not. <br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
As mentioned previously<sup>2</sup>, there is a lack of biliverdin reductases available for cloning into bacterial systems, especially mammalian. Recently the entire genome of <i>Synechocystis sp.</i> PP6803 was sequenced, along with a cyanobacterial biliverdin reductase homologue. This enzyme is used in the production of light-harvesting pigments called phycobilins which is a product in the breakdown of heme<sup>10</sup>. With more research into extraction of this gene from blue-green algae, a very useful biliverdin reductase BioBrick could be contributed the registry, allowing full degradation of heme to bilirubin. This would enable the microbe as well as the system to which it is contributing, to reap the strong antioxidant properties of this molecule.<br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
References:<br />
<br/><br />
<br/><br />
1. Moffet, D.A., Foley J. & Hecht M.H. (2003). Midpoint reduction potentials and heme binding stoichiometries of de novo proteins from designed combinatorial libraries. <i>Biophysical Chemistry</i>. 105:231-239.<br />
<br/><br />
<br/><br />
2. Frankenberg-Dinkel, N. (2004). Forum review: bacterial Heme Oxygenases. Antioxidants and Redox Signalling. 6:825 - 834.<br />
<br/><br />
<br/><br />
3. Huber W. & Backes W. (2008). Quantitation of heme oxygenase 1: Heme titration increases yield of purified protein. <i>Analytical Biochemistry</i>. 373:167-169.<br />
<br/><br />
<br/><br />
4. Migita C.T., Zhang X. & Yoshida T. (2003). Expression and characterization of cyanobacterium heme oxygenase, a key enzyme in the phycobilin synthesis. <i>European Journal of Biochemistry</i>. 270:687 – 698.<br />
<br/><br />
<br/><br />
5. Wang N., Zhao X. & Lu Y. (2005). Role of Heme Types in Heme-Copper Oxidases: Effects of Replacing a Heme b with a Heme to Mimic in an Engineered Heme-Copper Center in Myoglobin. <i>Journal of American Chemical Society</i>. 127:16541 – 16547.<br />
<br/><br />
<br/><br />
6. Yoshida T. & Kikuchi G. (1979). Purification and Properties of Heme Oxygenase from Rat Liver Microsomes. The <i>Journal of Biological Chemistry</i>. 254:4487 – 4491.<br />
<br/><br />
<br/><br />
7. Ding Z.K. & Xu Y.Q. (2002). Purification and characterization of biliverdin IXalpha from Atlantic salmon (Salmo salar) bile. <i>Biochemistry</i>. 67:927 – 932. <br />
<br/><br />
<br/><br />
8. Ollinger R., Yamashita K., Bilban M., Erat A., Kogler P., Thomas M., Csizmadia E., Usheva A., Margreiter R. & Bach F. H. (2007). Bilirubin and Biliverdin Treatment of Atherosclerotic Diseases. <i>Cell Cycle</i>. 6:39 – 43. <br />
<br/><br />
<br/><br />
9. Ishikawa K., Navab M., Leitinger N., Fogelman A.M. & Lusis A. J. (1997). Induction of heme oxygenase-1 inhibits the monocyte transmigration induced by mildly oxidized LDL. <i>Journal of Clinical Investigation</i>. 100:1209-1216.<br />
<br/><br />
<br/><br />
10. Cornejo J., Willows R. D. & Beale S. I. (1998). Phytobilin biosynthesis: cloning and expression of a gene encoding ferredoxin-dependent heme oxygenase from Synechocystis sp. PCC 6803. <i>The Plant Journal</i>. 15:99 – 107.<br />
</p><br />
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<a name="Part4"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Four: Future Directions</p></a><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Due to the delay of the synthesis of our binding construct, we were unable to test the presentation of the ITGA-4 chain of the VLA-4 antigen on <i>E. coli</i> outer membrane and the binding of the <i>E. coli</i> to endothelial cells expressing VCAM-1. In the future, we are planning to pursue this project further in the following areas.<br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"> <br />
<u>Construction</u><br />
</p><br />
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
1. Complete round 3 of PCR stitching and insert the binding construct into BioBrick backbone.<br />
<br />
2. Sequence the binding construct and the SAA construct.<br />
</pre><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<u>Endothelial Adhesion Assay</u><br />
</p><br />
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
1. Transform the binding construct and constitutive GFP expression construct into E. coli cells. <br />
<br />
2. Plate C166 murine endothelial cells on a 6-well plate. Incubate overnight. <br />
<br />
3. Prepare serial dilutions of the binding E. coli and control cells (containing plasmid backbone). <br />
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4. Incubate C166 cells with E. coli for two hours and then wash wells 3X with PBS. <br />
<br />
5. Quantify GFP emission using fluorimeter. <br />
<br />
6. Alternative test: ELISA binding assay with recombinant VCAM-1 protein. <br />
</pre><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<u>Atrial Natriuretic Peptide</u><br />
</p><br />
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
1. Transform <i>E. coli</i> cells with ANP construct (P<sub>tet</sub>-RBS-Lpp-OmpA-Linker-TEVx2-Linker-ANP)<br />
and constitutive GFP expression construct. <br />
<br />
2. Use the same protocol as endothelial adhesion assay to test the activation of membrane-bound <br />
guanylate cyclase (GCA) on endothelial cells by ANP. <br />
<br />
3. Harvest the endothelial cells and prepare whole cell lysates. <br />
<br />
4. Run SDS-PAGE and Western blotting analysis with antibodies recognizing whole and <br />
phosphorylated Vasodilator Stimulated Phosphoprotein (VASP). VASP is phosphorylated <br />
by cGMP dependent kinase, which is activated by ANP. <br />
</pre><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<u>Inducible Effector System</u><br />
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In order to make our <i>E. coli</i> cells releasing the effectors at the site of plaque, we plan to put the SAA and HO-1 gene under the control of the P<sub>lux</sub> promoter, which will be activated by a threshold concentration of AHL at the site of plaque.<br />
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<i>Fig. 7</i> Planned inducible effector construct.<br />
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We plan to transform <i>E. coli</i> cells with the effector construct and the binding construct, and conduct the endothelial adhesion assay. We will then analyze the growth medium of the endothelial culture by SDS-PAGE and Western blot analysis to determine the concentration of AHL, SAA, and biliverdin in the medium. We will also determine the expression level of TEV protease, which is located downstream of a double terminator (BBa_B0015) and RBS (BBa_B0034) on the effector construct. Expression level of TEV protease directly contributes to the efficiency of detachment from the plaque and termination by the kill switch.<br />
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Please note that Results are also available in PDF Format. <br />
Please click on the document you wish to view.<br />
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<a href="https://static.igem.org/mediawiki/2009/2/26/QueensResultsKateMike.pdf"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">Laboratory Two: Kate, Mike</p></a><br />
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</html></div>ParthivAhttp://2009.igem.org/Team:Queens/ProjectTeam:Queens/Project2009-10-21T02:03:15Z<p>ParthivA: </p>
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The QGEM project centres on the treatment of atherosclerosis by targeted drug delivery from <i>E.coli</i>. The project is broken down into three major components. The first, and the main, component is the cell membrane protein that allows <i>E.coli</i> to bind to the site of atherosclerotic plaque. The second component is the inducible effector system that produces factors to treat the plaque. The last component is the terminator system that detaches <i>E.coli</i> from the plaque and inhibits proliferation of <i>E.coli</i> in the blood stream.<br />
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Part One: Binding System<br />
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Part Two: Effector System<br />
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Part Three: Cleavage and Termination<br />
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In order to target <i>E.coli</i> cells to atherosclerotic plaques, we selected the VLA-4/VCAM-1 binding system. VCAM-1 (vascular cell adhesion molecule-1) is a specific marker that is commonly found at sites of damaged endothelium, such as the site of an atherosclerotic plaque. VLA-4 (Integrin α4β1 or very late antigen-4) is normally expressed on leukocyte membranes, and it directs the leukocytes to damaged sites in the vascular system. Studies have shown that the ITGA-4 chain of VLA-4 antigen can sufficient bind to VCAM-1.<br />
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Our goal is to express a fragment of ITGA-4 on the surface of the E.coli plasma membrane. To do this, we have modified the attachment system previously explored by NYMU-Taipei team 2008. Below is a schematic diagram of the binding construct. <br />
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Pconst – RBS – LppOmpA – linker - TEV cleavage site X2 – linker – VLA-4 – STOP <br />
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The Lpp (Lipoprotein signal peptide) and OmpA (Outer membrane protein A) fusion results in presentation of the protein at the outer membrane of E.coli. Two cleavage sites for Tobacco-Etch Virus (TEV) protease are inserted between two linker regions. The TEV protease is a part of the terminator system that detaches E.coli from the endothelial cells. The length of the linker sequence between TEV cut site and VLA-4 can be adjusted in order to optimize binding efficiency.<br />
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Ideally, our effector system should only begin to produce the effectors once a threshold density of <i>E.coli</i> cells is reached at the plaque site. Since the system is in a prokaryotic chassis, signal transduction via the VLA-4/VCAM binding is not a realistic approach. Instead, we choose to employ the highly characterized LuxI/LuxR quorum sensing system in bacteria. Briefly, the effector proteins to be released at the site of plaque are placed under the control of pLux promoter, activated by a threshold concentration of AHL. AHL is constitutively produced by our cells and it will reach the threshold concentration once a sufficient amount of E.coli cells is bound to the plaque. <br />
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In terms of treatment options we explored a number of routes; however, the following three proved to be the most feasible and advantageous choices. While we are not medical professionals and have not addressed the concept of dosage, the effectors we have chosen are such that mild overdoses would not be of great concern (i.e. beneficial effects far outweigh negative effects). We have also chosen these effectors for having minimal side effects. <br />
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<b> 1. Serum Amyloid A</b><br />
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SAA converts cholesterol stored in plaques into a form more accessible by High Density Lipoprotein (HDL), which is the body’s natural mechanism for returning cholesterol to the liver for packaging, metabolism and/or excretion. It is our hope that this effector, when released specifically at the site of atherosclerosis, will induce plaque regression. <br />
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However, the clinical effectiveness of SAA is still in doubt for several reasons:<br />
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* Not all atherosclerotic plaques are associated with cholesterol.<br />
* The body can naturally produce SAA at an astoundingly high rate, given the correct signals.<br />
* We are unsure if the molecule will reach the area of the plaque it is effective in.<br />
* We are unsure if the body produces enough HDL to clear atherosclerotic plaques.<br />
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<b> 2. Heme Oxygenase 1 (HO-1)</b><br />
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The HO-1 effector system involves the production of heme and HO-1. HO-1 catalyzes the degradation of heme into carbon monoxide (CO), biliverdin (BV), and free iron (Fe++), all of which have therapeutically beneficial effects on atherosclerotic plaques. CO acts as a local vasodilator, which may minimize chances of plaque rupture, as well as retard plaque growth. BV inhibits the proliferation of vascular smooth muscle cells, which has been shown to lead to stenosis of blood vessels. Fe++ induces the production of ferritin, which has an antioxidant effect that may protect the surrounding tissues from free radical attack and reduce the chance of plaque rupture. <br />
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<b> 3. Atrial Natriuretic Peptide (ANP)</b><br />
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ANP activates membrane-bound guanylate cyclase (GCA), which increases the level of intracellular cGMP, a signalling molecule that mediates vasodilation. The effect of ANP is similar to that of carbon monoxide. cGMP also possess an anti-clotting effect, although it is unlikely that we can take advantage of this since platelets do not have membrane-bound guanylate cyclase.<br />
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After our chassis has been bound to the site of the plaque for a period of time, it should begin to produce DNases and proteases. The DNases will function to sheer the bacterial genome, making it unable to proliferate in the blood; this functions as a safe-guard against bacteremia. The proteases serve to detach the chassis from the plaque site, so as to avoid a build-up of dead cell debris in an already inflamed area.<br />
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Last Updated: October 20, 2009 by Fr<sub>3</sub>P</center></font><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Parthiv Amin</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Nickname: francium phosphide (Fr<sub>3</sub>P) / fatal attrACTION</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Program: B.Sc. BioChemical Engineering 2011</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">QGEM Position: Student Volunteer/Webmaster</P><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
About: I am currently entering my third year in BioChemical Engineering at Queen's University. I garnered an interest in iGEM due to my fascination of the field of biofuels, especially the use of engineered algal strains for biodiesel production. As a volunteer member of the QGEM, my focus is mostly on the technological aspects of iGEM: maintaining the Wiki, compiling data, and animations. Outside of QGEM I'll be working on the Queen's BAJA SAE car, as well as getting some of the paperwork filed for a new chemical engineering design team a few of my friends and I are developing. In my free time I'll be playing my guitar & Halo 2, surfing the net, and of course doing a metric tonne of homework.</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Michael Freeman</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Nickname: The Naked Sasquatch / Microbicurious</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Program: B.Sc. BioChemical Engineering 2010</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">QGEM Position: Student Employee</P><br />
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About: I am in my fourth year at Queen's after transferring degree programs from chemistry to biochemical engineering. iGEM is a competition that grabbed my interest immediately because of the unbelievable possibilities synthetic biology makes available. Solving problems in general is incredibly interesting to me, and solving them in unconventional or obscure ways is even more exciting. I came into iGEM with a few project ideas like including engineered microbes in specific waste water treatment processes, creating a more conclusive indicator test for aggressive prostate cancers, and creating some microbial suspension which could be ingested and used to break down cellulose in the human stomach, a hugely abundant source of food and energy. For iGEM this year I was a 'wet labber' responsible for creating, engineering and testing our Heme/Heme-Oxygenase system which would be used as one of our plaque degrading effectors. In order to keep from going nuts from talking with bacteria all day I bar tend at night, I compete in triathlons and varsity rowing for Queen's, I am deeply in love with traveling and hosting travelers, and I have just entered the wonderful (and expensive) world of skydiving! Ever touched a cloud? This guy has.</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Jonas Elliott Gerson</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Nickname: Sex Pili</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Program:</p><br />
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About: More to come later</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">James MacLeod</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Nickname: Bond</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Program: B.Sc.H. Biology</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">QGEM Position: Co-Founder</P><br />
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About: I am currently in my third year at Queen's University pursuing a Bachelor's of Science (BScH). I was born in Britain, lived in the Middle East for a period, and now reside in Canada. I love the skiing that Canada has to offer, and I'm very excited that Queen's will be participating in the 2009 Jamboree and am looking forward to meeting teams from across the world! </p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Bogdan Momciu</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Nickname: The Count </p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Program: B.Sc.H. Life Sciences SSP 2010</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">QGEM Position: Student Employee</P><br />
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About: I am 4th year student in the Queen's University Life Sciences program with a focus towards research and biomedical applications. Once I heard about iGEM I was greatly enthusiastic about the idea due to the sheer number of innovations this kind of cooperative initiative could end up producing. During this past summer I worked on the design and synthesis of our SAA and Endothelial Cell Binding Constructs as well as testing and troubleshooting.<br />
In my spare time I like to play a variety of sports including hockey, soccer, and volleyball. </p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Chris Palmer</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Nickname: FRECsecutioner</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Program: B.Sc. Engineering Chemistry 2012</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">QGEM Position: Student Volunteer</P><br />
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About: I am a second year student in the Engineering Chemistry program at Queen's university, but I have always had a strong interest in microbiology and biochemistry as well. As a result, I was intrigued by the opportunities presented by the iGEM competition. As soon as I heard that Queen's was forming an iGEM team, I knew that I wanted to be a part of it and help get it off the ground in its first year. I have thoroughly enjoyed my time this summer working with everyone else on the team, and I look forward to meeting people and hearing about everyone's exciting projects at the jamboree!</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Bryant Shum</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Nickname: Blossom</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Program: Life Science SSP 2011</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">QGEM Position: Student Employee</P><br />
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About: I am currently a third year undergraduate student at Queen's, with a concentration in Life Science. Given the nature of my studies, I have always been fascinated by the sheer variety of phenotypes that can arise from a simple genetic code; therefore, the chance to play "Lego" with these complex molecules of life was an irresistible opportunity for me. Specifically, my role on the team this year was to help construct the genetic components required for plaque binding, as well as to conduct background research on the pathogenesis of atherosclerosis. And, so far, this has proven to be a challenging but unforgettable project.</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Kate Turner</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Nickname: Chicken Joe / The Octagon</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Program: B.Sc. BioChemical Engineering 2010</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">QGEM Position: Student Employee</P><br />
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About: I am in my fourth year of a chemical engineering degree at Queen’s, focusing on the biomedical stream. I spent last summer working as a field engineer in the Alberta oil patch, but this year I wanted to broaden my horizons and focus on biotechnology or biomedical research. I was especially interested in iGEM due to the wide-ranging applications of synthetic biology – from smog cleanup to combating tumours to degrading plastics – and now I hope to continue in this field after graduation. During the summer I worked in one of the labs testing the heme/HO-1 effector system and researching the amelioration of atherosclerotic plaques by CO, HO-1, and biliverdin. Outside of QGEM, I spent my summer camping, BBQing, and reading like a fiend.</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Christopher Yan</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Nickname: YanTASTIC</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Program: B.Sc.H. Life Sciences 2012</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">QGEM Position: Student Employee</P><br />
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About: I am a second year Life Sciences student at Queen's University. This is my first time participating in an event of such grand scale and am very much looking forward to the Jamboree.</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Harry Zhou</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Nickname: Bubbles</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Program: B.Sc.H. Biochemistry 2011</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">QGEM Position: Co-Founder</P><br />
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About: I am a third-year Biochemistry student at Queen’s University. Starting in high school, I had a strong interest in biotechnology that has the potential of benefiting health care and environment. When I was in grade 12, a professor at University of Calgary introduced me to synthetic biology and the iGEM competition and I volunteered for the UCalgary team in 2007. I thoroughly enjoyed the experience and was fascinated by the iGEM program, which provides a platform for students to tackle important problems concerning the environment, health care, and new technology. This was why I decided to put together a multidisciplinary team of undergraduate students at Queen’s to participate in this year’s iGEM competition. I had tons of fun this summer planning and carrying out experiments for the project and working with students from other discipline. In my spare time, I like to play sports, guitar, and read. </p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Ian D. Chin-Sang, Ph.D.</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Faculty: Arts & Science</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Department: Biology</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Position: Associate Professor and CCS/NCIC Research Scientist</P><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Research: Molecular Genetics of <i>C. elegans</i> Development</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Peter A. Greer, Ph.D.</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Faculty: Arts & Science</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Department: BioChemistry</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Position: Professor of Biochemistry and Pathology & Molecular Medicine</P><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Research: Cancer Signal Transduction</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">David P. Lebrun, MD</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Faculty: Medicine</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Department: Pathology and Molecular Medicine</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Position: Associate Professor of Pathology & Molecular Medicine</P><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Research: Molecular Mechanisms of Leukemia & Clinicopathological Correlations in Malignant Lymphoma</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Nancy Martin, Ph.D.</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Faculty: Arts & Science</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Department: Microbiology and Immunology</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Position: Associate Professor</P><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Research: Sensing and Adaptation to Environmental Changes in <i>Salmonella typhimurium</i></p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Ronald J. Neufeld, Ph.D.</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Faculty: Applied Science</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Department: Chemical Engineering</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Position: Professor</P><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Research: Bioencapsulation & Bioactives Processing and Controlled Release</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Waheed Sangrar, Ph.D.</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Faculty: Medicine</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Department: Pathology and Molecular Medicine</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Position: Assistant Professor</P><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Research: Breast Cancer</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Virginia K. Walker, Ph.D.</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Faculty: Arts & Science</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Department: Biology</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Position: Professor</P><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Research: Genetics and Molecular Biology of Resistance</p><br />
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Last Updated: October 20, 2009 by Fr<subs>3</subs>P</p></center><br />
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The Queen's Genetically Engineered Machine Team (QGEM for short), is a brand new team at Queen's University this year. We all hail from different parts of the country and different educational backgrounds, with the exception of all being in the sciences. Our team is comprised of Biology, Life Sciences, Biochemical Engineering, and Engineering Chemistry students, along with an equally varied lineup of faculty advisors. While, we are new to iGEM this year, we are putting forth our best efforts with our project and are looking forward to presenting our results at the Jamboree this year.<br />
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This year, the Queen’s iGEM team is exploring a new synthetic biology approach to treat atherosclerosis. The purpose of our project is to engineer <i>E.coli</i> cells to target and deliver drugs to the site of atherosclerotic plaques. This will be accomplished by designing a binding system that allows <i>E.coli</i> to adhere to plaque, an inducible effector system that produces and releases specific drugs to site of plaques, and a termination system that detaches the <i>E.coli</i> from the plaque and triggers self-destruction. For a more detailed explanation and breakdown please see our Project page.<br />
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Last Updated: October 20, 2009 by Fr<sub>3</sub>P</p></center><br />
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<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
All Part Documentation has been entered on the appropriate BioBrick Registry Part Page.<br />
The QGEM Team has submitted three parts this year. <br />
Please click on the part you wish to view more information for.<br />
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<a href="http://partsregistry.org/Part:BBa_K214001"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">BBa K214001 - ANP Outer Membrane Expression Construct</p></a><br />
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<a href="http://partsregistry.org/Part:BBa_K214002"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">BBa K214002 - Mature Atrial Natriuretic Peptide (ANP)</p></a><br />
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<a href="http://partsregistry.org/Part:BBa_K214003"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">BBa K214003 - Integrin Alpha 4 (ITGA4) Beta Epitope</p></a><br />
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All Part Documentation has been entered on the appropriate BioBrick Registry Part Page.<br />
The QGEM Team has submitted three parts this year. <br />
Please click on the part you wish to view more information for.<br />
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<a href="http://partsregistry.org/Part:BBa_K214001"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">BBa K214001 - ANP Outer Membrane Expression Construct</p></a><br />
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<a href="http://partsregistry.org/Part:BBa_K214002"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">BBa K214001 - Mature Atrial Natriuretic Peptide (ANP)</p></a><br />
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<a href="http://partsregistry.org/Part:BBa_K214003"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">BBa K214003 - Integrin Alpha 4 (ITGA4) Beta Epitope</p></a><br />
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All Part Documentation has been entered on the appropriate BioBrick Registry Part Page. The QGEM Team has submitted three parts this year. Please click on the part you wish to view more information for.<br />
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<a href="http://partsregistry.org/Part:BBa_K214001"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">BBa K214001 - ANP Outer Membrane Expression Construct</p></a><br />
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<a href="http://partsregistry.org/Part:BBa_K214002"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">BBa K214001 - Mature Atrial Natriuretic Peptide (ANP)</p></a><br />
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The following summarizes the results of the various aspects of this year's QGEM Project. <br />
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Part One: SAA<br />
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<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Two: Binding Construct<br />
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<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Three: Heme and HO-1<br />
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<a href="#Part4"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Four: Future Directions<br />
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<a name="Part1"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part One: SAA</p></a><br />
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Serum amyloid A (SAA) featured prominently on our list of possible effectors to release at the site of atherosclerotic plaques due to research done at Queen’s University by Tam et al. (2005). This research showed that treatment of SAA caused macrophages to reverse and prevent esterification of cholesterol, thereby allowing it to be exported out of plaques by high-density lipoprotein (HDL). In order to get the SAA to be picked up by the macrophages we needed to produce large quantities of the molecule, and then secrete it extracellularly. In order to secrete the protein we decided on using the twin-arginine translocase (TAT) system, which can transport fully folded proteins outside the cell (Sargent et al., 2006) and is found in the majority of prokaryotes. We then fused the TAT signal sequence to the front of our SAA sequence in order to produce a construct that should be able produce and secrete the protein, allowing it to be taken up by macrophages.<br />
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<i>Fig. 1</i> SAA expression and secretion by <i>E. coli</i> cells. Cultures of <i>E. coli</i> cells containing either the SAA construct or control plasmid were spun down at 0, 4, and 12 hours after seeding. The culture medium and whole cell lysates were analyzed by SDS-PAGE and Western blot analysis with a polyclonal antibody recognizing whole SAA protein. <br />
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Our <i>E. coli</i> cells transformed with SAA construct did not appear to express or secrete SAA into the growth medium even after growing for 12 hours in optimum condition. This might be due to defective ligation of the Ptet-RBS fragment to the SAA-encoding gene. We plan to sequence the construct in the future. <br />
</p><br />
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References:<br />
<br/><br />
<br/><br />
Benditt, E.P., Hoffman, J.S. & Eriksen, N. (1982). SAA, an apoprotein of HDL: its structure and function. <i> New York Academy of Sciences. </i> 82:183-189.<br />
<br/><br />
<br/><br />
Ancsin, J.B. & Kisilevsky, R. (1999). The heparin/heparin sulfate-binding site on apo-serum amyloid A. <i>Journal of Biological Chemistry.</i> Vol. 274, 11:7172-7181.<br />
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<a name="Part2"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Two: Binding Construct</p></a><br />
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In our original design of the binding construct, constitutive promoter BBa_J23119 was used to control the expression of the chimeric protein receptor consisted of the Lpp-OmpA fusion, TEV protease cut sites and ITGA4 fragment. We ordered our construct to be synthesized by Mr. GENE in early June. However, Mr. GENE notified us in August that our construct appeared to be unstable in and toxic to <i>E. coli</i> cells. The reason might be that high expression level of Lpp-OmpA might interfere with bacterial physiology, causing severe growth inhibition and reduced viability. (Daugherty et al, 1999) Mr. Gene was able to give us a sequence-confirmed PCR product of the synthesized construct. Thus, we decided to replace P¬const with P¬tet (BBa_R0040) by PCR. Below is a schematic diagram of the construction process. <br />
</p><br />
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<i>Fig. 2</i> Flowchart of PCR stitching for constructing the binding construct. <br />
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Due to time constraint, we were only able to confirm the stitching of Ptet-RBS fragment to Lpp-OmpA-Linker-TEVx2-Linker fragment (PCR round 2) using Agarose gel electrophoresis. The third round of PCR stitching did not yield expected band. Construction of the binding construct is ongoing. <br />
</p><br />
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<i>Fig. 3</i> <i>In silico</i> model of the binding construct. <br />
</p><br />
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References:<br />
<br/><br />
<br/><br />
Daugherty, P.S., Olsen, M.J., Iverson, B.L. & Georgiou, G. (1999). Development of an optimized expression system for the screening of antibody libraries displayed on the <i>Escherichia coli</i> surface. <i>Protein Engineering.</i> Vol. 12, 7:613-621.<br />
<br/><br />
<br/><br />
Yang, Z., Lui, Q., Wang, Q. & Zhang, Y. (2008). Novel bacterial surface display systems based on outer membrane anchoring elements from the marine bacterium <i>Vibrio anguillarum</i>. <i> American Society for Microbiology.</i> Vol. 74, 14: 4359-4365.<br />
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<a name="Part3"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Three: Heme and HO-1</p></a><br />
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<br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Before presenting our results some useful parts have been outlined briefly.<br />
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BioBrick and Construct Legend:<br />
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BioBrick I716390: Plasmid containing a composite of Heme A, B C & D with T7-promoter and RBS. Amp Resistance.<br />
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BioBrick K098010: HO and phycocaynobilin:ferredoxin oxidoreductase. KAN Resistance.<br />
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BioBrick I15009: HO-1 from <i>Synechocystis</i>. Amp Resistance.<br />
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NLM 350: Rough <i> E. coli</i> strain containing T7-polymerase. Queen's University Research<br />
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In order to determine whether or not the BioBrick I716390 submitted by the Berkeley iGEM team in 2007 was producing heme, NLM350. Overnight cultures containing 100 μg/mL ampicillin were set up and inoculated with NLM350 containing I716390. The culture was analyzed daily by wavelength scan in a spectrometer. We ran the wavelength scan twice, once with a blank of LB and once with a blank of LB culture containing NLM350 without the BioBrick. Both of the scan results were identical, which indicates that the peaks present are due to the BioBrick plasmid and not resulting from compounds produced by NLM350. The results from day three with a blank of LB are shown in Figure 4.<br />
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<i>Fig. 4</i> Results of a wavelength scan between 350 and 550nm of the NLM350 + I716390 culture three days after inoculation. Strongest peaks at 412nm. <br />
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In order to check for the presence of the heme/HO-1 complex, NLM350 + I716390 cells were made electrocompetent and two sets of electroporations were performed. The BioBricks K098010 and I15008 were taken up by NLM350 + I716390 and the resulting colonies were used to inoculate cultures containing 40 μg/mL kanamycin and 100 μg/mL ampicillin, respectively. Triplicate testing of NLM350 + 176390 + KO98010 as well as NLM350 + 176390 + I15008 was conducted over five days. Both tests produced similar strong peaks for heme/HO-1 complexes, ranging from 407-409 nm. Again, we ran the scans twice with blanks of LB and LB inoculated with NLM350 and found that results were identical. The results from day five with blanks of LB are shown in Figures 5 and 6.<br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 5</i> Wavelength scan between 350 and 550 nm of the NLM350 + I716390 + K098010 culture 5 days after inoculation. Strong peak of 407.5 nm. <br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 6</i> Wavelength scan between 350 and 550 nm of the NLM350 + I716390 + I15008 culture 5 days after inoculation. Strong peak of 407.5 nm. <br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
As can be seen from our results above, the peaks in the cultures containing heme and HO-1 producing BioBricks become clearer and occur at a lower absorbance, around 408nm, than the peaks from cultures containing only heme-producing BioBricks. Cultures containing only the heme-producing BioBrick has the strongest peaks at 412nm, which corresponds to the peak found by Moffet et al. in 2003 <sup>1</sup>. Since HO-1 does not have an absorbance peak of its own <sup>2,3,4</sup>, the presence of HO-1 can only be inferred by identifying the heme/HO-1 complexes via spectroscopy. According to several authors <sup>2,4</sup>, Soret bands for the oxy-bound form of the heme/HO-1 complex occur at 410nm. The peaks seen in Figures 5 and 6 are slightly blue-shifted compared to the literature values, which may be the result of different biochemical subunits present in the heme protein<sup>5</sup>. However, the difference between the spectra of Figures 4, 5, and 6 indicates that HO-1 is being produced and is binding with heme to form a complex which peaks at a different wavelength than heme alone.<br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
It was expected to see a peak for biliverdin as well, which would have occurred between 440 – 460nm according to the literature <sup>6,7</sup>, though we did not. This might have been due to the fact that the cultures containing heme and HO-1 producing BioBricks had some exposure to light. Researchers have noted in their methods that they had avoided light when handling biliverdin <sup>8,9</sup>. If biliverdin is photosensitive, this might be why these peaks did not appear in our wavelength scans.<br />
</p><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In order to continue on this project track, the genetic effector system would have to be streamlined and optimized. More thorough kinetics testing for the production of heme as well as its metabolism by HO-1 would be required to maximize effector quality and concentration. Though many of the products of heme metabolism were likely produced, bilirubin was almost certainly not. <br />
</p><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
As mentioned previously<sup>2</sup>, there is a lack of biliverdin reductases available for cloning into bacterial systems, especially mammalian. Recently the entire genome of <i>Synechocystis sp.</i> PP6803 was sequenced, along with a cyanobacterial biliverdin reductase homologue. This enzyme is used in the production of light-harvesting pigments called phycobilins which is a product in the breakdown of heme<sup>10</sup>. With more research into extraction of this gene from blue-green algae, a very useful biliverdin reductase BioBrick could be contributed the registry, allowing full degradation of heme to bilirubin. This would enable the microbe as well as the system to which it is contributing, to reap the strong antioxidant properties of this molecule.<br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
References:<br />
<br/><br />
<br/><br />
1. Moffet, D.A., Foley J. & Hecht M.H. (2003). Midpoint reduction potentials and heme binding stoichiometries of de novo proteins from designed combinatorial libraries. <i>Biophysical Chemistry</i>. 105:231-239.<br />
<br/><br />
<br/><br />
2. Frankenberg-Dinkel, N. (2004). Forum review: bacterial Heme Oxygenases. Antioxidants and Redox Signalling. 6:825 - 834.<br />
<br/><br />
<br/><br />
3. Huber W. & Backes W. (2008). Quantitation of heme oxygenase 1: Heme titration increases yield of purified protein. <i>Analytical Biochemistry</i>. 373:167-169.<br />
<br/><br />
<br/><br />
4. Migita C.T., Zhang X. & Yoshida T. (2003). Expression and characterization of cyanobacterium heme oxygenase, a key enzyme in the phycobilin synthesis. <i>European Journal of Biochemistry</i>. 270:687 – 698.<br />
<br/><br />
<br/><br />
5. Wang N., Zhao X. & Lu Y. (2005). Role of Heme Types in Heme-Copper Oxidases: Effects of Replacing a Heme b with a Heme to Mimic in an Engineered Heme-Copper Center in Myoglobin. <i>Journal of American Chemical Society</i>. 127:16541 – 16547.<br />
<br/><br />
<br/><br />
6. Yoshida T. & Kikuchi G. (1979). Purification and Properties of Heme Oxygenase from Rat Liver Microsomes. The <i>Journal of Biological Chemistry</i>. 254:4487 – 4491.<br />
<br/><br />
<br/><br />
7. Ding Z.K. & Xu Y.Q. (2002). Purification and characterization of biliverdin IXalpha from Atlantic salmon (Salmo salar) bile. <i>Biochemistry</i>. 67:927 – 932. <br />
<br/><br />
<br/><br />
8. Ollinger R., Yamashita K., Bilban M., Erat A., Kogler P., Thomas M., Csizmadia E., Usheva A., Margreiter R. & Bach F. H. (2007). Bilirubin and Biliverdin Treatment of Atherosclerotic Diseases. <i>Cell Cycle</i>. 6:39 – 43. <br />
<br/><br />
<br/><br />
9. Ishikawa K., Navab M., Leitinger N., Fogelman A.M. & Lusis A. J. (1997). Induction of heme oxygenase-1 inhibits the monocyte transmigration induced by mildly oxidized LDL. <i>Journal of Clinical Investigation</i>. 100:1209-1216.<br />
<br/><br />
<br/><br />
10. Cornejo J., Willows R. D. & Beale S. I. (1998). Phytobilin biosynthesis: cloning and expression of a gene encoding ferredoxin-dependent heme oxygenase from Synechocystis sp. PCC 6803. <i>The Plant Journal</i>. 15:99 – 107.<br />
</p><br />
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<a name="Part4"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Four: Future Directions</p></a><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Due to the delay of the synthesis of our binding construct, we were unable to test the presentation of the ITGA-4 chain of the VLA-4 antigen on <i>E. coli</i> outer membrane and the binding of the <i>E. coli</i> to endothelial cells expressing VCAM-1. In the future, we are planning to pursue this project further in the following areas.<br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"> <br />
<u>Construction</u><br />
</p><br />
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
1. Complete round 3 of PCR stitching and insert the binding construct into BioBrick backbone.<br />
<br />
2. Sequence the binding construct and the SAA construct.<br />
</pre><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<u>Endothelial Adhesion Assay</u><br />
</p><br />
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
1. Transform the binding construct and constitutive GFP expression construct into E. coli cells. <br />
<br />
2. Plate C166 murine endothelial cells on a 6-well plate. Incubate overnight. <br />
<br />
3. Prepare serial dilutions of the binding E. coli and control cells (containing plasmid backbone). <br />
<br />
4. Incubate C166 cells with E. coli for two hours and then wash wells 3X with PBS. <br />
<br />
5. Quantify GFP emission using fluorimeter. <br />
<br />
6. Alternative test: ELISA binding assay with recombinant VCAM-1 protein. <br />
</pre><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<u>Atrial Natriuretic Peptide</u><br />
</p><br />
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
1. Transform <i>E. coli</i> cells with ANP construct (P<sub>tet</sub>-RBS-Lpp-OmpA-Linker-TEVx2-Linker-ANP)<br />
and constitutive GFP expression construct. <br />
<br />
2. Use the same protocol as endothelial adhesion assay to test the activation of membrane-bound <br />
guanylate cyclase (GCA) on endothelial cells by ANP. <br />
<br />
3. Harvest the endothelial cells and prepare whole cell lysates. <br />
<br />
4. Run SDS-PAGE and Western blotting analysis with antibodies recognizing whole and <br />
phosphorylated Vasodilator Stimulated Phosphoprotein (VASP). VASP is phosphorylated <br />
by cGMP dependent kinase, which is activated by ANP. <br />
</pre><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<u>Inducible Effector System</u><br />
<br/><br />
<br/><br />
In order to make our <i>E. coli</i> cells releasing the effectors at the site of plaque, we plan to put the SAA and HO-1 gene under the control of the P<sub>lux</sub> promoter, which will be activated by a threshold concentration of AHL at the site of plaque.<br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/2/27/QueensFigure7.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 7</i> Planned inducible effector construct.<br />
</p><br />
<br/><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
We plan to transform <i>E. coli</i> cells with the effector construct and the binding construct, and conduct the endothelial adhesion assay. We will then analyze the growth medium of the endothelial culture by SDS-PAGE and Western blot analysis to determine the concentration of AHL, SAA, and biliverdin in the medium. We will also determine the expression level of TEV protease, which is located downstream of a double terminator (BBa_B0015) and RBS (BBa_B0034) on the effector construct. Expression level of TEV protease directly contributes to the efficiency of detachment from the plaque and termination by the kill switch.<br />
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Please note that Results are also available in PDF Format. <br />
Please click on the document you wish to view.<br />
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<a href="https://static.igem.org/mediawiki/2009/f/fc/QueensResultsJamesBogdanBryantHarry.pdf"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">Laboratory One: Harry, Bogdan, James, Bryant</p></a><br />
<a href="https://static.igem.org/mediawiki/2009/2/26/QueensResultsKateMike.pdf"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">Laboratory Two: Kate, Mike</p></a><br />
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<td align="left"><p style="font-size:175%;font-family:corbel;color:#172C4E;font-weight:bold"><i>Results</i><br />
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The following summarizes the results of the various aspects of this year's QGEM Project. <br />
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<a href="#Part1"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part One: SAA<br />
</p></a><br />
<a href="#Part2"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Two: Binding Construct<br />
</p></a><br />
<a href="#Part3"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Three: Heme and HO-1<br />
</p></a><br />
<a href="#Part4"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Four: Future Directions<br />
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<a name="Part1"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part One: SAA</p></a><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Serum amyloid A (SAA) featured prominently on our list of possible effectors to release at the site of atherosclerotic plaques due to research done at Queen’s University by Tam et al. (2005). This research showed that treatment of SAA caused macrophages to reverse and prevent esterification of cholesterol, thereby allowing it to be exported out of plaques by high-density lipoprotein (HDL). In order to get the SAA to be picked up by the macrophages we needed to produce large quantities of the molecule, and then secrete it extracellularly. In order to secrete the protein we decided on using the twin-arginine translocase (TAT) system, which can transport fully folded proteins outside the cell (Sargent et al., 2006) and is found in the majority of prokaryotes. We then fused the TAT signal sequence to the front of our SAA sequence in order to produce a construct that should be able produce and secrete the protein, allowing it to be taken up by macrophages.<br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/c/cb/QueensFigure1.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 1</i> SAA expression and secretion by <i>E. coli</i> cells. Cultures of <i>E. coli</i> cells containing either the SAA construct or control plasmid were spun down at 0, 4, and 12 hours after seeding. The culture medium and whole cell lysates were analyzed by SDS-PAGE and Western blot analysis with a polyclonal antibody recognizing whole SAA protein. <br />
</p><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Our <i>E. coli</i> cells transformed with SAA construct did not appear to express or secrete SAA into the growth medium even after growing for 12 hours in optimum condition. This might be due to defective ligation of the Ptet-RBS fragment to the SAA-encoding gene. We plan to sequence the construct in the future. <br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
References:<br />
<br/><br />
<br/><br />
Benditt, E.P., Hoffman, J.S. & Eriksen, N. (1982). SAA, an apoprotein of HDL: its structure and function. <i> New York Academy of Sciences. </i> 82:183-189.<br />
<br/><br />
<br/><br />
Ancsin, J.B. & Kisilevsky, R. (1999). The heparin/heparin sulfate-binding site on apo-serum amyloid A. <i>Journal of Biological Chemistry.</i> Vol. 274, 11:7172-7181.<br />
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<a name="Part2"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Two: Binding Construct</p></a><br />
<br/><br />
<br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In our original design of the binding construct, constitutive promoter BBa_J23119 was used to control the expression of the chimeric protein receptor consisted of the Lpp-OmpA fusion, TEV protease cut sites and ITGA4 fragment. We ordered our construct to be synthesized by Mr. GENE in early June. However, Mr. GENE notified us in August that our construct appeared to be unstable in and toxic to <i>E. coli</i> cells. The reason might be that high expression level of Lpp-OmpA might interfere with bacterial physiology, causing severe growth inhibition and reduced viability. (Daugherty et al, 1999) Mr. Gene was able to give us a sequence-confirmed PCR product of the synthesized construct. Thus, we decided to replace P¬const with P¬tet (BBa_R0040) by PCR. Below is a schematic diagram of the construction process. <br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/0/08/QueensFigure2.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 2</i> Flowchart of PCR stitching for constructing the binding construct. <br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Due to time constraint, we were only able to confirm the stitching of Ptet-RBS fragment to Lpp-OmpA-Linker-TEVx2-Linker fragment (PCR round 2) using Agarose gel electrophoresis. The third round of PCR stitching did not yield expected band. Construction of the binding construct is ongoing. <br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/1/14/QueensFigure3.png"></center><br />
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<i>Fig. 3</i> <i>In silico</i> model of the binding construct. <br />
</p><br />
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References:<br />
<br/><br />
<br/><br />
Daugherty, P.S., Olsen, M.J., Iverson, B.L. & Georgiou, G. (1999). Development of an optimized expression system for the screening of antibody libraries displayed on the <i>Escherichia coli</i> surface. <i>Protein Engineering.</i> Vol. 12, 7:613-621.<br />
<br/><br />
<br/><br />
Yang, Z., Lui, Q., Wang, Q. & Zhang, Y. (2008). Novel bacterial surface display systems based on outer membrane anchoring elements from the marine bacterium <i>Vibrio anguillarum</i>. <i> American Society for Microbiology.</i> Vol. 74, 14: 4359-4365.<br />
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<a name="Part3"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Three: Heme and HO-1</p></a><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Before presenting our results some useful parts have been outlined briefly.<br />
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BioBrick and Construct Legend:<br />
<br/><br />
<br/><br />
BioBrick I716390: Plasmid containing a composite of Heme A, B C & D with T7-promoter and RBS. Amp Resistance.<br />
<br/><br />
BioBrick K098010: HO and phycocaynobilin:ferredoxin oxidoreductase. KAN Resistance.<br />
<br/><br />
BioBrick I15009: HO-1 from <i>Synechocystis</i>. Amp Resistance.<br />
<br/><br />
NLM 350: Rough <i> E. coli</i> strain containing T7-polymerase. Queen's University Research<br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In order to determine whether or not the BioBrick I716390 submitted by the Berkeley iGEM team in 2007 was producing heme, NLM350. Overnight cultures containing 100 μg/mL ampicillin were set up and inoculated with NLM350 containing I716390. The culture was analyzed daily by wavelength scan in a spectrometer. We ran the wavelength scan twice, once with a blank of LB and once with a blank of LB culture containing NLM350 without the BioBrick. Both of the scan results were identical, which indicates that the peaks present are due to the BioBrick plasmid and not resulting from compounds produced by NLM350. The results from day three with a blank of LB are shown in Figure 4.<br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/c/ce/QueensFigure4.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 4</i> Results of a wavelength scan between 350 and 550nm of the NLM350 + I716390 culture three days after inoculation. Strongest peaks at 412nm. <br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In order to check for the presence of the heme/HO-1 complex, NLM350 + I716390 cells were made electrocompetent and two sets of electroporations were performed. The BioBricks K098010 and I15008 were taken up by NLM350 + I716390 and the resulting colonies were used to inoculate cultures containing 40 μg/mL kanamycin and 100 μg/mL ampicillin, respectively. Triplicate testing of NLM350 + 176390 + KO98010 as well as NLM350 + 176390 + I15008 was conducted over five days. Both tests produced similar strong peaks for heme/HO-1 complexes, ranging from 407-409 nm. Again, we ran the scans twice with blanks of LB and LB inoculated with NLM350 and found that results were identical. The results from day five with blanks of LB are shown in Figures 5 and 6.<br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/2/2b/QueensFigure5.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 5</i> Wavelength scan between 350 and 550 nm of the NLM350 + I716390 + K098010 culture 5 days after inoculation. Strong peak of 407.5 nm. <br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/7/74/QueensFigure6.png"></center><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 6</i> Wavelength scan between 350 and 550 nm of the NLM350 + I716390 + I15008 culture 5 days after inoculation. Strong peak of 407.5 nm. <br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
As can be seen from our results above, the peaks in the cultures containing heme and HO-1 producing BioBricks become clearer and occur at a lower absorbance, around 408nm, than the peaks from cultures containing only heme-producing BioBricks. Cultures containing only the heme-producing BioBrick has the strongest peaks at 412nm, which corresponds to the peak found by Moffet et al. in 2003 <sup>1</sup>. Since HO-1 does not have an absorbance peak of its own <sup>2,3,4</sup>, the presence of HO-1 can only be inferred by identifying the heme/HO-1 complexes via spectroscopy. According to several authors <sup>2,4</sup>, Soret bands for the oxy-bound form of the heme/HO-1 complex occur at 410nm. The peaks seen in Figures 5 and 6 are slightly blue-shifted compared to the literature values, which may be the result of different biochemical subunits present in the heme protein<sup>5</sup>. However, the difference between the spectra of Figures 4, 5, and 6 indicates that HO-1 is being produced and is binding with heme to form a complex which peaks at a different wavelength than heme alone.<br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
It was expected to see a peak for biliverdin as well, which would have occurred between 440 – 460nm according to the literature <sup>6,7</sup>, though we did not. This might have been due to the fact that the cultures containing heme and HO-1 producing BioBricks had some exposure to light. Researchers have noted in their methods that they had avoided light when handling biliverdin <sup>8,9</sup>. If biliverdin is photosensitive, this might be why these peaks did not appear in our wavelength scans.<br />
</p><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In order to continue on this project track, the genetic effector system would have to be streamlined and optimized. More thorough kinetics testing for the production of heme as well as its metabolism by HO-1 would be required to maximize effector quality and concentration. Though many of the products of heme metabolism were likely produced, bilirubin was almost certainly not. <br />
</p><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
As mentioned previously<sup>2</sup>, there is a lack of biliverdin reductases available for cloning into bacterial systems, especially mammalian. Recently the entire genome of <i>Synechocystis sp.</i> PP6803 was sequenced, along with a cyanobacterial biliverdin reductase homologue. This enzyme is used in the production of light-harvesting pigments called phycobilins which is a product in the breakdown of heme<sup>10</sup>. With more research into extraction of this gene from blue-green algae, a very useful biliverdin reductase BioBrick could be contributed the registry, allowing full degradation of heme to bilirubin. This would enable the microbe as well as the system to which it is contributing, to reap the strong antioxidant properties of this molecule.<br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
References:<br />
<br/><br />
<br/><br />
1. Moffet, D.A., Foley J. & Hecht M.H. (2003). Midpoint reduction potentials and heme binding stoichiometries of de novo proteins from designed combinatorial libraries. <i>Biophysical Chemistry</i>. 105:231-239.<br />
<br/><br />
<br/><br />
2. Frankenberg-Dinkel, N. (2004). Forum review: bacterial Heme Oxygenases. Antioxidants and Redox Signalling. 6:825 - 834.<br />
<br/><br />
<br/><br />
3. Huber W. & Backes W. (2008). Quantitation of heme oxygenase 1: Heme titration increases yield of purified protein. <i>Analytical Biochemistry</i>. 373:167-169.<br />
<br/><br />
<br/><br />
4. Migita C.T., Zhang X. & Yoshida T. (2003). Expression and characterization of cyanobacterium heme oxygenase, a key enzyme in the phycobilin synthesis. <i>European Journal of Biochemistry</i>. 270:687 – 698.<br />
<br/><br />
<br/><br />
5. Wang N., Zhao X. & Lu Y. (2005). Role of Heme Types in Heme-Copper Oxidases: Effects of Replacing a Heme b with a Heme to Mimic in an Engineered Heme-Copper Center in Myoglobin. <i>Journal of American Chemical Society</i>. 127:16541 – 16547.<br />
<br/><br />
<br/><br />
6. Yoshida T. & Kikuchi G. (1979). Purification and Properties of Heme Oxygenase from Rat Liver Microsomes. The <i>Journal of Biological Chemistry</i>. 254:4487 – 4491.<br />
<br/><br />
<br/><br />
7. Ding Z.K. & Xu Y.Q. (2002). Purification and characterization of biliverdin IXalpha from Atlantic salmon (Salmo salar) bile. <i>Biochemistry</i>. 67:927 – 932. <br />
<br/><br />
<br/><br />
8. Ollinger R., Yamashita K., Bilban M., Erat A., Kogler P., Thomas M., Csizmadia E., Usheva A., Margreiter R. & Bach F. H. (2007). Bilirubin and Biliverdin Treatment of Atherosclerotic Diseases. <i>Cell Cycle</i>. 6:39 – 43. <br />
<br/><br />
<br/><br />
9. Ishikawa K., Navab M., Leitinger N., Fogelman A.M. & Lusis A. J. (1997). Induction of heme oxygenase-1 inhibits the monocyte transmigration induced by mildly oxidized LDL. <i>Journal of Clinical Investigation</i>. 100:1209-1216.<br />
<br/><br />
<br/><br />
10. Cornejo J., Willows R. D. & Beale S. I. (1998). Phytobilin biosynthesis: cloning and expression of a gene encoding ferredoxin-dependent heme oxygenase from Synechocystis sp. PCC 6803. <i>The Plant Journal</i>. 15:99 – 107.<br />
</p><br />
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<a name="Part4"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Four: Future Directions</p></a><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Due to the delay of the synthesis of our binding construct, we were unable to test the presentation of the ITGA-4 chain of the VLA-4 antigen on <i>E. coli</i> outer membrane and the binding of the <i>E. coli</i> to endothelial cells expressing VCAM-1. In the future, we are planning to pursue this project further in the following areas.<br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"> <br />
<u>Construction</u><br />
</p><br />
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
1. Complete round 3 of PCR stitching and insert the binding construct into BioBrick backbone.<br />
<br />
2. Sequence the binding construct and the SAA construct.<br />
</pre><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<u>Endothelial Adhesion Assay</u><br />
</p><br />
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
1. Transform the binding construct and constitutive GFP expression construct into E. coli cells. <br />
<br />
2. Plate C166 murine endothelial cells on a 6-well plate. Incubate overnight. <br />
<br />
3. Prepare serial dilutions of the binding E. coli and control cells (containing plasmid backbone). <br />
<br />
4. Incubate C166 cells with E. coli for two hours and then wash wells 3X with PBS. <br />
<br />
5. Quantify GFP emission using fluorimeter. <br />
<br />
6. Alternative test: ELISA binding assay with recombinant VCAM-1 protein. <br />
</pre><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<u>Atrial Natriuretic Peptide</u><br />
</p><br />
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
1. Transform <i>E. coli</i> cells with ANP construct (P<sub>tet</sub>-RBS-Lpp-OmpA-Linker-TEVx2-Linker-ANP)<br />
and constitutive GFP expression construct. <br />
<br />
2. Use the same protocol as endothelial adhesion assay to test the activation of membrane-bound <br />
guanylate cyclase (GCA) on endothelial cells by ANP. <br />
<br />
3. Harvest the endothelial cells and prepare whole cell lysates. <br />
<br />
4. Run SDS-PAGE and Western blotting analysis with antibodies recognizing whole and <br />
phosphorylated Vasodilator Stimulated Phosphoprotein (VASP). VASP is phosphorylated <br />
by cGMP dependent kinase, which is activated by ANP. <br />
</pre><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<u>Inducible Effector System</u><br />
<br/><br />
<br/><br />
In order to make our <i>E. coli</i> cells releasing the effectors at the site of plaque, we plan to put the SAA and HO-1 gene under the control of the P<sub>lux</sub> promoter, which will be activated by a threshold concentration of AHL at the site of plaque.<br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/2/27/QueensFigure7.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 7</i> Planned inducible effector construct.<br />
</p><br />
<br/><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
We plan to transform <i>E. coli</i> cells with the effector construct and the binding construct, and conduct the endothelial adhesion assay. We will then analyze the growth medium of the endothelial culture by SDS-PAGE and Western blot analysis to determine the concentration of AHL, SAA, and biliverdin in the medium. We will also determine the expression level of TEV protease, which is located downstream of a double terminator (BBa_B0015) and RBS (BBa_B0034) on the effector construct. Expression level of TEV protease directly contributes to the efficiency of detachment from the plaque and termination by the kill switch.<br />
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Please note that Results are also available in PDF Format. <br />
Please click on the document you wish to view.<br />
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<a href="https://static.igem.org/mediawiki/2009/f/fc/QueensResultsJamesBogdanBryantHarry.pdf"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">Laboratory One: Harry, Bogdan, James, Bryant</p></a><br />
<a href="https://static.igem.org/mediawiki/2009/2/26/QueensResultsKateMike.pdf"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">Laboratory Two: Kate, Mike</p></a><br />
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<td align="left"><p style="font-size:175%;font-family:corbel;color:#172C4E;font-weight:bold"><i>Results</i><br />
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The following summarizes the results of the various aspects of this year's QGEM Project. <br />
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<a href="#Part1"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part One: SAA<br />
</p></a><br />
<a href="#Part2"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Two: Binding Construct<br />
</p></a><br />
<a href="#Part3"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Three: Heme and HO-1<br />
</p></a><br />
<a href="#Part4"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Four: Future Directions<br />
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<a name="Part1"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part One: SAA</p></a><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Serum amyloid A (SAA) featured prominently on our list of possible effectors to release at the site of atherosclerotic plaques due to research done at Queen’s University by Tam et al. (2005). This research showed that treatment of SAA caused macrophages to reverse and prevent esterification of cholesterol, thereby allowing it to be exported out of plaques by high-density lipoprotein (HDL). In order to get the SAA to be picked up by the macrophages we needed to produce large quantities of the molecule, and then secrete it extracellularly. In order to secrete the protein we decided on using the twin-arginine translocase (TAT) system, which can transport fully folded proteins outside the cell (Sargent et al., 2006) and is found in the majority of prokaryotes. We then fused the TAT signal sequence to the front of our SAA sequence in order to produce a construct that should be able produce and secrete the protein, allowing it to be taken up by macrophages.<br />
</p><br />
<br/><br />
<center><img src="https://static.igem.org/mediawiki/2009/c/cb/QueensFigure1.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 1</i> SAA expression and secretion by <i>E. coli</i> cells. Cultures of <i>E. coli</i> cells containing either the SAA construct or control plasmid were spun down at 0, 4, and 12 hours after seeding. The culture medium and whole cell lysates were analyzed by SDS-PAGE and Western blot analysis with a polyclonal antibody recognizing whole SAA protein. <br />
</p><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Our <i>E. coli</i> cells transformed with SAA construct did not appear to express or secrete SAA into the growth medium even after growing for 12 hours in optimum condition. This might be due to defective ligation of the Ptet-RBS fragment to the SAA-encoding gene. We plan to sequence the construct in the future. <br />
</p><br />
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<br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
References:<br />
<br/><br />
<br/><br />
Benditt, E.P., Hoffman, J.S. & Eriksen, N. (1982). SAA, an apoprotein of HDL: its structure and function. <i> New York Academy of Sciences. </i> 82:183-189.<br />
<br/><br />
<br/><br />
Ancsin, J.B. & Kisilevsky, R. (1999). The heparin/heparin sulfate-binding site on apo-serum amyloid A. <i>Journal of Biological Chemistry.</i> Vol. 274, 11:7172-7181.<br />
</p><br />
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<a name="Part2"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Two: Binding Construct</p></a><br />
<br/><br />
<br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In our original design of the binding construct, constitutive promoter BBa_J23119 was used to control the expression of the chimeric protein receptor consisted of the Lpp-OmpA fusion, TEV protease cut sites and ITGA4 fragment. We ordered our construct to be synthesized by Mr. GENE in early June. However, Mr. GENE notified us in August that our construct appeared to be unstable in and toxic to <i>E. coli</i> cells. The reason might be that high expression level of Lpp-OmpA might interfere with bacterial physiology, causing severe growth inhibition and reduced viability. (Daugherty et al, 1999) Mr. Gene was able to give us a sequence-confirmed PCR product of the synthesized construct. Thus, we decided to replace P¬const with P¬tet (BBa_R0040) by PCR. Below is a schematic diagram of the construction process. <br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/0/08/QueensFigure2.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 2</i> Flowchart of PCR stitching for constructing the binding construct. <br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Due to time constraint, we were only able to confirm the stitching of Ptet-RBS fragment to Lpp-OmpA-Linker-TEVx2-Linker fragment (PCR round 2) using Agarose gel electrophoresis. The third round of PCR stitching did not yield expected band. Construction of the binding construct is ongoing. <br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/1/14/QueensFigure3.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 3</i> <i>In silico</i> model of the binding construct. <br />
</p><br />
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References:<br />
<br/><br />
<br/><br />
Daugherty, P.S., Olsen, M.J., Iverson, B.L. & Georgiou, G. (1999). Development of an optimized expression system for the screening of antibody libraries displayed on the <i>Escherichia coli</i> surface. <i>Protein Engineering.</i> Vol. 12, 7:613-621.<br />
<br/><br />
<br/><br />
Yang, Z., Lui, Q., Wang, Q. & Zhang, Y. (2008). Novel bacterial surface display systems based on outer membrane anchoring elements from the marine bacterium <i>Vibrio anguillarum</i>. <i> American Society for Microbiology.</i> Vol. 74, 14: 4359-4365.<br />
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<a name="Part3"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Three: Heme and HO-1</p></a><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Before presenting our results some useful parts have been outlined briefly.<br />
<br/><br />
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BioBrick and Construct Legend:<br />
<br/><br />
<br/><br />
BioBrick I716390: Plasmid containing a composite of Heme A, B C & D with T7-promoter and RBS. Amp Resistance.<br />
<br/><br />
BioBrick K098010: HO and phycocaynobilin:ferredoxin oxidoreductase. KAN Resistance.<br />
<br/><br />
BioBrick I15009: HO-1 from <i>Synechocystis</i>. Amp Resistance.<br />
<br/><br />
NLM 350: Rough <i> E. coli</i> strain containing T7-polymerase. Queen's University Research<br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In order to determine whether or not the BioBrick I716390 submitted by the Berkeley iGEM team in 2007 was producing heme, NLM350. Overnight cultures containing 100 μg/mL ampicillin were set up and inoculated with NLM350 containing I716390. The culture was analyzed daily by wavelength scan in a spectrometer. We ran the wavelength scan twice, once with a blank of LB and once with a blank of LB culture containing NLM350 without the BioBrick. Both of the scan results were identical, which indicates that the peaks present are due to the BioBrick plasmid and not resulting from compounds produced by NLM350. The results from day three with a blank of LB are shown in Figure 4.<br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/c/ce/QueensFigure4.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 4</i> Results of a wavelength scan between 350 and 550nm of the NLM350 + I716390 culture three days after inoculation. Strongest peaks at 412nm. <br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In order to check for the presence of the heme/HO-1 complex, NLM350 + I716390 cells were made electrocompetent and two sets of electroporations were performed. The BioBricks K098010 and I15008 were taken up by NLM350 + I716390 and the resulting colonies were used to inoculate cultures containing 40 μg/mL kanamycin and 100 μg/mL ampicillin, respectively. Triplicate testing of NLM350 + 176390 + KO98010 as well as NLM350 + 176390 + I15008 was conducted over five days. Both tests produced similar strong peaks for heme/HO-1 complexes, ranging from 407-409 nm. Again, we ran the scans twice with blanks of LB and LB inoculated with NLM350 and found that results were identical. The results from day five with blanks of LB are shown in Figures 5 and 6.<br />
</p><br />
<br/><br />
<center><img src="https://static.igem.org/mediawiki/2009/2/2b/QueensFigure5.png"></center><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 5</i> Wavelength scan between 350 and 550 nm of the NLM350 + I716390 + K098010 culture 5 days after inoculation. Strong peak of 407.5 nm. <br />
</p><br />
<br/><br />
<center><img src="https://static.igem.org/mediawiki/2009/7/74/QueensFigure6.png"></center><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 6</i> Wavelength scan between 350 and 550 nm of the NLM350 + I716390 + I15008 culture 5 days after inoculation. Strong peak of 407.5 nm. <br />
</p><br />
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<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
As can be seen from our results above, the peaks in the cultures containing heme and HO-1 producing BioBricks become clearer and occur at a lower absorbance, around 408nm, than the peaks from cultures containing only heme-producing BioBricks. Cultures containing only the heme-producing BioBrick has the strongest peaks at 412nm, which corresponds to the peak found by Moffet et al. in 2003 <sup>1</sup>. Since HO-1 does not have an absorbance peak of its own <sup>2,3,4</sup>, the presence of HO-1 can only be inferred by identifying the heme/HO-1 complexes via spectroscopy. According to several authors <sup>2,4</sup>, Soret bands for the oxy-bound form of the heme/HO-1 complex occur at 410nm. The peaks seen in Figures 5 and 6 are slightly blue-shifted compared to the literature values, which may be the result of different biochemical subunits present in the heme protein<sup>5</sup>. However, the difference between the spectra of Figures 4, 5, and 6 indicates that HO-1 is being produced and is binding with heme to form a complex which peaks at a different wavelength than heme alone.<br />
</p><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
It was expected to see a peak for biliverdin as well, which would have occurred between 440 – 460nm according to the literature <sup>6,7</sup>, though we did not. This might have been due to the fact that the cultures containing heme and HO-1 producing BioBricks had some exposure to light. Researchers have noted in their methods that they had avoided light when handling biliverdin <sup>8,9</sup>. If biliverdin is photosensitive, this might be why these peaks did not appear in our wavelength scans.<br />
</p><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In order to continue on this project track, the genetic effector system would have to be streamlined and optimized. More thorough kinetics testing for the production of heme as well as its metabolism by HO-1 would be required to maximize effector quality and concentration. Though many of the products of heme metabolism were likely produced, bilirubin was almost certainly not. <br />
</p><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
As mentioned previously<sup>2</sup>, there is a lack of biliverdin reductases available for cloning into bacterial systems, especially mammalian. Recently the entire genome of <i>Synechocystis sp.</i> PP6803 was sequenced, along with a cyanobacterial biliverdin reductase homologue. This enzyme is used in the production of light-harvesting pigments called phycobilins which is a product in the breakdown of heme<sup>10</sup>. With more research into extraction of this gene from blue-green algae, a very useful biliverdin reductase BioBrick could be contributed the registry, allowing full degradation of heme to bilirubin. This would enable the microbe as well as the system to which it is contributing, to reap the strong antioxidant properties of this molecule.<br />
</p><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
References:<br />
<br/><br />
<br/><br />
1. Moffet, D.A., Foley J. & Hecht M.H. (2003). Midpoint reduction potentials and heme binding stoichiometries of de novo proteins from designed combinatorial libraries. <i>Biophysical Chemistry</i>. 105:231-239.<br />
<br/><br />
<br/><br />
2. Frankenberg-Dinkel, N. (2004). Forum review: bacterial Heme Oxygenases. Antioxidants and Redox Signalling. 6:825 - 834.<br />
<br/><br />
<br/><br />
3. Huber W. & Backes W. (2008). Quantitation of heme oxygenase 1: Heme titration increases yield of purified protein. <i>Analytical Biochemistry</i>. 373:167-169.<br />
<br/><br />
<br/><br />
4. Migita C.T., Zhang X. & Yoshida T. (2003). Expression and characterization of cyanobacterium heme oxygenase, a key enzyme in the phycobilin synthesis. <i>European Journal of Biochemistry</i>. 270:687 – 698.<br />
<br/><br />
<br/><br />
5. Wang N., Zhao X. & Lu Y. (2005). Role of Heme Types in Heme-Copper Oxidases: Effects of Replacing a Heme b with a Heme to Mimic in an Engineered Heme-Copper Center in Myoglobin. <i>Journal of American Chemical Society</i>. 127:16541 – 16547.<br />
<br/><br />
<br/><br />
6. Yoshida T. & Kikuchi G. (1979). Purification and Properties of Heme Oxygenase from Rat Liver Microsomes. The <i>Journal of Biological Chemistry</i>. 254:4487 – 4491.<br />
<br/><br />
<br/><br />
7. Ding Z.K. & Xu Y.Q. (2002). Purification and characterization of biliverdin IXalpha from Atlantic salmon (Salmo salar) bile. <i>Biochemistry</i>. 67:927 – 932. <br />
<br/><br />
<br/><br />
8. Ollinger R., Yamashita K., Bilban M., Erat A., Kogler P., Thomas M., Csizmadia E., Usheva A., Margreiter R. & Bach F. H. (2007). Bilirubin and Biliverdin Treatment of Atherosclerotic Diseases. <i>Cell Cycle</i>. 6:39 – 43. <br />
<br/><br />
<br/><br />
9. Ishikawa K., Navab M., Leitinger N., Fogelman A.M. & Lusis A. J. (1997). Induction of heme oxygenase-1 inhibits the monocyte transmigration induced by mildly oxidized LDL. <i>Journal of Clinical Investigation</i>. 100:1209-1216.<br />
<br/><br />
<br/><br />
10. Cornejo J., Willows R. D. & Beale S. I. (1998). Phytobilin biosynthesis: cloning and expression of a gene encoding ferredoxin-dependent heme oxygenase from Synechocystis sp. PCC 6803. <i>The Plant Journal</i>. 15:99 – 107.<br />
</p><br />
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<a name="Part4"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Four: Future Directions</p></a><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Due to the delay of the synthesis of our binding construct, we were unable to test the presentation of the ITGA-4 chain of the VLA-4 antigen on <i>E. coli</i> outer membrane and the binding of the <i>E. coli</i> to endothelial cells expressing VCAM-1. In the future, we are planning to pursue this project further in the following areas.<br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"> <br />
<u>Construction</u><br />
</p><br />
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
1. Complete round 3 of PCR stitching and insert the binding construct into BioBrick backbone.<br />
<br />
2. Sequence the binding construct and the SAA construct.<br />
</pre><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<u>Endothelial Adhesion Assay</u><br />
</p><br />
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
1. Transform the binding construct and constitutive GFP expression construct into E. coli cells. <br />
<br />
2. Plate C166 murine endothelial cells on a 6-well plate. Incubate overnight. <br />
<br />
3. Prepare serial dilutions of the binding E. coli and control cells (containing plasmid backbone). <br />
<br />
4. Incubate C166 cells with E. coli for two hours and then wash wells 3X with PBS. <br />
<br />
5. Quantify GFP emission using fluorimeter. <br />
<br />
6. Alternative test: ELISA binding assay with recombinant VCAM-1 protein. <br />
</pre><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<u>Atrial Natriuretic Peptide</u><br />
</p><br />
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
1. Transform <i>E. coli</i> cells with ANP construct (P<sub>tet</sub>-RBS-Lpp-OmpA-Linker-TEVx2-Linker-ANP)<br />
and constitutive GFP expression construct. <br />
<br />
2. Use the same protocol as endothelial adhesion assay to test the activation of membrane-bound guanylate <br />
cyclase (GCA) on endothelial cells by ANP. <br />
<br />
3. Harvest the endothelial cells and prepare whole cell lysates. <br />
<br />
4. Run SDS-PAGE and Western blotting analysis with antibodies recognizing whole and phosphorylated Vasodilator<br />
Stimulated Phosphoprotein (VASP). VASP is phosphorylated by cGMP dependent kinase, which is activated by ANP. <br />
</pre><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<u>Inducible Effector System</u><br />
<br/><br />
<br/><br />
In order to make our <i>E. coli</i> cells releasing the effectors at the site of plaque, we plan to put the SAA and HO-1 gene under the control of the P<sub>lux</sub> promoter, which will be activated by a threshold concentration of AHL at the site of plaque.<br />
</p><br />
<br/><br />
<center><img src="https://static.igem.org/mediawiki/2009/2/27/QueensFigure7.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 7</i> Planned inducible effector construct.<br />
</p><br />
<br/><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
We plan to transform <i>E. coli</i> cells with the effector construct and the binding construct, and conduct the endothelial adhesion assay. We will then analyze the growth medium of the endothelial culture by SDS-PAGE and Western blot analysis to determine the concentration of AHL, SAA, and biliverdin in the medium. We will also determine the expression level of TEV protease, which is located downstream of a double terminator (BBa_B0015) and RBS (BBa_B0034) on the effector construct. Expression level of TEV protease directly contributes to the efficiency of detachment from the plaque and termination by the kill switch.<br />
</p><br />
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Please note that Results are also available in PDF Format. <br />
Please click on the document you wish to view.<br />
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<a href="https://static.igem.org/mediawiki/2009/f/fc/QueensResultsJamesBogdanBryantHarry.pdf"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">Laboratory One: Harry, Bogdan, James, Bryant</p></a><br />
<a href="https://static.igem.org/mediawiki/2009/2/26/QueensResultsKateMike.pdf"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">Laboratory Two: Kate, Mike</p></a><br />
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<td align="left"><p style="font-size:175%;font-family:corbel;color:#172C4E;font-weight:bold"><i>Results</i><br />
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The following summarizes the results of the various aspects of this year's QGEM Project. <br />
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<a href="#Part1"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part One: SAA<br />
</p></a><br />
<a href="#Part2"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Two: Binding Construct<br />
</p></a><br />
<a href="#Part3"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Three: Heme and HO-1<br />
</p></a><br />
<a href="#Part4"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Four: Future Directions<br />
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<a name="Part1"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part One: SAA</p></a><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Serum amyloid A (SAA) featured prominently on our list of possible effectors to release at the site of atherosclerotic plaques due to research done at Queen’s University by Tam et al. (2005). This research showed that treatment of SAA caused macrophages to reverse and prevent esterification of cholesterol, thereby allowing it to be exported out of plaques by high-density lipoprotein (HDL). In order to get the SAA to be picked up by the macrophages we needed to produce large quantities of the molecule, and then secrete it extracellularly. In order to secrete the protein we decided on using the twin-arginine translocase (TAT) system, which can transport fully folded proteins outside the cell (Sargent et al., 2006) and is found in the majority of prokaryotes. We then fused the TAT signal sequence to the front of our SAA sequence in order to produce a construct that should be able produce and secrete the protein, allowing it to be taken up by macrophages.<br />
</p><br />
<br/><br />
<center><img src="https://static.igem.org/mediawiki/2009/c/cb/QueensFigure1.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 1</i> SAA expression and secretion by <i>E. coli</i> cells. Cultures of <i>E. coli</i> cells containing either the SAA construct or control plasmid were spun down at 0, 4, and 12 hours after seeding. The culture medium and whole cell lysates were analyzed by SDS-PAGE and Western blot analysis with a polyclonal antibody recognizing whole SAA protein. <br />
</p><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Our <i>E. coli</i> cells transformed with SAA construct did not appear to express or secrete SAA into the growth medium even after growing for 12 hours in optimum condition. This might be due to defective ligation of the Ptet-RBS fragment to the SAA-encoding gene. We plan to sequence the construct in the future. <br />
</p><br />
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<br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
References:<br />
<br/><br />
<br/><br />
Benditt, E.P., Hoffman, J.S. & Eriksen, N. (1982). SAA, an apoprotein of HDL: its structure and function. <i> New York Academy of Sciences. </i> 82:183-189.<br />
<br/><br />
<br/><br />
Ancsin, J.B. & Kisilevsky, R. (1999). The heparin/heparin sulfate-binding site on apo-serum amyloid A. <i>Journal of Biological Chemistry.</i> Vol. 274, 11:7172-7181.<br />
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<a name="Part2"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Two: Binding Construct</p></a><br />
<br/><br />
<br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In our original design of the binding construct, constitutive promoter BBa_J23119 was used to control the expression of the chimeric protein receptor consisted of the Lpp-OmpA fusion, TEV protease cut sites and ITGA4 fragment. We ordered our construct to be synthesized by Mr. GENE in early June. However, Mr. GENE notified us in August that our construct appeared to be unstable in and toxic to <i>E. coli</i> cells. The reason might be that high expression level of Lpp-OmpA might interfere with bacterial physiology, causing severe growth inhibition and reduced viability. (Daugherty et al, 1999) Mr. Gene was able to give us a sequence-confirmed PCR product of the synthesized construct. Thus, we decided to replace P¬const with P¬tet (BBa_R0040) by PCR. Below is a schematic diagram of the construction process. <br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/0/08/QueensFigure2.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 2</i> Flowchart of PCR stitching for constructing the binding construct. <br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Due to time constraint, we were only able to confirm the stitching of Ptet-RBS fragment to Lpp-OmpA-Linker-TEVx2-Linker fragment (PCR round 2) using Agarose gel electrophoresis. The third round of PCR stitching did not yield expected band. Construction of the binding construct is ongoing. <br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/1/14/QueensFigure3.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 3</i> <i>In silico</i> model of the binding construct. <br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
References:<br />
<br/><br />
<br/><br />
Daugherty, P.S., Olsen, M.J., Iverson, B.L. & Georgiou, G. (1999). Development of an optimized expression system for the screening of antibody libraries displayed on the <i>Escherichia coli</i> surface. <i>Protein Engineering.</i> Vol. 12, 7:613-621.<br />
<br/><br />
<br/><br />
Yang, Z., Lui, Q., Wang, Q. & Zhang, Y. (2008). Novel bacterial surface display systems based on outer membrane anchoring elements from the marine bacterium <i>Vibrio anguillarum</i>. <i> American Society for Microbiology.</i> Vol. 74, 14: 4359-4365.<br />
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<a name="Part3"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Three: Heme and HO-1</p></a><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Before presenting our results some useful parts have been outlined briefly.<br />
<br/><br />
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BioBrick and Construct Legend:<br />
<br/><br />
<br/><br />
BioBrick I716390: Plasmid containing a composite of Heme A, B C & D with T7-promoter and RBS. Amp Resistance.<br />
<br/><br />
BioBrick K098010: HO and phycocaynobilin:ferredoxin oxidoreductase. KAN Resistance.<br />
<br/><br />
BioBrick I15009: HO-1 from <i>Synechocystis</i>. Amp Resistance.<br />
<br/><br />
NLM 350: Rough <i> E. coli</i> strain containing T7-polymerase. Queen's University Research<br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In order to determine whether or not the BioBrick I716390 submitted by the Berkeley iGEM team in 2007 was producing heme, NLM350. Overnight cultures containing 100 μg/mL ampicillin were set up and inoculated with NLM350 containing I716390. The culture was analyzed daily by wavelength scan in a spectrometer. We ran the wavelength scan twice, once with a blank of LB and once with a blank of LB culture containing NLM350 without the BioBrick. Both of the scan results were identical, which indicates that the peaks present are due to the BioBrick plasmid and not resulting from compounds produced by NLM350. The results from day three with a blank of LB are shown in Figure 4.<br />
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<center><img src="https://static.igem.org/mediawiki/2009/c/ce/QueensFigure4.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 4</i> Results of a wavelength scan between 350 and 550nm of the NLM350 + I716390 culture three days after inoculation. Strongest peaks at 412nm. <br />
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In order to check for the presence of the heme/HO-1 complex, NLM350 + I716390 cells were made electrocompetent and two sets of electroporations were performed. The BioBricks K098010 and I15008 were taken up by NLM350 + I716390 and the resulting colonies were used to inoculate cultures containing 40 μg/mL kanamycin and 100 μg/mL ampicillin, respectively. Triplicate testing of NLM350 + 176390 + KO98010 as well as NLM350 + 176390 + I15008 was conducted over five days. Both tests produced similar strong peaks for heme/HO-1 complexes, ranging from 407-409 nm. Again, we ran the scans twice with blanks of LB and LB inoculated with NLM350 and found that results were identical. The results from day five with blanks of LB are shown in Figures 5 and 6.<br />
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<center><img src="https://static.igem.org/mediawiki/2009/2/2b/QueensFigure5.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 5</i> Wavelength scan between 350 and 550 nm of the NLM350 + I716390 + K098010 culture 5 days after inoculation. Strong peak of 407.5 nm. <br />
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<center><img src="https://static.igem.org/mediawiki/2009/7/74/QueensFigure6.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 6</i> Wavelength scan between 350 and 550 nm of the NLM350 + I716390 + I15008 culture 5 days after inoculation. Strong peak of 407.5 nm. <br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
As can be seen from our results above, the peaks in the cultures containing heme and HO-1 producing BioBricks become clearer and occur at a lower absorbance, around 408nm, than the peaks from cultures containing only heme-producing BioBricks. Cultures containing only the heme-producing BioBrick has the strongest peaks at 412nm, which corresponds to the peak found by Moffet et al. in 2003 <sup>1</sup>. Since HO-1 does not have an absorbance peak of its own <sup>2,3,4</sup>, the presence of HO-1 can only be inferred by identifying the heme/HO-1 complexes via spectroscopy. According to several authors <sup>2,4</sup>, Soret bands for the oxy-bound form of the heme/HO-1 complex occur at 410nm. The peaks seen in Figures 5 and 6 are slightly blue-shifted compared to the literature values, which may be the result of different biochemical subunits present in the heme protein<sup>5</sup>. However, the difference between the spectra of Figures 4, 5, and 6 indicates that HO-1 is being produced and is binding with heme to form a complex which peaks at a different wavelength than heme alone.<br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
It was expected to see a peak for biliverdin as well, which would have occurred between 440 – 460nm according to the literature <sup>6,7</sup>, though we did not. This might have been due to the fact that the cultures containing heme and HO-1 producing BioBricks had some exposure to light. Researchers have noted in their methods that they had avoided light when handling biliverdin <sup>8,9</sup>. If biliverdin is photosensitive, this might be why these peaks did not appear in our wavelength scans.<br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In order to continue on this project track, the genetic effector system would have to be streamlined and optimized. More thorough kinetics testing for the production of heme as well as its metabolism by HO-1 would be required to maximize effector quality and concentration. Though many of the products of heme metabolism were likely produced, bilirubin was almost certainly not. <br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
As mentioned previously<sup>2</sup>, there is a lack of biliverdin reductases available for cloning into bacterial systems, especially mammalian. Recently the entire genome of <i>Synechocystis sp.</i> PP6803 was sequenced, along with a cyanobacterial biliverdin reductase homologue. This enzyme is used in the production of light-harvesting pigments called phycobilins which is a product in the breakdown of heme<sup>10</sup>. With more research into extraction of this gene from blue-green algae, a very useful biliverdin reductase BioBrick could be contributed the registry, allowing full degradation of heme to bilirubin. This would enable the microbe as well as the system to which it is contributing, to reap the strong antioxidant properties of this molecule.<br />
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References:<br />
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1. Moffet, D.A., Foley J. & Hecht M.H. (2003). Midpoint reduction potentials and heme binding stoichiometries of de novo proteins from designed combinatorial libraries. <i>Biophysical Chemistry</i>. 105:231-239.<br />
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2. Frankenberg-Dinkel, N. (2004). Forum review: bacterial Heme Oxygenases. Antioxidants and Redox Signalling. 6:825 - 834.<br />
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3. Huber W. & Backes W. (2008). Quantitation of heme oxygenase 1: Heme titration increases yield of purified protein. <i>Analytical Biochemistry</i>. 373:167-169.<br />
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<br/><br />
4. Migita C.T., Zhang X. & Yoshida T. (2003). Expression and characterization of cyanobacterium heme oxygenase, a key enzyme in the phycobilin synthesis. <i>European Journal of Biochemistry</i>. 270:687 – 698.<br />
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5. Wang N., Zhao X. & Lu Y. (2005). Role of Heme Types in Heme-Copper Oxidases: Effects of Replacing a Heme b with a Heme to Mimic in an Engineered Heme-Copper Center in Myoglobin. <i>Journal of American Chemical Society</i>. 127:16541 – 16547.<br />
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6. Yoshida T. & Kikuchi G. (1979). Purification and Properties of Heme Oxygenase from Rat Liver Microsomes. The <i>Journal of Biological Chemistry</i>. 254:4487 – 4491.<br />
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7. Ding Z.K. & Xu Y.Q. (2002). Purification and characterization of biliverdin IXalpha from Atlantic salmon (Salmo salar) bile. <i>Biochemistry</i>. 67:927 – 932. <br />
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8. Ollinger R., Yamashita K., Bilban M., Erat A., Kogler P., Thomas M., Csizmadia E., Usheva A., Margreiter R. & Bach F. H. (2007). Bilirubin and Biliverdin Treatment of Atherosclerotic Diseases. <i>Cell Cycle</i>. 6:39 – 43. <br />
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9. Ishikawa K., Navab M., Leitinger N., Fogelman A.M. & Lusis A. J. (1997). Induction of heme oxygenase-1 inhibits the monocyte transmigration induced by mildly oxidized LDL. <i>Journal of Clinical Investigation</i>. 100:1209-1216.<br />
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10. Cornejo J., Willows R. D. & Beale S. I. (1998). Phytobilin biosynthesis: cloning and expression of a gene encoding ferredoxin-dependent heme oxygenase from Synechocystis sp. PCC 6803. <i>The Plant Journal</i>. 15:99 – 107.<br />
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<a name="Part4"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Four: Future Directions</p></a><br />
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Due to the delay of the synthesis of our binding construct, we were unable to test the presentation of the ITGA-4 chain of the VLA-4 antigen on <i>E. coli</i> outer membrane and the binding of the <i>E. coli</i> to endothelial cells expressing VCAM-1. In the future, we are planning to pursue this project further in the following areas.<br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"> <br />
<u>Construction</u><br />
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<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
1. Complete round 3 of PCR stitching and insert the binding construct into BioBrick backbone.<br />
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2. Sequence the binding construct and the SAA construct.<br />
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<u>Endothelial Adhesion Assay</u><br />
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<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
1. Transform the binding construct and constitutive GFP expression construct into E. coli cells. <br />
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2. Plate C166 murine endothelial cells on a 6-well plate. Incubate overnight. <br />
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3. Prepare serial dilutions of the binding E. coli and control cells (containing plasmid backbone). <br />
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4. Incubate C166 cells with E. coli for two hours and then wash wells 3X with PBS. <br />
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5. Quantify GFP emission using fluorimeter. <br />
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6. Alternative test: ELISA binding assay with recombinant VCAM-1 protein. <br />
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<u>Atrial Natriuretic Peptide</u><br />
</p><br />
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
1. Transform <i>E. coli</i> cells with ANP construct (P<sub>tet</sub>-RBS-Lpp-OmpA-Linker-TEVx2-Linker-ANP) and constitutive GFP expression construct. <br />
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2. Use the same protocol as endothelial adhesion assay to test the activation of membrane-bound guanylate cyclase (GCA) on endothelial cells by ANP. <br />
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3. Harvest the endothelial cells and prepare whole cell lysates. <br />
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4. Run SDS-PAGE and Western blotting analysis with antibodies recognizing whole and phosphorylated Vasodilator Stimulated Phosphoprotein (VASP). VASP is phosphorylated by cGMP dependent kinase, which is activated by ANP. <br />
</pre><br />
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<u>Inducible Effector System</u><br />
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In order to make our <i>E. coli</i> cells releasing the effectors at the site of plaque, we plan to put the SAA and HO-1 gene under the control of the P<sub>lux</sub> promoter, which will be activated by a threshold concentration of AHL at the site of plaque.<br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/2/27/QueensFigure7.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 7</i> Planned inducible effector construct.<br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
We plan to transform <i>E. coli</i> cells with the effector construct and the binding construct, and conduct the endothelial adhesion assay. We will then analyze the growth medium of the endothelial culture by SDS-PAGE and Western blot analysis to determine the concentration of AHL, SAA, and biliverdin in the medium. We will also determine the expression level of TEV protease, which is located downstream of a double terminator (BBa_B0015) and RBS (BBa_B0034) on the effector construct. Expression level of TEV protease directly contributes to the efficiency of detachment from the plaque and termination by the kill switch.<br />
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Please note that Results are also available in PDF Format. <br />
Please click on the document you wish to view.<br />
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<a href="https://static.igem.org/mediawiki/2009/f/fc/QueensResultsJamesBogdanBryantHarry.pdf"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">Laboratory One: Harry, Bogdan, James, Bryant</p></a><br />
<a href="https://static.igem.org/mediawiki/2009/2/26/QueensResultsKateMike.pdf"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">Laboratory Two: Kate, Mike</p></a><br />
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<td align="left"><p style="font-size:175%;font-family:corbel;color:#172C4E;font-weight:bold"><i>Results<i><br />
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The following summarizes the results of the various aspects of this year's QGEM Project. <br />
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Part One: SAA<br />
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Part Two: Binding Construct<br />
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Part Three: Heme and HO-1<br />
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<a href="#Part4"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Four: Future Directions<br />
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<a name="Part1"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part One: SAA</p></a><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Serum amyloid A (SAA) featured prominently on our list of possible effectors to release at the site of atherosclerotic plaques due to research done at Queen’s University by Tam et al. (2005). This research showed that treatment of SAA caused macrophages to reverse and prevent esterification of cholesterol, thereby allowing it to be exported out of plaques by high-density lipoprotein (HDL). In order to get the SAA to be picked up by the macrophages we needed to produce large quantities of the molecule, and then secrete it extracellularly. In order to secrete the protein we decided on using the twin-arginine translocase (TAT) system, which can transport fully folded proteins outside the cell (Sargent et al., 2006) and is found in the majority of prokaryotes. We then fused the TAT signal sequence to the front of our SAA sequence in order to produce a construct that should be able produce and secrete the protein, allowing it to be taken up by macrophages.<br />
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<i>Fig. 1</i> SAA expression and secretion by <i>E. coli</i> cells. Cultures of <i>E. coli</i> cells containing either the SAA construct or control plasmid were spun down at 0, 4, and 12 hours after seeding. The culture medium and whole cell lysates were analyzed by SDS-PAGE and Western blot analysis with a polyclonal antibody recognizing whole SAA protein. <br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Our <i>E. coli</i> cells transformed with SAA construct did not appear to express or secrete SAA into the growth medium even after growing for 12 hours in optimum condition. This might be due to defective ligation of the Ptet-RBS fragment to the SAA-encoding gene. We plan to sequence the construct in the future. <br />
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References:<br />
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Benditt, E.P., Hoffman, J.S. & Eriksen, N. (1982). SAA, an apoprotein of HDL: its structure and function. <i> New York Academy of Sciences. </i> 82:183-189.<br />
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Ancsin, J.B. & Kisilevsky, R. (1999). The heparin/heparin sulfate-binding site on apo-serum amyloid A. <i>Journal of Biological Chemistry.</i> Vol. 274, 11:7172-7181.<br />
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<a name="Part2"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Two: Binding Construct</p></a><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In our original design of the binding construct, constitutive promoter BBa_J23119 was used to control the expression of the chimeric protein receptor consisted of the Lpp-OmpA fusion, TEV protease cut sites and ITGA4 fragment. We ordered our construct to be synthesized by Mr. GENE in early June. However, Mr. GENE notified us in August that our construct appeared to be unstable in and toxic to <i>E. coli</i> cells. The reason might be that high expression level of Lpp-OmpA might interfere with bacterial physiology, causing severe growth inhibition and reduced viability. (Daugherty et al, 1999) Mr. Gene was able to give us a sequence-confirmed PCR product of the synthesized construct. Thus, we decided to replace P¬const with P¬tet (BBa_R0040) by PCR. Below is a schematic diagram of the construction process. <br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 2</i> Flowchart of PCR stitching for constructing the binding construct. <br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Due to time constraint, we were only able to confirm the stitching of Ptet-RBS fragment to Lpp-OmpA-Linker-TEVx2-Linker fragment (PCR round 2) using Agarose gel electrophoresis. The third round of PCR stitching did not yield expected band. Construction of the binding construct is ongoing. <br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 3</i> <i>In silico</i> model of the binding construct. <br />
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References:<br />
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Daugherty, P.S., Olsen, M.J., Iverson, B.L. & Georgiou, G. (1999). Development of an optimized expression system for the screening of antibody libraries displayed on the <i>Escherichia coli</i> surface. <i>Protein Engineering.</i> Vol. 12, 7:613-621.<br />
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<br/><br />
Yang, Z., Lui, Q., Wang, Q. & Zhang, Y. (2008). Novel bacterial surface display systems based on outer membrane anchoring elements from the marine bacterium <i>Vibrio anguillarum</i>. <i> American Society for Microbiology.</i> Vol. 74, 14: 4359-4365.<br />
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<a name="Part3"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Three: Heme and HO-1</p></a><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Before presenting our results some useful parts have been outlined briefly.<br />
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BioBrick and Construct Legend:<br />
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BioBrick I716390: Plasmid containing a composite of Heme A, B C & D with T7-promoter and RBS. Amp Resistance.<br />
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BioBrick K098010: HO and phycocaynobilin:ferredoxin oxidoreductase. KAN Resistance.<br />
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BioBrick I15009: HO-1 from <i>Synechocystis</i>. Amp Resistance.<br />
<br/><br />
NLM 350: Rough <i> E. coli</i> strain containing T7-polymerase. Queen's University Research<br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In order to determine whether or not the BioBrick I716390 submitted by the Berkeley iGEM team in 2007 was producing heme, NLM350. Overnight cultures containing 100 μg/mL ampicillin were set up and inoculated with NLM350 containing I716390. The culture was analyzed daily by wavelength scan in a spectrometer. We ran the wavelength scan twice, once with a blank of LB and once with a blank of LB culture containing NLM350 without the BioBrick. Both of the scan results were identical, which indicates that the peaks present are due to the BioBrick plasmid and not resulting from compounds produced by NLM350. The results from day three with a blank of LB are shown in Figure 4.<br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/c/ce/QueensFigure4.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 4</i> Results of a wavelength scan between 350 and 550nm of the NLM350 + I716390 culture three days after inoculation. Strongest peaks at 412nm. <br />
</p><br />
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In order to check for the presence of the heme/HO-1 complex, NLM350 + I716390 cells were made electrocompetent and two sets of electroporations were performed. The BioBricks K098010 and I15008 were taken up by NLM350 + I716390 and the resulting colonies were used to inoculate cultures containing 40 μg/mL kanamycin and 100 μg/mL ampicillin, respectively. Triplicate testing of NLM350 + 176390 + KO98010 as well as NLM350 + 176390 + I15008 was conducted over five days. Both tests produced similar strong peaks for heme/HO-1 complexes, ranging from 407-409 nm. Again, we ran the scans twice with blanks of LB and LB inoculated with NLM350 and found that results were identical. The results from day five with blanks of LB are shown in Figures 5 and 6.<br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/2/2b/QueensFigure5.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 5</i> Wavelength scan between 350 and 550 nm of the NLM350 + I716390 + K098010 culture 5 days after inoculation. Strong peak of 407.5 nm. <br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/7/74/QueensFigure6.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 6</i> Wavelength scan between 350 and 550 nm of the NLM350 + I716390 + I15008 culture 5 days after inoculation. Strong peak of 407.5 nm. <br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
As can be seen from our results above, the peaks in the cultures containing heme and HO-1 producing BioBricks become clearer and occur at a lower absorbance, around 408nm, than the peaks from cultures containing only heme-producing BioBricks. Cultures containing only the heme-producing BioBrick has the strongest peaks at 412nm, which corresponds to the peak found by Moffet et al. in 2003 <sup>1</sup>. Since HO-1 does not have an absorbance peak of its own <sup>2,3,4</sup>, the presence of HO-1 can only be inferred by identifying the heme/HO-1 complexes via spectroscopy. According to several authors <sup>2,4</sup>, Soret bands for the oxy-bound form of the heme/HO-1 complex occur at 410nm. The peaks seen in Figures 5 and 6 are slightly blue-shifted compared to the literature values, which may be the result of different biochemical subunits present in the heme protein<sup>5</sup>. However, the difference between the spectra of Figures 4, 5, and 6 indicates that HO-1 is being produced and is binding with heme to form a complex which peaks at a different wavelength than heme alone.<br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
It was expected to see a peak for biliverdin as well, which would have occurred between 440 – 460nm according to the literature <sup>6,7</sup>, though we did not. This might have been due to the fact that the cultures containing heme and HO-1 producing BioBricks had some exposure to light. Researchers have noted in their methods that they had avoided light when handling biliverdin <sup>8,9</sup>. If biliverdin is photosensitive, this might be why these peaks did not appear in our wavelength scans.<br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In order to continue on this project track, the genetic effector system would have to be streamlined and optimized. More thorough kinetics testing for the production of heme as well as its metabolism by HO-1 would be required to maximize effector quality and concentration. Though many of the products of heme metabolism were likely produced, bilirubin was almost certainly not. <br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
As mentioned previously<sup>2</sup>, there is a lack of biliverdin reductases available for cloning into bacterial systems, especially mammalian. Recently the entire genome of <i>Synechocystis sp.</i> PP6803 was sequenced, along with a cyanobacterial biliverdin reductase homologue. This enzyme is used in the production of light-harvesting pigments called phycobilins which is a product in the breakdown of heme<sup>10</sup>. With more research into extraction of this gene from blue-green algae, a very useful biliverdin reductase BioBrick could be contributed the registry, allowing full degradation of heme to bilirubin. This would enable the microbe as well as the system to which it is contributing, to reap the strong antioxidant properties of this molecule.<br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
References:<br />
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<br/><br />
1. Moffet, D.A., Foley J. & Hecht M.H. (2003). Midpoint reduction potentials and heme binding stoichiometries of de novo proteins from designed combinatorial libraries. <i>Biophysical Chemistry</i>. 105:231-239.<br />
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2. Frankenberg-Dinkel, N. (2004). Forum review: bacterial Heme Oxygenases. Antioxidants and Redox Signalling. 6:825 - 834.<br />
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3. Huber W. & Backes W. (2008). Quantitation of heme oxygenase 1: Heme titration increases yield of purified protein. <i>Analytical Biochemistry</i>. 373:167-169.<br />
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<br/><br />
4. Migita C.T., Zhang X. & Yoshida T. (2003). Expression and characterization of cyanobacterium heme oxygenase, a key enzyme in the phycobilin synthesis. <i>European Journal of Biochemistry</i>. 270:687 – 698.<br />
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<br/><br />
5. Wang N., Zhao X. & Lu Y. (2005). Role of Heme Types in Heme-Copper Oxidases: Effects of Replacing a Heme b with a Heme to Mimic in an Engineered Heme-Copper Center in Myoglobin. <i>Journal of American Chemical Society</i>. 127:16541 – 16547.<br />
<br/><br />
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6. Yoshida T. & Kikuchi G. (1979). Purification and Properties of Heme Oxygenase from Rat Liver Microsomes. The <i>Journal of Biological Chemistry</i>. 254:4487 – 4491.<br />
<br/><br />
<br/><br />
7. Ding Z.K. & Xu Y.Q. (2002). Purification and characterization of biliverdin IXalpha from Atlantic salmon (Salmo salar) bile. <i>Biochemistry</i>. 67:927 – 932. <br />
<br/><br />
<br/><br />
8. Ollinger R., Yamashita K., Bilban M., Erat A., Kogler P., Thomas M., Csizmadia E., Usheva A., Margreiter R. & Bach F. H. (2007). Bilirubin and Biliverdin Treatment of Atherosclerotic Diseases. <i>Cell Cycle</i>. 6:39 – 43. <br />
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9. Ishikawa K., Navab M., Leitinger N., Fogelman A.M. & Lusis A. J. (1997). Induction of heme oxygenase-1 inhibits the monocyte transmigration induced by mildly oxidized LDL. <i>Journal of Clinical Investigation</i>. 100:1209-1216.<br />
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<br/><br />
10. Cornejo J., Willows R. D. & Beale S. I. (1998). Phytobilin biosynthesis: cloning and expression of a gene encoding ferredoxin-dependent heme oxygenase from Synechocystis sp. PCC 6803. <i>The Plant Journal</i>. 15:99 – 107.<br />
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<a name="Part4"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Four: Future Directions</p></a><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Due to the delay of the synthesis of our binding construct, we were unable to test the presentation of the ITGA-4 chain of the VLA-4 antigen on <i>E. coli</i> outer membrane and the binding of the <i>E. coli</i> to endothelial cells expressing VCAM-1. In the future, we are planning to pursue this project further in the following areas.<br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"> <br />
<u>Construction</u><br />
</p><br />
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
1. Complete round 3 of PCR stitching and insert the binding construct into BioBrick backbone.<br />
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2. Sequence the binding construct and the SAA construct.<br />
</pre><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<u>Endothelial Adhesion Assay</u><br />
</p><br />
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
1. Transform the binding construct and constitutive GFP expression construct into E. coli cells. <br />
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2. Plate C166 murine endothelial cells on a 6-well plate. Incubate overnight. <br />
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3. Prepare serial dilutions of the binding E. coli and control cells (containing plasmid backbone). <br />
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4. Incubate C166 cells with E. coli for two hours and then wash wells 3X with PBS. <br />
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5. Quantify GFP emission using fluorimeter. <br />
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6. Alternative test: ELISA binding assay with recombinant VCAM-1 protein. <br />
</pre><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<u>Atrial Natriuretic Peptide</u><br />
</p><br />
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
1. Transform <i>E. coli</i> cells with ANP construct (P<sub>tet</sub>-RBS-Lpp-OmpA-Linker-TEVx2-Linker-ANP) and constitutive GFP expression construct. <br />
<br />
2. Use the same protocol as endothelial adhesion assay to test the activation of membrane-bound guanylate cyclase (GCA) on endothelial cells by ANP. <br />
<br />
3. Harvest the endothelial cells and prepare whole cell lysates. <br />
<br />
4. Run SDS-PAGE and Western blotting analysis with antibodies recognizing whole and phosphorylated Vasodilator Stimulated Phosphoprotein (VASP). VASP is phosphorylated by cGMP dependent kinase, which is activated by ANP. <br />
</pre><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<u>Inducible Effector System</u><br />
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In order to make our <i>E. coli</i> cells releasing the effectors at the site of plaque, we plan to put the SAA and HO-1 gene under the control of the P<sub>lux</sub> promoter, which will be activated by a threshold concentration of AHL at the site of plaque.<br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/2/27/QueensFigure7.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 7</i> Planned inducible effector construct.<br />
</p><br />
</br><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
We plan to transform <i>E. coli</i> cells with the effector construct and the binding construct, and conduct the endothelial adhesion assay. We will then analyze the growth medium of the endothelial culture by SDS-PAGE and Western blot analysis to determine the concentration of AHL, SAA, and biliverdin in the medium. We will also determine the expression level of TEV protease, which is located downstream of a double terminator (BBa_B0015) and RBS (BBa_B0034) on the effector construct. Expression level of TEV protease directly contributes to the efficiency of detachment from the plaque and termination by the kill switch.<br />
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Please note that Results are also available in PDF Format. <br />
Please click on the document you wish to view.<br />
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<a href="https://static.igem.org/mediawiki/2009/f/fc/QueensResultsJamesBogdanBryantHarry.pdf"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">Laboratory One: Harry, Bogdan, James, Bryant</p></a><br />
<a href="https://static.igem.org/mediawiki/2009/2/26/QueensResultsKateMike.pdf"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">Laboratory Two: Kate, Mike</p></a><br />
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<td align="left"><p style="font-size:175%;font-family:corbel;color:#172C4E;font-weight:bold"><i>Results<i><br />
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The following summarizes the results of the various aspects of this year's QGEM Project. <br />
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<a href="#Part1"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part One: SAA<br />
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<a href="#Part2"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Two: Binding Construct<br />
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<a href="#Part3"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Three: Heme and HO-1<br />
</p></a><br />
<a href="#Part4"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Four: Future Directions<br />
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<a name="Part1"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part One: SAA</p></a><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Serum amyloid A (SAA) featured prominently on our list of possible effectors to release at the site of atherosclerotic plaques due to research done at Queen’s University by Tam et al. (2005). This research showed that treatment of SAA caused macrophages to reverse and prevent esterification of cholesterol, thereby allowing it to be exported out of plaques by high-density lipoprotein (HDL). In order to get the SAA to be picked up by the macrophages we needed to produce large quantities of the molecule, and then secrete it extracellularly. In order to secrete the protein we decided on using the twin-arginine translocase (TAT) system, which can transport fully folded proteins outside the cell (Sargent et al., 2006) and is found in the majority of prokaryotes. We then fused the TAT signal sequence to the front of our SAA sequence in order to produce a construct that should be able produce and secrete the protein, allowing it to be taken up by macrophages.<br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 1</i> SAA expression and secretion by <i>E. coli</i> cells. Cultures of <i>E. coli</i> cells containing either the SAA construct or control plasmid were spun down at 0, 4, and 12 hours after seeding. The culture medium and whole cell lysates were analyzed by SDS-PAGE and Western blot analysis with a polyclonal antibody recognizing whole SAA protein. <br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Our <i>E. coli</i> cells transformed with SAA construct did not appear to express or secrete SAA into the growth medium even after growing for 12 hours in optimum condition. This might be due to defective ligation of the Ptet-RBS fragment to the SAA-encoding gene. We plan to sequence the construct in the future. <br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
References:<br />
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Benditt, E.P., Hoffman, J.S. & Eriksen, N. (1982). SAA, an apoprotein of HDL: its structure and function. <i> New York Academy of Sciences. </i> 82:183-189.<br />
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Ancsin, J.B. & Kisilevsky, R. (1999). The heparin/heparin sulfate-binding site on apo-serum amyloid A. <i>Journal of Biological Chemistry.</i> Vol. 274, 11:7172-7181.<br />
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<a name="Part2"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Two: Binding Construct</p></a><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In our original design of the binding construct, constitutive promoter BBa_J23119 was used to control the expression of the chimeric protein receptor consisted of the Lpp-OmpA fusion, TEV protease cut sites and ITGA4 fragment. We ordered our construct to be synthesized by Mr. GENE in early June. However, Mr. GENE notified us in August that our construct appeared to be unstable in and toxic to <i>E. coli</i> cells. The reason might be that high expression level of Lpp-OmpA might interfere with bacterial physiology, causing severe growth inhibition and reduced viability. (Daugherty et al, 1999) Mr. Gene was able to give us a sequence-confirmed PCR product of the synthesized construct. Thus, we decided to replace P¬const with P¬tet (BBa_R0040) by PCR. Below is a schematic diagram of the construction process. <br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 2</i> Flowchart of PCR stitching for constructing the binding construct. <br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Due to time constraint, we were only able to confirm the stitching of Ptet-RBS fragment to Lpp-OmpA-Linker-TEVx2-Linker fragment (PCR round 2) using Agarose gel electrophoresis. The third round of PCR stitching did not yield expected band. Construction of the binding construct is ongoing. <br />
</p><br />
<br/><br />
<center><img src="https://static.igem.org/mediawiki/2009/1/14/QueensFigure3.png"></center><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 3</i> <i>In silico</i> model of the binding construct. <br />
</p><br />
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<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
References:<br />
<br/><br />
<br/><br />
Daugherty, P.S., Olsen, M.J., Iverson, B.L. & Georgiou, G. (1999). Development of an optimized expression system for the screening of antibody libraries displayed on the <i>Escherichia coli</i> surface. <i>Protein Engineering.</i> Vol. 12, 7:613-621.<br />
<br/><br />
<br/><br />
Yang, Z., Lui, Q., Wang, Q. & Zhang, Y. (2008). Novel bacterial surface display systems based on outer membrane anchoring elements from the marine bacterium <i>Vibrio anguillarum</i>. <i> American Society for Microbiology.</i> Vol. 74, 14: 4359-4365.<br />
</p><br />
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<a name="Part3"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Three: Heme and HO-1</p></a><br />
<br/><br />
<br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Before presenting our results some useful parts have been outlined briefly.<br />
<br/><br />
<br/><br />
BioBrick and Construct Legend:<br />
<br/><br />
<br/><br />
BioBrick I716390: Plasmid containing a composite of Heme A, B C & D with T7-promoter and RBS. Amp Resistance.<br />
<br/><br />
BioBrick K098010: HO and phycocaynobilin:ferredoxin oxidoreductase. KAN Resistance.<br />
<br/><br />
BioBrick I15009: HO-1 from <i>Synechocystis</i>. Amp Resistance.<br />
<br/><br />
NLM 350: Rough <i> E. coli</i> strain containing T7-polymerase. Queen's University Research<br />
</p><br />
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<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In order to determine whether or not the BioBrick I716390 submitted by the Berkeley iGEM team in 2007 was producing heme, NLM350. Overnight cultures containing 100 μg/mL ampicillin were set up and inoculated with NLM350 containing I716390. The culture was analyzed daily by wavelength scan in a spectrometer. We ran the wavelength scan twice, once with a blank of LB and once with a blank of LB culture containing NLM350 without the BioBrick. Both of the scan results were identical, which indicates that the peaks present are due to the BioBrick plasmid and not resulting from compounds produced by NLM350. The results from day three with a blank of LB are shown in Figure 4.<br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/c/ce/QueensFigure4.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 4</i> Results of a wavelength scan between 350 and 550nm of the NLM350 + I716390 culture three days after inoculation. Strongest peaks at 412nm. <br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In order to check for the presence of the heme/HO-1 complex, NLM350 + I716390 cells were made electrocompetent and two sets of electroporations were performed. The BioBricks K098010 and I15008 were taken up by NLM350 + I716390 and the resulting colonies were used to inoculate cultures containing 40 μg/mL kanamycin and 100 μg/mL ampicillin, respectively. Triplicate testing of NLM350 + 176390 + KO98010 as well as NLM350 + 176390 + I15008 was conducted over five days. Both tests produced similar strong peaks for heme/HO-1 complexes, ranging from 407-409 nm. Again, we ran the scans twice with blanks of LB and LB inoculated with NLM350 and found that results were identical. The results from day five with blanks of LB are shown in Figures 5 and 6.<br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/2/2b/QueensFigure5.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 5</i> Wavelength scan between 350 and 550 nm of the NLM350 + I716390 + K098010 culture 5 days after inoculation. Strong peak of 407.5 nm. <br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/7/74/QueensFigure6.png"></center><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 6</i> Wavelength scan between 350 and 550 nm of the NLM350 + I716390 + I15008 culture 5 days after inoculation. Strong peak of 407.5 nm. <br />
</p><br />
<br/><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
As can be seen from our results above, the peaks in the cultures containing heme and HO-1 producing BioBricks become clearer and occur at a lower absorbance, around 408nm, than the peaks from cultures containing only heme-producing BioBricks. Cultures containing only the heme-producing BioBrick has the strongest peaks at 412nm, which corresponds to the peak found by Moffet et al. in 2003 <sup>1</sup>. Since HO-1 does not have an absorbance peak of its own <sup>2,3,4</sup>, the presence of HO-1 can only be inferred by identifying the heme/HO-1 complexes via spectroscopy. According to several authors <sup>2,4</sup>, Soret bands for the oxy-bound form of the heme/HO-1 complex occur at 410nm. The peaks seen in Figures 5 and 6 are slightly blue-shifted compared to the literature values, which may be the result of different biochemical subunits present in the heme protein<sup>5</sup>. However, the difference between the spectra of Figures 4, 5, and 6 indicates that HO-1 is being produced and is binding with heme to form a complex which peaks at a different wavelength than heme alone.<br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
It was expected to see a peak for biliverdin as well, which would have occurred between 440 – 460nm according to the literature <sup>6,7</sup>, though we did not. This might have been due to the fact that the cultures containing heme and HO-1 producing BioBricks had some exposure to light. Researchers have noted in their methods that they had avoided light when handling biliverdin <sup>8,9</sup>. If biliverdin is photosensitive, this might be why these peaks did not appear in our wavelength scans.<br />
</p><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In order to continue on this project track, the genetic effector system would have to be streamlined and optimized. More thorough kinetics testing for the production of heme as well as its metabolism by HO-1 would be required to maximize effector quality and concentration. Though many of the products of heme metabolism were likely produced, bilirubin was almost certainly not. <br />
</p><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
As mentioned previously<sup>2</sup>, there is a lack of biliverdin reductases available for cloning into bacterial systems, especially mammalian. Recently the entire genome of <i>Synechocystis sp.</i> PP6803 was sequenced, along with a cyanobacterial biliverdin reductase homologue. This enzyme is used in the production of light-harvesting pigments called phycobilins which is a product in the breakdown of heme<sup>10</sup>. With more research into extraction of this gene from blue-green algae, a very useful biliverdin reductase BioBrick could be contributed the registry, allowing full degradation of heme to bilirubin. This would enable the microbe as well as the system to which it is contributing, to reap the strong antioxidant properties of this molecule.<br />
</p><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
References:<br />
<br/><br />
<br/><br />
1. Moffet, D.A., Foley J. & Hecht M.H. (2003). Midpoint reduction potentials and heme binding stoichiometries of de novo proteins from designed combinatorial libraries. <i>Biophysical Chemistry</i>. 105:231-239.<br />
<br/><br />
<br/><br />
2. Frankenberg-Dinkel, N. (2004). Forum review: bacterial Heme Oxygenases. Antioxidants and Redox Signalling. 6:825 - 834.<br />
<br/><br />
<br/><br />
3. Huber W. & Backes W. (2008). Quantitation of heme oxygenase 1: Heme titration increases yield of purified protein. <i>Analytical Biochemistry</i>. 373:167-169.<br />
<br/><br />
<br/><br />
4. Migita C.T., Zhang X. & Yoshida T. (2003). Expression and characterization of cyanobacterium heme oxygenase, a key enzyme in the phycobilin synthesis. <i>European Journal of Biochemistry</i>. 270:687 – 698.<br />
<br/><br />
<br/><br />
5. Wang N., Zhao X. & Lu Y. (2005). Role of Heme Types in Heme-Copper Oxidases: Effects of Replacing a Heme b with a Heme to Mimic in an Engineered Heme-Copper Center in Myoglobin. <i>Journal of American Chemical Society</i>. 127:16541 – 16547.<br />
<br/><br />
<br/><br />
6. Yoshida T. & Kikuchi G. (1979). Purification and Properties of Heme Oxygenase from Rat Liver Microsomes. The <i>Journal of Biological Chemistry</i>. 254:4487 – 4491.<br />
<br/><br />
<br/><br />
7. Ding Z.K. & Xu Y.Q. (2002). Purification and characterization of biliverdin IXalpha from Atlantic salmon (Salmo salar) bile. <i>Biochemistry</i>. 67:927 – 932. <br />
<br/><br />
<br/><br />
8. Ollinger R., Yamashita K., Bilban M., Erat A., Kogler P., Thomas M., Csizmadia E., Usheva A., Margreiter R. & Bach F. H. (2007). Bilirubin and Biliverdin Treatment of Atherosclerotic Diseases. <i>Cell Cycle</i>. 6:39 – 43. <br />
<br/><br />
<br/><br />
9. Ishikawa K., Navab M., Leitinger N., Fogelman A.M. & Lusis A. J. (1997). Induction of heme oxygenase-1 inhibits the monocyte transmigration induced by mildly oxidized LDL. <i>Journal of Clinical Investigation</i>. 100:1209-1216.<br />
<br/><br />
<br/><br />
10. Cornejo J., Willows R. D. & Beale S. I. (1998). Phytobilin biosynthesis: cloning and expression of a gene encoding ferredoxin-dependent heme oxygenase from Synechocystis sp. PCC 6803. <i>The Plant Journal</i>. 15:99 – 107.<br />
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<a name="Part4"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Four: Future Directions</p></a><br />
<br/><br />
<br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Due to the delay of the synthesis of our binding construct, we were unable to test the presentation of the ITGA-4 chain of the VLA-4 antigen on <i>E. coli</i> outer membrane and the binding of the <i>E. coli</i> to endothelial cells expressing VCAM-1. In the future, we are planning to pursue this project further in the following areas.<br />
</p><br />
<br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"> <br />
<u>Construction</u><br />
</p><br />
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
1. Complete round 3 of PCR stitching and insert the binding construct into BioBrick backbone.<br />
<br />
2. Sequence the binding construct and the SAA construct.<br />
</pre><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<u>Endothelial Adhesion Assay</u><br />
</p><br />
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
1. Transform the binding construct and constitutive GFP expression construct into E. coli cells. <br />
<br />
2. Plate C166 murine endothelial cells on a 6-well plate. Incubate overnight. <br />
<br />
3. Prepare serial dilutions of the binding E. coli and control cells (containing plasmid backbone). <br />
<br />
4. Incubate C166 cells with E. coli for two hours and then wash wells 3X with PBS. <br />
<br />
5. Quantify GFP emission using fluorimeter. <br />
<br />
6. Alternative test: ELISA binding assay with recombinant VCAM-1 protein. <br />
</pre><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<u>Atrial Natriuretic Peptide</u><br />
</p><br />
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
1. Transform <i>E. coli</i> cells with ANP construct (P<sub>tet</sub>-RBS-Lpp-OmpA-Linker-TEVx2-Linker-ANP) and constitutive GFP expression construct. <br />
<br />
2. Use the same protocol as endothelial adhesion assay to test the activation of membrane-bound guanylate cyclase (GCA) on endothelial cells by ANP. <br />
<br />
3. Harvest the endothelial cells and prepare whole cell lysates. <br />
<br />
4. Run SDS-PAGE and Western blotting analysis with antibodies recognizing whole and phosphorylated Vasodilator Stimulated Phosphoprotein (VASP). VASP is phosphorylated by cGMP dependent kinase, which is activated by ANP. <br />
</pre><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<u>Inducible Effector System</u><br />
<br/><br />
<br/><br />
In order to make our <i>E. coli</i> cells releasing the effectors at the site of plaque, we plan to put the SAA and HO-1 gene under the control of the P<sub>lux</sub> promoter, which will be activated by a threshold concentration of AHL at the site of plaque.<br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/2/27/QueensFigure7.png"></center><br />
<br/> <br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 7</i> Planned inducible effector construct.<br />
</p><br />
</br><br />
</br><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
We plan to transform <i>E. coli</i> cells with the effector construct and the binding construct, and conduct the endothelial adhesion assay. We will then analyze the growth medium of the endothelial culture by SDS-PAGE and Western blot analysis to determine the concentration of AHL, SAA, and biliverdin in the medium. We will also determine the expression level of TEV protease, which is located downstream of a double terminator (BBa_B0015) and RBS (BBa_B0034) on the effector construct. Expression level of TEV protease directly contributes to the efficiency of detachment from the plaque and termination by the kill switch.<br />
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Please note that Results are also available in PDF Format. <br />
Please click on the document you wish to view.<br />
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<a href="https://static.igem.org/mediawiki/2009/f/fc/QueensResultsJamesBogdanBryantHarry.pdf"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">Laboratory One: Harry, Bogdan, James, Bryant</p></a><br />
<a href="https://static.igem.org/mediawiki/2009/2/26/QueensResultsKateMike.pdf"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">Laboratory Two: Kate, Mike</p></a><br />
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<td align="left"><p style="font-size:175%;font-family:corbel;color:#172C4E;font-weight:bold"><i>Results<i><br />
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The following summarizes the results of the various aspects of this year's QGEM Project. <br />
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<a href="#Part1"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part One: SAA<br />
</p></a><br />
<a href="#Part2"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Two: Binding Construct<br />
</p></a><br />
<a href="#Part3"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Three: Heme and HO-1<br />
</p></a><br />
<a href="#Part4"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Four: Future Directions<br />
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<a name="Part1"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part One: SAA</p></a><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Serum amyloid A (SAA) featured prominently on our list of possible effectors to release at the site of atherosclerotic plaques due to research done at Queen’s University by Tam et al. (2005). This research showed that treatment of SAA caused macrophages to reverse and prevent esterification of cholesterol, thereby allowing it to be exported out of plaques by high-density lipoprotein (HDL). In order to get the SAA to be picked up by the macrophages we needed to produce large quantities of the molecule, and then secrete it extracellularly. In order to secrete the protein we decided on using the twin-arginine translocase (TAT) system, which can transport fully folded proteins outside the cell (Sargent et al., 2006) and is found in the majority of prokaryotes. We then fused the TAT signal sequence to the front of our SAA sequence in order to produce a construct that should be able produce and secrete the protein, allowing it to be taken up by macrophages.<br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/c/cb/QueensFigure1.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 1</i> SAA expression and secretion by <i>E. coli</i> cells. Cultures of <i>E. coli</i> cells containing either the SAA construct or control plasmid were spun down at 0, 4, and 12 hours after seeding. The culture medium and whole cell lysates were analyzed by SDS-PAGE and Western blot analysis with a polyclonal antibody recognizing whole SAA protein. <br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Our <i>E. coli</i> cells transformed with SAA construct did not appear to express or secrete SAA into the growth medium even after growing for 12 hours in optimum condition. This might be due to defective ligation of the Ptet-RBS fragment to the SAA-encoding gene. We plan to sequence the construct in the future. <br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
References:<br />
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<br/><br />
Benditt, E.P., Hoffman, J.S. & Eriksen, N. (1982). SAA, an apoprotein of HDL: its structure and function. <i> New York Academy of Sciences. </i> 82:183-189.<br />
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<br/><br />
Ancsin, J.B. & Kisilevsky, R. (1999). The heparin/heparin sulfate-binding site on apo-serum amyloid A. <i>Journal of Biological Chemistry.</i> Vol. 274, 11:7172-7181.<br />
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<a name="Part2"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Two: Binding Construct</p></a><br />
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<br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In our original design of the binding construct, constitutive promoter BBa_J23119 was used to control the expression of the chimeric protein receptor consisted of the Lpp-OmpA fusion, TEV protease cut sites and ITGA4 fragment. We ordered our construct to be synthesized by Mr. GENE in early June. However, Mr. GENE notified us in August that our construct appeared to be unstable in and toxic to <i>E. coli</i> cells. The reason might be that high expression level of Lpp-OmpA might interfere with bacterial physiology, causing severe growth inhibition and reduced viability. (Daugherty et al, 1999) Mr. Gene was able to give us a sequence-confirmed PCR product of the synthesized construct. Thus, we decided to replace P¬const with P¬tet (BBa_R0040) by PCR. Below is a schematic diagram of the construction process. <br />
</p><br />
<br/><br />
<center><img src="https://static.igem.org/mediawiki/2009/0/08/QueensFigure2.png"></center><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 2</i> Flowchart of PCR stitching for constructing the binding construct. <br />
</p><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Due to time constraint, we were only able to confirm the stitching of Ptet-RBS fragment to Lpp-OmpA-Linker-TEVx2-Linker fragment (PCR round 2) using Agarose gel electrophoresis. The third round of PCR stitching did not yield expected band. Construction of the binding construct is ongoing. <br />
</p><br />
<br/><br />
<center><img src="https://static.igem.org/mediawiki/2009/1/14/QueensFigure3.png"></center><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 3</i> <i>In silico</i> model of the binding construct. <br />
</p><br />
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<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
References:<br />
<br/><br />
<br/><br />
Daugherty, P.S., Olsen, M.J., Iverson, B.L. & Georgiou, G. (1999). Development of an optimized expression system for the screening of antibody libraries displayed on the <i>Escherichia coli</i> surface. <i>Protein Engineering.</i> Vol. 12, 7:613-621.<br />
<br/><br />
<br/><br />
Yang, Z., Lui, Q., Wang, Q. & Zhang, Y. (2008). Novel bacterial surface display systems based on outer membrane anchoring elements from the marine bacterium <i>Vibrio anguillarum</i>. <i> American Society for Microbiology.</i> Vol. 74, 14: 4359-4365.<br />
</p><br />
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<a name="Part3"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Three: Heme and HO-1</p></a><br />
<br/><br />
<br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Before presenting our results some useful parts have been outlined briefly.<br />
<br/><br />
<br/><br />
BioBrick and Construct Legend:<br />
<br/><br />
<br/><br />
BioBrick I716390: Plasmid containing a composite of Heme A, B C & D with T7-promoter and RBS. Amp Resistance.<br />
<br/><br />
BioBrick K098010: HO and phycocaynobilin:ferredoxin oxidoreductase. KAN Resistance.<br />
<br/><br />
BioBrick I15009: HO-1 from <i>Synechocystis</i>. Amp Resistance.<br />
<br/><br />
NLM 350: Rough <i> E. coli</i> strain containing T7-polymerase. Queen's University Research<br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In order to determine whether or not the BioBrick I716390 submitted by the Berkeley iGEM team in 2007 was producing heme, NLM350. Overnight cultures containing 100 μg/mL ampicillin were set up and inoculated with NLM350 containing I716390. The culture was analyzed daily by wavelength scan in a spectrometer. We ran the wavelength scan twice, once with a blank of LB and once with a blank of LB culture containing NLM350 without the BioBrick. Both of the scan results were identical, which indicates that the peaks present are due to the BioBrick plasmid and not resulting from compounds produced by NLM350. The results from day three with a blank of LB are shown in Figure 4.<br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/c/ce/QueensFigure4.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 4</i> Results of a wavelength scan between 350 and 550nm of the NLM350 + I716390 culture three days after inoculation. Strongest peaks at 412nm. <br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In order to check for the presence of the heme/HO-1 complex, NLM350 + I716390 cells were made electrocompetent and two sets of electroporations were performed. The BioBricks K098010 and I15008 were taken up by NLM350 + I716390 and the resulting colonies were used to inoculate cultures containing 40 μg/mL kanamycin and 100 μg/mL ampicillin, respectively. Triplicate testing of NLM350 + 176390 + KO98010 as well as NLM350 + 176390 + I15008 was conducted over five days. Both tests produced similar strong peaks for heme/HO-1 complexes, ranging from 407-409 nm. Again, we ran the scans twice with blanks of LB and LB inoculated with NLM350 and found that results were identical. The results from day five with blanks of LB are shown in Figures 5 and 6.<br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/2/2b/QueensFigure5.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 5</i> Wavelength scan between 350 and 550 nm of the NLM350 + I716390 + K098010 culture 5 days after inoculation. Strong peak of 407.5 nm. <br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/7/74/QueensFigure6.png"></center><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 6</i> Wavelength scan between 350 and 550 nm of the NLM350 + I716390 + I15008 culture 5 days after inoculation. Strong peak of 407.5 nm. <br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
As can be seen from our results above, the peaks in the cultures containing heme and HO-1 producing BioBricks become clearer and occur at a lower absorbance, around 408nm, than the peaks from cultures containing only heme-producing BioBricks. Cultures containing only the heme-producing BioBrick has the strongest peaks at 412nm, which corresponds to the peak found by Moffet et al. in 2003 <sup>1</sup>. Since HO-1 does not have an absorbance peak of its own <sup>2,3,4</sup>, the presence of HO-1 can only be inferred by identifying the heme/HO-1 complexes via spectroscopy. According to several authors <sup>2,4</sup>, Soret bands for the oxy-bound form of the heme/HO-1 complex occur at 410nm. The peaks seen in Figures 5 and 6 are slightly blue-shifted compared to the literature values, which may be the result of different biochemical subunits present in the heme protein<sup>5</sup>. However, the difference between the spectra of Figures 4, 5, and 6 indicates that HO-1 is being produced and is binding with heme to form a complex which peaks at a different wavelength than heme alone.<br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
It was expected to see a peak for biliverdin as well, which would have occurred between 440 – 460nm according to the literature <sup>6,7</sup>, though we did not. This might have been due to the fact that the cultures containing heme and HO-1 producing BioBricks had some exposure to light. Researchers have noted in their methods that they had avoided light when handling biliverdin <sup>8,9</sup>. If biliverdin is photosensitive, this might be why these peaks did not appear in our wavelength scans.<br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In order to continue on this project track, the genetic effector system would have to be streamlined and optimized. More thorough kinetics testing for the production of heme as well as its metabolism by HO-1 would be required to maximize effector quality and concentration. Though many of the products of heme metabolism were likely produced, bilirubin was almost certainly not. <br />
</p><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
As mentioned previously<sup>2</sup>, there is a lack of biliverdin reductases available for cloning into bacterial systems, especially mammalian. Recently the entire genome of <i>Synechocystis sp.</i> PP6803 was sequenced, along with a cyanobacterial biliverdin reductase homologue. This enzyme is used in the production of light-harvesting pigments called phycobilins which is a product in the breakdown of heme<sup>10</sup>. With more research into extraction of this gene from blue-green algae, a very useful biliverdin reductase BioBrick could be contributed the registry, allowing full degradation of heme to bilirubin. This would enable the microbe as well as the system to which it is contributing, to reap the strong antioxidant properties of this molecule.<br />
</p><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
References:<br />
<br/><br />
<br/><br />
1. Moffet, D.A., Foley J. & Hecht M.H. (2003). Midpoint reduction potentials and heme binding stoichiometries of de novo proteins from designed combinatorial libraries. <i>Biophysical Chemistry</i>. 105:231-239.<br />
<br/><br />
<br/><br />
2. Frankenberg-Dinkel, N. (2004). Forum review: bacterial Heme Oxygenases. Antioxidants and Redox Signalling. 6:825 - 834.<br />
<br/><br />
<br/><br />
3. Huber W. & Backes W. (2008). Quantitation of heme oxygenase 1: Heme titration increases yield of purified protein. <i>Analytical Biochemistry</i>. 373:167-169.<br />
<br/><br />
<br/><br />
4. Migita C.T., Zhang X. & Yoshida T. (2003). Expression and characterization of cyanobacterium heme oxygenase, a key enzyme in the phycobilin synthesis. <i>European Journal of Biochemistry</i>. 270:687 – 698.<br />
<br/><br />
<br/><br />
5. Wang N., Zhao X. & Lu Y. (2005). Role of Heme Types in Heme-Copper Oxidases: Effects of Replacing a Heme b with a Heme to Mimic in an Engineered Heme-Copper Center in Myoglobin. <i>Journal of American Chemical Society</i>. 127:16541 – 16547.<br />
<br/><br />
<br/><br />
6. Yoshida T. & Kikuchi G. (1979). Purification and Properties of Heme Oxygenase from Rat Liver Microsomes. The <i>Journal of Biological Chemistry</i>. 254:4487 – 4491.<br />
<br/><br />
<br/><br />
7. Ding Z.K. & Xu Y.Q. (2002). Purification and characterization of biliverdin IXalpha from Atlantic salmon (Salmo salar) bile. <i>Biochemistry</i>. 67:927 – 932. <br />
<br/><br />
<br/><br />
8. Ollinger R., Yamashita K., Bilban M., Erat A., Kogler P., Thomas M., Csizmadia E., Usheva A., Margreiter R. & Bach F. H. (2007). Bilirubin and Biliverdin Treatment of Atherosclerotic Diseases. <i>Cell Cycle</i>. 6:39 – 43. <br />
<br/><br />
<br/><br />
9. Ishikawa K., Navab M., Leitinger N., Fogelman A.M. & Lusis A. J. (1997). Induction of heme oxygenase-1 inhibits the monocyte transmigration induced by mildly oxidized LDL. <i>Journal of Clinical Investigation</i>. 100:1209-1216.<br />
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<br/><br />
10. Cornejo J., Willows R. D. & Beale S. I. (1998). Phytobilin biosynthesis: cloning and expression of a gene encoding ferredoxin-dependent heme oxygenase from Synechocystis sp. PCC 6803. <i>The Plant Journal</i>. 15:99 – 107.<br />
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<a name="Part4"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Four: Future Directions</p></a><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Due to the delay of the synthesis of our binding construct, we were unable to test the presentation of the ITGA-4 chain of the VLA-4 antigen on <i>E. coli</i> outer membrane and the binding of the <i>E. coli</i> to endothelial cells expressing VCAM-1. In the future, we are planning to pursue this project further in the following areas.<br />
</p><br />
<br />
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"> <br />
<u>Construction</u><br />
<br />
1. Complete round 3 of PCR stitching and insert the binding construct into BioBrick backbone.<br />
<br />
2. Sequence the binding construct and the SAA construct.<br />
<br />
<u>Endothelial Adhesion Assay</u><br />
<br />
1. Transform the binding construct and constitutive GFP expression construct into E. coli cells. <br />
<br />
2. Plate C166 murine endothelial cells on a 6-well plate. Incubate overnight. <br />
<br />
3. Prepare serial dilutions of the binding E. coli and control cells (containing plasmid backbone). <br />
<br />
4. Incubate C166 cells with E. coli for two hours and then wash wells 3X with PBS. <br />
<br />
5. Quantify GFP emission using fluorimeter. <br />
<br />
6. Alternative test: ELISA binding assay with recombinant VCAM-1 protein. <br />
<br />
<u>Atrial Natriuretic Peptide</u><br />
<br />
1. Transform <i>E. coli</i> cells with ANP construct (P<sub>tet</sub>-RBS-Lpp-OmpA-Linker-TEVx2-Linker-ANP) and constitutive GFP expression construct. <br />
<br />
2. Use the same protocol as endothelial adhesion assay to test the activation of membrane-bound guanylate cyclase (GCA) on endothelial cells by ANP. <br />
<br />
3. Harvest the endothelial cells and prepare whole cell lysates. <br />
<br />
4. Run SDS-PAGE and Western blotting analysis with antibodies recognizing whole and phosphorylated Vasodilator Stimulated Phosphoprotein (VASP). VASP is phosphorylated by cGMP dependent kinase, which is activated by ANP. <br />
</pre><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<u>Inducible Effector System</u><br />
<br/><br />
<br/><br />
In order to make our <i>E. coli</i> cells releasing the effectors at the site of plaque, we plan to put the SAA and HO-1 gene under the control of the P<sub>lux</sub> promoter, which will be activated by a threshold concentration of AHL at the site of plaque.<br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/2/27/QueensFigure7.png"></center><br />
<br/> <br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 7</i> Planned inducible effector construct.<br />
</p><br />
</br><br />
</br><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
We plan to transform <i>E. coli</i> cells with the effector construct and the binding construct, and conduct the endothelial adhesion assay. We will then analyze the growth medium of the endothelial culture by SDS-PAGE and Western blot analysis to determine the concentration of AHL, SAA, and biliverdin in the medium. We will also determine the expression level of TEV protease, which is located downstream of a double terminator (BBa_B0015) and RBS (BBa_B0034) on the effector construct. Expression level of TEV protease directly contributes to the efficiency of detachment from the plaque and termination by the kill switch.<br />
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Please note that Results are also available in PDF Format. <br />
Please click on the document you wish to view.<br />
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<a href="https://static.igem.org/mediawiki/2009/f/fc/QueensResultsJamesBogdanBryantHarry.pdf"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">Laboratory One: Harry, Bogdan, James, Bryant</p></a><br />
<a href="https://static.igem.org/mediawiki/2009/2/26/QueensResultsKateMike.pdf"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">Laboratory Two: Kate, Mike</p></a><br />
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<td align="left"><p style="font-size:175%;font-family:corbel;color:#172C4E;font-weight:bold"><i>Results<i><br />
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The following summarizes the results of the various aspects of this year's QGEM Project. <br />
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Part One: SAA<br />
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<a href="#Part2"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Two: Binding Construct<br />
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<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Three: Heme and HO-1<br />
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<a href="#Part4"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Four: Future Directions<br />
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<a name="Part1"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part One: SAA</p></a><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Serum amyloid A (SAA) featured prominently on our list of possible effectors to release at the site of atherosclerotic plaques due to research done at Queen’s University by Tam et al. (2005). This research showed that treatment of SAA caused macrophages to reverse and prevent esterification of cholesterol, thereby allowing it to be exported out of plaques by high-density lipoprotein (HDL). In order to get the SAA to be picked up by the macrophages we needed to produce large quantities of the molecule, and then secrete it extracellularly. In order to secrete the protein we decided on using the twin-arginine translocase (TAT) system, which can transport fully folded proteins outside the cell (Sargent et al., 2006) and is found in the majority of prokaryotes. We then fused the TAT signal sequence to the front of our SAA sequence in order to produce a construct that should be able produce and secrete the protein, allowing it to be taken up by macrophages.<br />
</p><br />
<br/><br />
<center><img src="https://static.igem.org/mediawiki/2009/c/cb/QueensFigure1.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 1</i> SAA expression and secretion by <i>E. coli</i> cells. Cultures of <i>E. coli</i> cells containing either the SAA construct or control plasmid were spun down at 0, 4, and 12 hours after seeding. The culture medium and whole cell lysates were analyzed by SDS-PAGE and Western blot analysis with a polyclonal antibody recognizing whole SAA protein. <br />
</p><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Our <i>E. coli</i> cells transformed with SAA construct did not appear to express or secrete SAA into the growth medium even after growing for 12 hours in optimum condition. This might be due to defective ligation of the Ptet-RBS fragment to the SAA-encoding gene. We plan to sequence the construct in the future. <br />
</p><br />
<br/><br />
<br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
References:<br />
<br/><br />
<br/><br />
Benditt, E.P., Hoffman, J.S. & Eriksen, N. (1982). SAA, an apoprotein of HDL: its structure and function. <i> New York Academy of Sciences. </i> 82:183-189.<br />
<br/><br />
<br/><br />
Ancsin, J.B. & Kisilevsky, R. (1999). The heparin/heparin sulfate-binding site on apo-serum amyloid A. <i>Journal of Biological Chemistry.</i> Vol. 274, 11:7172-7181.<br />
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<a name="Part2"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Two: Binding Construct</p></a><br />
<br/><br />
<br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In our original design of the binding construct, constitutive promoter BBa_J23119 was used to control the expression of the chimeric protein receptor consisted of the Lpp-OmpA fusion, TEV protease cut sites and ITGA4 fragment. We ordered our construct to be synthesized by Mr. GENE in early June. However, Mr. GENE notified us in August that our construct appeared to be unstable in and toxic to <i>E. coli</i> cells. The reason might be that high expression level of Lpp-OmpA might interfere with bacterial physiology, causing severe growth inhibition and reduced viability. (Daugherty et al, 1999) Mr. Gene was able to give us a sequence-confirmed PCR product of the synthesized construct. Thus, we decided to replace P¬const with P¬tet (BBa_R0040) by PCR. Below is a schematic diagram of the construction process. <br />
</p><br />
<br/><br />
<center><img src="https://static.igem.org/mediawiki/2009/0/08/QueensFigure2.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 2</i> Flowchart of PCR stitching for constructing the binding construct. <br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Due to time constraint, we were only able to confirm the stitching of Ptet-RBS fragment to Lpp-OmpA-Linker-TEVx2-Linker fragment (PCR round 2) using Agarose gel electrophoresis. The third round of PCR stitching did not yield expected band. Construction of the binding construct is ongoing. <br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/1/14/QueensFigure3.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 3</i> <i>In silico</i> model of the binding construct. <br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
References:<br />
<br/><br />
<br/><br />
Daugherty, P.S., Olsen, M.J., Iverson, B.L. & Georgiou, G. (1999). Development of an optimized expression system for the screening of antibody libraries displayed on the <i>Escherichia coli</i> surface. <i>Protein Engineering.</i> Vol. 12, 7:613-621.<br />
<br/><br />
<br/><br />
Yang, Z., Lui, Q., Wang, Q. & Zhang, Y. (2008). Novel bacterial surface display systems based on outer membrane anchoring elements from the marine bacterium <i>Vibrio anguillarum</i>. <i> American Society for Microbiology.</i> Vol. 74, 14: 4359-4365.<br />
</p><br />
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<a name="Part3"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Three: Heme and HO-1</p></a><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Before presenting our results some useful parts have been outlined briefly.<br />
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BioBrick and Construct Legend:<br />
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<br/><br />
BioBrick I716390: Plasmid containing a composite of Heme A, B C & D with T7-promoter and RBS. Amp Resistance.<br />
<br/><br />
BioBrick K098010: HO and phycocaynobilin:ferredoxin oxidoreductase. KAN Resistance.<br />
<br/><br />
BioBrick I15009: HO-1 from <i>Synechocystis</i>. Amp Resistance.<br />
<br/><br />
NLM 350: Rough <i> E. coli</i> strain containing T7-polymerase. Queen's University Research<br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In order to determine whether or not the BioBrick I716390 submitted by the Berkeley iGEM team in 2007 was producing heme, NLM350. Overnight cultures containing 100 μg/mL ampicillin were set up and inoculated with NLM350 containing I716390. The culture was analyzed daily by wavelength scan in a spectrometer. We ran the wavelength scan twice, once with a blank of LB and once with a blank of LB culture containing NLM350 without the BioBrick. Both of the scan results were identical, which indicates that the peaks present are due to the BioBrick plasmid and not resulting from compounds produced by NLM350. The results from day three with a blank of LB are shown in Figure 4.<br />
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<center><img src="https://static.igem.org/mediawiki/2009/c/ce/QueensFigure4.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 4</i> Results of a wavelength scan between 350 and 550nm of the NLM350 + I716390 culture three days after inoculation. Strongest peaks at 412nm. <br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In order to check for the presence of the heme/HO-1 complex, NLM350 + I716390 cells were made electrocompetent and two sets of electroporations were performed. The BioBricks K098010 and I15008 were taken up by NLM350 + I716390 and the resulting colonies were used to inoculate cultures containing 40 μg/mL kanamycin and 100 μg/mL ampicillin, respectively. Triplicate testing of NLM350 + 176390 + KO98010 as well as NLM350 + 176390 + I15008 was conducted over five days. Both tests produced similar strong peaks for heme/HO-1 complexes, ranging from 407-409 nm. Again, we ran the scans twice with blanks of LB and LB inoculated with NLM350 and found that results were identical. The results from day five with blanks of LB are shown in Figures 5 and 6.<br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/2/2b/QueensFigure5.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 5</i> Wavelength scan between 350 and 550 nm of the NLM350 + I716390 + K098010 culture 5 days after inoculation. Strong peak of 407.5 nm. <br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/7/74/QueensFigure6.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 6</i> Wavelength scan between 350 and 550 nm of the NLM350 + I716390 + I15008 culture 5 days after inoculation. Strong peak of 407.5 nm. <br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
As can be seen from our results above, the peaks in the cultures containing heme and HO-1 producing BioBricks become clearer and occur at a lower absorbance, around 408nm, than the peaks from cultures containing only heme-producing BioBricks. Cultures containing only the heme-producing BioBrick has the strongest peaks at 412nm, which corresponds to the peak found by Moffet et al. in 2003 <super>1</super>. Since HO-1 does not have an absorbance peak of its own <super>2,3,4</super>, the presence of HO-1 can only be inferred by identifying the heme/HO-1 complexes via spectroscopy. According to several authors <super>2,4</super>, Soret bands for the oxy-bound form of the heme/HO-1 complex occur at 410nm. The peaks seen in Figures 5 and 6 are slightly blue-shifted compared to the literature values, which may be the result of different biochemical subunits present in the heme protein<super>5</super>. However, the difference between the spectra of Figures 4, 5, and 6 indicates that HO-1 is being produced and is binding with heme to form a complex which peaks at a different wavelength than heme alone.<br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
It was expected to see a peak for biliverdin as well, which would have occurred between 440 – 460nm according to the literature <super>6,7</super>, though we did not. This might have been due to the fact that the cultures containing heme and HO-1 producing BioBricks had some exposure to light. Researchers have noted in their methods that they had avoided light when handling biliverdin <super>8,9</super>. If biliverdin is photosensitive, this might be why these peaks did not appear in our wavelength scans.<br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In order to continue on this project track, the genetic effector system would have to be streamlined and optimized. More thorough kinetics testing for the production of heme as well as its metabolism by HO-1 would be required to maximize effector quality and concentration. Though many of the products of heme metabolism were likely produced, bilirubin was almost certainly not. <br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
As mentioned previously<super>2</super>, there is a lack of biliverdin reductases available for cloning into bacterial systems, especially mammalian. Recently the entire genome of <i>Synechocystis sp.</i> PP6803 was sequenced, along with a cyanobacterial biliverdin reductase homologue. This enzyme is used in the production of light-harvesting pigments called phycobilins which is a product in the breakdown of heme<super>10</super>. With more research into extraction of this gene from blue-green algae, a very useful biliverdin reductase BioBrick could be contributed the registry, allowing full degradation of heme to bilirubin. This would enable the microbe as well as the system to which it is contributing, to reap the strong antioxidant properties of this molecule.<br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
References:<br />
<br/><br />
<br/><br />
1. Moffet, D.A., Foley J. & Hecht M.H. (2003). Midpoint reduction potentials and heme binding stoichiometries of de novo proteins from designed combinatorial libraries. <i>Biophysical Chemistry</i>. 105:231-239.<br />
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<br/><br />
2. Frankenberg-Dinkel, N. (2004). Forum review: bacterial Heme Oxygenases. Antioxidants and Redox Signalling. 6:825 - 834.<br />
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<br/><br />
3. Huber W. & Backes W. (2008). Quantitation of heme oxygenase 1: Heme titration increases yield of purified protein. <i>Analytical Biochemistry</i>. 373:167-169.<br />
<br/><br />
<br/><br />
4. Migita C.T., Zhang X. & Yoshida T. (2003). Expression and characterization of cyanobacterium heme oxygenase, a key enzyme in the phycobilin synthesis. <i>European Journal of Biochemistry</i>. 270:687 – 698.<br />
<br/><br />
<br/><br />
5. Wang N., Zhao X. & Lu Y. (2005). Role of Heme Types in Heme-Copper Oxidases: Effects of Replacing a Heme b with a Heme to Mimic in an Engineered Heme-Copper Center in Myoglobin. <i>Journal of American Chemical Society</i>. 127:16541 – 16547.<br />
<br/><br />
<br/><br />
6. Yoshida T. & Kikuchi G. (1979). Purification and Properties of Heme Oxygenase from Rat Liver Microsomes. The <i>Journal of Biological Chemistry</i>. 254:4487 – 4491.<br />
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<br/><br />
7. Ding Z.K. & Xu Y.Q. (2002). Purification and characterization of biliverdin IXalpha from Atlantic salmon (Salmo salar) bile. <i>Biochemistry</i>. 67:927 – 932. <br />
<br/><br />
<br/><br />
8. Ollinger R., Yamashita K., Bilban M., Erat A., Kogler P., Thomas M., Csizmadia E., Usheva A., Margreiter R. & Bach F. H. (2007). Bilirubin and Biliverdin Treatment of Atherosclerotic Diseases. <i>Cell Cycle</i>. 6:39 – 43. <br />
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<br/><br />
9. Ishikawa K., Navab M., Leitinger N., Fogelman A.M. & Lusis A. J. (1997). Induction of heme oxygenase-1 inhibits the monocyte transmigration induced by mildly oxidized LDL. <i>Journal of Clinical Investigation</i>. 100:1209-1216.<br />
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<br/><br />
10. Cornejo J., Willows R. D. & Beale S. I. (1998). Phytobilin biosynthesis: cloning and expression of a gene encoding ferredoxin-dependent heme oxygenase from Synechocystis sp. PCC 6803. <i>The Plant Journal</i>. 15:99 – 107.<br />
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<a name="Part4"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Four: Future Directions</p></a><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Due to the delay of the synthesis of our binding construct, we were unable to test the presentation of the ITGA-4 chain of the VLA-4 antigen on <i>E. coli</i> outer membrane and the binding of the <i>E. coli</i> to endothelial cells expressing VCAM-1. In the future, we are planning to pursue this project further in the following areas.<br />
</p><br />
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<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"> <br />
<u>Construction</u><br />
<br />
1. Complete round 3 of PCR stitching and insert the binding construct into BioBrick backbone.<br />
<br />
2. Sequence the binding construct and the SAA construct.<br />
<br />
<br />
<u>Endothelial Adhesion Assay</u><br />
<br />
1. Transform the binding construct and constitutive GFP expression construct into E. coli cells. <br />
<br />
2. Plate C166 murine endothelial cells on a 6-well plate. Incubate overnight. <br />
<br />
3. Prepare serial dilutions of the binding E. coli and control cells (containing plasmid backbone). <br />
<br />
4. Incubate C166 cells with E. coli for two hours and then wash wells 3X with PBS. <br />
<br />
5. Quantify GFP emission using fluorimeter. <br />
<br />
6. Alternative test: ELISA binding assay with recombinant VCAM-1 protein. <br />
<br />
<br />
<u>Atrial Natriuretic Peptide</u><br />
<br />
1. Transform <i>E. coli</i> cells with ANP construct (P<sub>tet</sub>-RBS-Lpp-OmpA-Linker-TEVx2-Linker-ANP) and constitutive GFP expression construct. <br />
<br />
2. Use the same protocol as endothelial adhesion assay to test the activation of membrane-bound guanylate cyclase (GCA) on endothelial cells by ANP. <br />
<br />
3. Harvest the endothelial cells and prepare whole cell lysates. <br />
<br />
4. Run SDS-PAGE and Western blotting analysis with antibodies recognizing whole and phosphorylated Vasodilator Stimulated Phosphoprotein (VASP). VASP is phosphorylated by cGMP dependent kinase, which is activated by ANP. <br />
</pre><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<u>Inducible Effector System</u><br />
<br/><br />
<br/><br />
In order to make our <i>E. coli</i> cells releasing the effectors at the site of plaque, we plan to put the SAA and HO-1 gene under the control of the P<sub>lux</sub> promoter, which will be activated by a threshold concentration of AHL at the site of plaque.<br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/2/27/QueensFigure7.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 7</i> Planned inducible effector construct.<br />
</p><br />
</br><br />
</br><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
We plan to transform <i>E. coli</i> cells with the effector construct and the binding construct, and conduct the endothelial adhesion assay. We will then analyze the growth medium of the endothelial culture by SDS-PAGE and Western blot analysis to determine the concentration of AHL, SAA, and biliverdin in the medium. We will also determine the expression level of TEV protease, which is located downstream of a double terminator (BBa_B0015) and RBS (BBa_B0034) on the effector construct. Expression level of TEV protease directly contributes to the efficiency of detachment from the plaque and termination by the kill switch.<br />
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Please note that Results are also available in PDF Format. <br />
Please click on the document you wish to view.<br />
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<a href="https://static.igem.org/mediawiki/2009/f/fc/QueensResultsJamesBogdanBryantHarry.pdf"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">Laboratory One: Harry, Bogdan, James, Bryant</p></a><br />
<a href="https://static.igem.org/mediawiki/2009/2/26/QueensResultsKateMike.pdf"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">Laboratory Two: Kate, Mike</p></a><br />
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The following summarizes the results of the various aspects of this year's QGEM Project. <br />
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Part One: SAA<br />
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Part Two: Binding Construct<br />
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Part Three: Heme and HO-1<br />
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<a href="#Part4"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Four: Future Directions<br />
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<a name="Part1"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part One: SAA</p></a><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Serum amyloid A (SAA) featured prominently on our list of possible effectors to release at the site of atherosclerotic plaques due to research done at Queen’s University by Tam et al. (2005). This research showed that treatment of SAA caused macrophages to reverse and prevent esterification of cholesterol, thereby allowing it to be exported out of plaques by high-density lipoprotein (HDL). In order to get the SAA to be picked up by the macrophages we needed to produce large quantities of the molecule, and then secrete it extracellularly. In order to secrete the protein we decided on using the twin-arginine translocase (TAT) system, which can transport fully folded proteins outside the cell (Sargent et al., 2006) and is found in the majority of prokaryotes. We then fused the TAT signal sequence to the front of our SAA sequence in order to produce a construct that should be able produce and secrete the protein, allowing it to be taken up by macrophages.<br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 1</i> SAA expression and secretion by <i>E. coli</i> cells. Cultures of <i>E. coli</i> cells containing either the SAA construct or control plasmid were spun down at 0, 4, and 12 hours after seeding. The culture medium and whole cell lysates were analyzed by SDS-PAGE and Western blot analysis with a polyclonal antibody recognizing whole SAA protein. <br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Our <i>E. coli</i> cells transformed with SAA construct did not appear to express or secrete SAA into the growth medium even after growing for 12 hours in optimum condition. This might be due to defective ligation of the Ptet-RBS fragment to the SAA-encoding gene. We plan to sequence the construct in the future. <br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
References:<br />
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Benditt, E.P., Hoffman, J.S. & Eriksen, N. (1982). SAA, an apoprotein of HDL: its structure and function. <i> New York Academy of Sciences. </i> 82:183-189.<br />
<br/><br />
<br/><br />
Ancsin, J.B. & Kisilevsky, R. (1999). The heparin/heparin sulfate-binding site on apo-serum amyloid A. <i>Journal of Biological Chemistry.</i> Vol. 274, 11:7172-7181.<br />
</p><br />
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<a name="Part2"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Two: Binding Construct</p></a><br />
<br/><br />
<br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In our original design of the binding construct, constitutive promoter BBa_J23119 was used to control the expression of the chimeric protein receptor consisted of the Lpp-OmpA fusion, TEV protease cut sites and ITGA4 fragment. We ordered our construct to be synthesized by Mr. GENE in early June. However, Mr. GENE notified us in August that our construct appeared to be unstable in and toxic to <i>E. coli</i> cells. The reason might be that high expression level of Lpp-OmpA might interfere with bacterial physiology, causing severe growth inhibition and reduced viability. (Daugherty et al, 1999) Mr. Gene was able to give us a sequence-confirmed PCR product of the synthesized construct. Thus, we decided to replace P¬const with P¬tet (BBa_R0040) by PCR. Below is a schematic diagram of the construction process. <br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/0/08/QueensFigure2.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 2</i> Flowchart of PCR stitching for constructing the binding construct. <br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Due to time constraint, we were only able to confirm the stitching of Ptet-RBS fragment to Lpp-OmpA-Linker-TEVx2-Linker fragment (PCR round 2) using Agarose gel electrophoresis. The third round of PCR stitching did not yield expected band. Construction of the binding construct is ongoing. <br />
</p><br />
<br/><br />
<center><img src="https://static.igem.org/mediawiki/2009/1/14/QueensFigure3.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 3</i> <i>In silico</i> model of the binding construct. <br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
References:<br />
<br/><br />
<br/><br />
Daugherty, P.S., Olsen, M.J., Iverson, B.L. & Georgiou, G. (1999). Development of an optimized expression system for the screening of antibody libraries displayed on the <i>Escherichia coli</i> surface. <i>Protein Engineering.</i> Vol. 12, 7:613-621.<br />
<br/><br />
<br/><br />
Yang, Z., Lui, Q., Wang, Q. & Zhang, Y. (2008). Novel bacterial surface display systems based on outer membrane anchoring elements from the marine bacterium <i>Vibrio anguillarum</i>. <i> American Society for Microbiology.</i> Vol. 74, 14: 4359-4365.<br />
</p><br />
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<a name="Part3"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Three: Heme and HO-1</p></a><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Before presenting our results some useful parts have been outlined briefly.<br />
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BioBrick and Construct Legend:<br />
<br/><br />
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BioBrick I716390: Plasmid containing a composite of Heme A, B C & D with T7-promoter and RBS. Amp Resistance.<br />
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BioBrick K098010: HO and phycocaynobilin:ferredoxin oxidoreductase. KAN Resistance.<br />
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BioBrick I15009: HO-1 from <i>Synechocystis</i>. Amp Resistance.<br />
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NLM 350: Rough <i> E. coli</i> strain containing T7-polymerase. Queen's University Research<br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In order to determine whether or not the BioBrick I716390 submitted by the Berkeley iGEM team in 2007 was producing heme, NLM350. Overnight cultures containing 100 μg/mL ampicillin were set up and inoculated with NLM350 containing I716390. The culture was analyzed daily by wavelength scan in a spectrometer. We ran the wavelength scan twice, once with a blank of LB and once with a blank of LB culture containing NLM350 without the BioBrick. Both of the scan results were identical, which indicates that the peaks present are due to the BioBrick plasmid and not resulting from compounds produced by NLM350. The results from day three with a blank of LB are shown in Figure 4.<br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/c/ce/QueensFigure4.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 4</i> Results of a wavelength scan between 350 and 550nm of the NLM350 + I716390 culture three days after inoculation. Strongest peaks at 412nm. <br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In order to check for the presence of the heme/HO-1 complex, NLM350 + I716390 cells were made electrocompetent and two sets of electroporations were performed. The BioBricks K098010 and I15008 were taken up by NLM350 + I716390 and the resulting colonies were used to inoculate cultures containing 40 μg/mL kanamycin and 100 μg/mL ampicillin, respectively. Triplicate testing of NLM350 + 176390 + KO98010 as well as NLM350 + 176390 + I15008 was conducted over five days. Both tests produced similar strong peaks for heme/HO-1 complexes, ranging from 407-409 nm. Again, we ran the scans twice with blanks of LB and LB inoculated with NLM350 and found that results were identical. The results from day five with blanks of LB are shown in Figures 5 and 6.<br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/2/2b/QueensFigure5.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 5</i> Wavelength scan between 350 and 550 nm of the NLM350 + I716390 + K098010 culture 5 days after inoculation. Strong peak of 407.5 nm. <br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/7/74/QueensFigure6.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 4</i> Wavelength scan between 350 and 550 nm of the NLM350 + I716390 + I15008 culture 5 days after inoculation. Strong peak of 407.5 nm. <br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
As can be seen from our results above, the peaks in the cultures containing heme and HO-1 producing BioBricks become clearer and occur at a lower absorbance, around 408nm, than the peaks from cultures containing only heme-producing BioBricks. Cultures containing only the heme-producing BioBrick has the strongest peaks at 412nm, which corresponds to the peak found by Moffet et al. in 2003 <super>1</super>. Since HO-1 does not have an absorbance peak of its own <super>2,3,4</super>, the presence of HO-1 can only be inferred by identifying the heme/HO-1 complexes via spectroscopy. According to several authors <super>2,4</super>, Soret bands for the oxy-bound form of the heme/HO-1 complex occur at 410nm. The peaks seen in Figures 5 and 6 are slightly blue-shifted compared to the literature values, which may be the result of different biochemical subunits present in the heme protein<super>5</super>. However, the difference between the spectra of Figures 4, 5, and 6 indicates that HO-1 is being produced and is binding with heme to form a complex which peaks at a different wavelength than heme alone.<br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
It was expected to see a peak for biliverdin as well, which would have occurred between 440 – 460nm according to the literature <super>6,7</super>, though we did not. This might have been due to the fact that the cultures containing heme and HO-1 producing BioBricks had some exposure to light. Researchers have noted in their methods that they had avoided light when handling biliverdin <super>8,9</super>. If biliverdin is photosensitive, this might be why these peaks did not appear in our wavelength scans.<br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In order to continue on this project track, the genetic effector system would have to be streamlined and optimized. More thorough kinetics testing for the production of heme as well as its metabolism by HO-1 would be required to maximize effector quality and concentration. Though many of the products of heme metabolism were likely produced, bilirubin was almost certainly not. <br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
As mentioned previously<super>2</super>, there is a lack of biliverdin reductases available for cloning into bacterial systems, especially mammalian. Recently the entire genome of <i>Synechocystis sp.</i> PP6803 was sequenced, along with a cyanobacterial biliverdin reductase homologue. This enzyme is used in the production of light-harvesting pigments called phycobilins which is a product in the breakdown of heme<super>10</super>. With more research into extraction of this gene from blue-green algae, a very useful biliverdin reductase BioBrick could be contributed the registry, allowing full degradation of heme to bilirubin. This would enable the microbe as well as the system to which it is contributing, to reap the strong antioxidant properties of this molecule.<br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
References:<br />
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1. Moffet, D.A., Foley J. & Hecht M.H. (2003). Midpoint reduction potentials and heme binding stoichiometries of de novo proteins from designed combinatorial libraries. <i>Biophysical Chemistry</i>. 105:231-239.<br />
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2. Frankenberg-Dinkel, N. (2004). Forum review: bacterial Heme Oxygenases. Antioxidants and Redox Signalling. 6:825 - 834.<br />
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<br/><br />
3. Huber W. & Backes W. (2008). Quantitation of heme oxygenase 1: Heme titration increases yield of purified protein. <i>Analytical Biochemistry</i>. 373:167-169.<br />
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<br/><br />
4. Migita C.T., Zhang X. & Yoshida T. (2003). Expression and characterization of cyanobacterium heme oxygenase, a key enzyme in the phycobilin synthesis. <i>European Journal of Biochemistry</i>. 270:687 – 698.<br />
<br/><br />
<br/><br />
5. Wang N., Zhao X. & Lu Y. (2005). Role of Heme Types in Heme-Copper Oxidases: Effects of Replacing a Heme b with a Heme to Mimic in an Engineered Heme-Copper Center in Myoglobin. <i>Journal of American Chemical Society</i>. 127:16541 – 16547.<br />
<br/><br />
<br/><br />
6. Yoshida T. & Kikuchi G. (1979). Purification and Properties of Heme Oxygenase from Rat Liver Microsomes. The <i>Journal of Biological Chemistry</i>. 254:4487 – 4491.<br />
<br/><br />
<br/><br />
7. Ding Z.K. & Xu Y.Q. (2002). Purification and characterization of biliverdin IXalpha from Atlantic salmon (Salmo salar) bile. <i>Biochemistry</i>. 67:927 – 932. <br />
<br/><br />
<br/><br />
8. Ollinger R., Yamashita K., Bilban M., Erat A., Kogler P., Thomas M., Csizmadia E., Usheva A., Margreiter R. & Bach F. H. (2007). Bilirubin and Biliverdin Treatment of Atherosclerotic Diseases. <i>Cell Cycle</i>. 6:39 – 43. <br />
<br/><br />
<br/><br />
9. Ishikawa K., Navab M., Leitinger N., Fogelman A.M. & Lusis A. J. (1997). Induction of heme oxygenase-1 inhibits the monocyte transmigration induced by mildly oxidized LDL. <i>Journal of Clinical Investigation</i>. 100:1209-1216.<br />
<br/><br />
<br/><br />
10. Cornejo J., Willows R. D. & Beale S. I. (1998). Phytobilin biosynthesis: cloning and expression of a gene encoding ferredoxin-dependent heme oxygenase from Synechocystis sp. PCC 6803. <i>The Plant Journal</i>. 15:99 – 107.<br />
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<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Please note that Results are also available in PDF Format. <br />
Please click on the document you wish to view.<br />
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<a href="https://static.igem.org/mediawiki/2009/f/fc/QueensResultsJamesBogdanBryantHarry.pdf"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">Laboratory One: Harry, Bogdan, James, Bryant</p></a><br />
<a href="https://static.igem.org/mediawiki/2009/2/26/QueensResultsKateMike.pdf"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">Laboratory Two: Kate, Mike</p></a><br />
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<td align="left"><p style="font-size:175%;font-family:corbel;color:#172C4E;font-weight:bold"><i>Results<i><br />
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The following summarizes the results of the various aspects of this year's QGEM Project. <br />
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<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part One: SAA<br />
</p></a><br />
<a href="#Part2"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Two: Binding Construct<br />
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<a href="#Part3"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Three: Heme and HO-1<br />
</p></a><br />
<a href="#Part4"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Four: Future Directions<br />
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<a name="Part1"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part One: SAA</p></a><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Serum amyloid A (SAA) featured prominently on our list of possible effectors to release at the site of atherosclerotic plaques due to research done at Queen’s University by Tam et al. (2005). This research showed that treatment of SAA caused macrophages to reverse and prevent esterification of cholesterol, thereby allowing it to be exported out of plaques by high-density lipoprotein (HDL). In order to get the SAA to be picked up by the macrophages we needed to produce large quantities of the molecule, and then secrete it extracellularly. In order to secrete the protein we decided on using the twin-arginine translocase (TAT) system, which can transport fully folded proteins outside the cell (Sargent et al., 2006) and is found in the majority of prokaryotes. We then fused the TAT signal sequence to the front of our SAA sequence in order to produce a construct that should be able produce and secrete the protein, allowing it to be taken up by macrophages.<br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/c/cb/QueensFigure1.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 1</i> SAA expression and secretion by <i>E. coli</i> cells. Cultures of <i>E. coli</i> cells containing either the SAA construct or control plasmid were spun down at 0, 4, and 12 hours after seeding. The culture medium and whole cell lysates were analyzed by SDS-PAGE and Western blot analysis with a polyclonal antibody recognizing whole SAA protein. <br />
</p><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Our <i>E. coli</i> cells transformed with SAA construct did not appear to express or secrete SAA into the growth medium even after growing for 12 hours in optimum condition. This might be due to defective ligation of the Ptet-RBS fragment to the SAA-encoding gene. We plan to sequence the construct in the future. <br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
References:<br />
<br/><br />
<br/><br />
Benditt, E.P., Hoffman, J.S. & Eriksen, N. (1982). SAA, an apoprotein of HDL: its structure and function. <i> New York Academy of Sciences. </i> 82:183-189.<br />
<br/><br />
<br/><br />
Ancsin, J.B. & Kisilevsky, R. (1999). The heparin/heparin sulfate-binding site on apo-serum amyloid A. <i>Journal of Biological Chemistry.</i> Vol. 274, 11:7172-7181.<br />
</p><br />
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<a name="Part2"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Two: Binding Construct</p></a><br />
<br/><br />
<br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In our original design of the binding construct, constitutive promoter BBa_J23119 was used to control the expression of the chimeric protein receptor consisted of the Lpp-OmpA fusion, TEV protease cut sites and ITGA4 fragment. We ordered our construct to be synthesized by Mr. GENE in early June. However, Mr. GENE notified us in August that our construct appeared to be unstable in and toxic to <i>E. coli</i> cells. The reason might be that high expression level of Lpp-OmpA might interfere with bacterial physiology, causing severe growth inhibition and reduced viability. (Daugherty et al, 1999) Mr. Gene was able to give us a sequence-confirmed PCR product of the synthesized construct. Thus, we decided to replace P¬const with P¬tet (BBa_R0040) by PCR. Below is a schematic diagram of the construction process. <br />
</p><br />
<br/><br />
<center><img src="https://static.igem.org/mediawiki/2009/0/08/QueensFigure2.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 2</i> Flowchart of PCR stitching for constructing the binding construct. <br />
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<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Due to time constraint, we were only able to confirm the stitching of Ptet-RBS fragment to Lpp-OmpA-Linker-TEVx2-Linker fragment (PCR round 2) using Agarose gel electrophoresis. The third round of PCR stitching did not yield expected band. Construction of the binding construct is ongoing. <br />
</p><br />
<br/><br />
<center><img src="https://static.igem.org/mediawiki/2009/1/14/QueensFigure3.png"></center><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 3</i> <i>In silico</i> model of the binding construct. <br />
</p><br />
<br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
References:<br />
<br/><br />
<br/><br />
Daugherty, P.S., Olsen, M.J., Iverson, B.L. & Georgiou, G. (1999). Development of an optimized expression system for the screening of antibody libraries displayed on the <i>Escherichia coli</i> surface. <i>Protein Engineering.</i> Vol. 12, 7:613-621.<br />
<br/><br />
<br/><br />
Yang, Z., Lui, Q., Wang, Q. & Zhang, Y. (2008). Novel bacterial surface display systems based on outer membrane anchoring elements from the marine bacterium <i>Vibrio anguillarum</i>. <i> American Society for Microbiology.</i> Vol. 74, 14: 4359-4365.<br />
</p><br />
<br/><br />
</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td align="left"><br />
<a name="Part3"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Three: Cleavage and Termination</p></a><br />
<br/><br />
<br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
After our chassis has been bound to the site of the plaque for a period of time, it should begin to produce DNases and proteases. The DNases will function to sheer the bacterial genome, making it unable to proliferate in the blood; this functions as a safe-guard against bacteremia. The proteases serve to detach the chassis from the plaque site, so as to avoid a build-up of dead cell debris in an already inflamed area.<br />
</p><br />
<br/><br />
</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td align="left"><br />
<p style="font-size:120%;font-family:palatino linotype;color:#172C4E;"><br />
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Please note that Results are also available in PDF Format. <br />
Please click on the document you wish to view.<br />
</pre></p></td><br />
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<a href="https://static.igem.org/mediawiki/2009/f/fc/QueensResultsJamesBogdanBryantHarry.pdf"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">Laboratory One: Harry, Bogdan, James, Bryant</p></a><br />
<a href="https://static.igem.org/mediawiki/2009/2/26/QueensResultsKateMike.pdf"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">Laboratory Two: Kate, Mike</p></a><br />
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</html></div>ParthivAhttp://2009.igem.org/Team:Queens/ResultsTeam:Queens/Results2009-10-21T00:25:00Z<p>ParthivA: </p>
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<td align="left"><p style="font-size:175%;font-family:corbel;color:#172C4E;font-weight:bold"><i>Results<i><br />
</p></td><br />
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<td><br />
<br/><br />
</td><br />
</tr><br />
<br />
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<p style="font-size=120%;font-family:palatino linotype;color:#ECB528"><br />
The following summarizes the results of the various aspects of this year's QGEM Project. <br />
</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<br/><br />
</td><br />
</tr><br />
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<tr><br />
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<a href="#Part1"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part One: SAA<br />
</p></a><br />
<a href="#Part2"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Two: Binding Construct<br />
</p></a><br />
<a href="#Part3"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Three: Heme and HO-1<br />
</p></a><br />
<a href="#Part4"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Four: Future Directions<br />
</p></a><br />
</td><br />
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<td><br />
<br/><br />
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<a name="Part1"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part One: SAA</p></a><br />
<br/><br />
<br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Serum amyloid A (SAA) featured prominently on our list of possible effectors to release at the site of atherosclerotic plaques due to research done at Queen’s University by Tam et al. (2005). This research showed that treatment of SAA caused macrophages to reverse and prevent esterification of cholesterol, thereby allowing it to be exported out of plaques by high-density lipoprotein (HDL). In order to get the SAA to be picked up by the macrophages we needed to produce large quantities of the molecule, and then secrete it extracellularly. In order to secrete the protein we decided on using the twin-arginine translocase (TAT) system, which can transport fully folded proteins outside the cell (Sargent et al., 2006) and is found in the majority of prokaryotes. We then fused the TAT signal sequence to the front of our SAA sequence in order to produce a construct that should be able produce and secrete the protein, allowing it to be taken up by macrophages.<br />
</p><br />
<br/><br />
<center><img src="https://static.igem.org/mediawiki/2009/c/cb/QueensFigure1.png"></center><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 1</i> SAA expression and secretion by <i>E. coli</i> cells. Cultures of <i>E. coli</i> cells containing either the SAA construct or control plasmid were spun down at 0, 4, and 12 hours after seeding. The culture medium and whole cell lysates were analyzed by SDS-PAGE and Western blot analysis with a polyclonal antibody recognizing whole SAA protein. <br />
</p><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Our <i>E. coli</i> cells transformed with SAA construct did not appear to express or secrete SAA into the growth medium even after growing for 12 hours in optimum condition. This might be due to defective ligation of the Ptet-RBS fragment to the SAA-encoding gene. We plan to sequence the construct in the future. <br />
</p><br />
<br/><br />
<br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
References:<br />
<br/><br />
<br/><br />
Benditt, E.P., Hoffman, J.S. & Eriksen, N. (1982). SAA, an apoprotein of HDL: its structure and function. <i> New York Academy of Sciences. </i> 82:183-189.<br />
<br/><br />
Ancsin, J.B. & Kisilevsky, R. (1999). The heparin/heparin sulfate-binding site on apo-serum amyloid A. <i>Journal of Biological Chemistry.</i> Vol. 274, 11:7172-7181.<br />
</p><br />
<br/><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td align="left"><br />
<a name="Part2"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Two: Binding Construct</p></a><br />
<br/><br />
<br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In our original design of the binding construct, constitutive promoter BBa_J23119 was used to control the expression of the chimeric protein receptor consisted of the Lpp-OmpA fusion, TEV protease cut sites and ITGA4 fragment. We ordered our construct to be synthesized by Mr. GENE in early June. However, Mr. GENE notified us in August that our construct appeared to be unstable in and toxic to <i>E. coli</i> cells. The reason might be that high expression level of Lpp-OmpA might interfere with bacterial physiology, causing severe growth inhibition and reduced viability. (Daugherty et al, 1999) Mr. Gene was able to give us a sequence-confirmed PCR product of the synthesized construct. Thus, we decided to replace P¬const with P¬tet (BBa_R0040) by PCR. Below is a schematic diagram of the construction process. <br />
</p><br />
<br/><br />
<center><img src="https://static.igem.org/mediawiki/2009/0/08/QueensFigure2.png"></center><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 2</i> Flowchart of PCR stitching for constructing the binding construct. <br />
</p><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Due to time constraint, we were only able to confirm the stitching of Ptet-RBS fragment to Lpp-OmpA-Linker-TEVx2-Linker fragment (PCR round 2) using Agarose gel electrophoresis. The third round of PCR stitching did not yield expected band. Construction of the binding construct is ongoing. <br />
</p><br />
<br/><br />
<center><img src="https://static.igem.org/mediawiki/2009/1/14/QueensFigure3.png"></center><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 3</i> <i>In silico</i> model of the binding construct. <br />
</p><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
References:<br />
<br/><br />
<br/><br />
Daugherty, P.S., Olsen, M.J., Iverson, B.L. & Georgiou, G. (1999). Development of an optimized expression system for the screening of antibody libraries displayed on the <i>Escherichia coli</i> surface. <i>Protein Engineering.</i> Vol. 12, 7:613-621.<br />
<br/><br />
Yang, Z., Lui, Q., Wang, Q. & Zhang, Y. (2008). Novel bacterial surface display systems based on outer membrane anchoring elements from the marine bacterium <i>Vibrio anguillarum</i>. <i> American Society for Microbiology.</i> Vol. 74, 14: 4359-4365.<br />
</p><br />
<br/><br />
</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td align="left"><br />
<a name="Part3"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Three: Cleavage and Termination</p></a><br />
<br/><br />
<br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
After our chassis has been bound to the site of the plaque for a period of time, it should begin to produce DNases and proteases. The DNases will function to sheer the bacterial genome, making it unable to proliferate in the blood; this functions as a safe-guard against bacteremia. The proteases serve to detach the chassis from the plaque site, so as to avoid a build-up of dead cell debris in an already inflamed area.<br />
</p><br />
<br/><br />
</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td align="left"><br />
<p style="font-size:120%;font-family:palatino linotype;color:#172C4E;"><br />
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Please note that Results are also available in PDF Format. <br />
Please click on the document you wish to view.<br />
</pre></p></td><br />
</tr><br />
<br />
<br/><br />
<br />
<tr><br />
<td align="left"><br />
<a href="https://static.igem.org/mediawiki/2009/f/fc/QueensResultsJamesBogdanBryantHarry.pdf"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">Laboratory One: Harry, Bogdan, James, Bryant</p></a><br />
<a href="https://static.igem.org/mediawiki/2009/2/26/QueensResultsKateMike.pdf"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">Laboratory Two: Kate, Mike</p></a><br />
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<font color="#172C4E"><center><br />
Last Updated: October 19, 2009 by Fr<sub>3</sub>P</center></font><br />
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</footer><br />
</html></div>ParthivAhttp://2009.igem.org/File:QueensFigure3.pngFile:QueensFigure3.png2009-10-21T00:20:51Z<p>ParthivA: </p>
<hr />
<div></div>ParthivAhttp://2009.igem.org/File:QueensFigure2.pngFile:QueensFigure2.png2009-10-21T00:17:59Z<p>ParthivA: </p>
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<div></div>ParthivAhttp://2009.igem.org/Team:Queens/ResultsTeam:Queens/Results2009-10-21T00:12:12Z<p>ParthivA: </p>
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<td align="left"><p style="font-size:175%;font-family:corbel;color:#172C4E;font-weight:bold"><i>Results<i><br />
</p></td><br />
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<br />
<tr><br />
<td><br />
<br/><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td align="left"><br />
<p style="font-size=120%;font-family:palatino linotype;color:#ECB528"><br />
The following summarizes the results of the various aspects of this year's QGEM Project. <br />
</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<br/><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td align="left"><br />
<a href="#Part1"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part One: SAA<br />
</p></a><br />
<a href="#Part2"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Two: Binding Construct<br />
</p></a><br />
<a href="#Part3"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Three: Heme and HO-1<br />
</p></a><br />
<a href="#Part4"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Four: Future Directions<br />
</p></a><br />
</td><br />
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<br />
<tr><br />
<td><br />
<br/><br />
<br/><br />
</td><br />
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<br />
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<td align="left"><br />
<a name="Part1"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part One: SAA</p></a><br />
<br/><br />
<br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Serum amyloid A (SAA) featured prominently on our list of possible effectors to release at the site of atherosclerotic plaques due to research done at Queen’s University by Tam et al. (2005). This research showed that treatment of SAA caused macrophages to reverse and prevent esterification of cholesterol, thereby allowing it to be exported out of plaques by high-density lipoprotein (HDL). In order to get the SAA to be picked up by the macrophages we needed to produce large quantities of the molecule, and then secrete it extracellularly. In order to secrete the protein we decided on using the twin-arginine translocase (TAT) system, which can transport fully folded proteins outside the cell (Sargent et al., 2006) and is found in the majority of prokaryotes. We then fused the TAT signal sequence to the front of our SAA sequence in order to produce a construct that should be able produce and secrete the protein, allowing it to be taken up by macrophages.<br />
</p><br />
<br/><br />
<center><img src="https://static.igem.org/mediawiki/2009/c/cb/QueensFigure1.png"></center><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 1</i> SAA expression and secretion by <i>E. coli</i> cells. Cultures of <i>E. coli</i> cells containing either the SAA construct or control plasmid were spun down at 0, 4, and 12 hours after seeding. The culture medium and whole cell lysates were analyzed by SDS-PAGE and Western blot analysis with a polyclonal antibody recognizing whole SAA protein. <br />
</p><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Our <i>E. coli</i> cells transformed with SAA construct did not appear to express or secrete SAA into the growth medium even after growing for 12 hours in optimum condition. This might be due to defective ligation of the Ptet-RBS fragment to the SAA-encoding gene. We plan to sequence the construct in the future. <br />
</p><br />
<br/><br />
<br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
References:<br />
<br/><br />
<br/><br />
Benditt, E.P., Hoffman, J.S. & Eriksen, N. (1982). SAA, an apoprotein of HDL: its structure and function. <i> New York Academy of Sciences. </i> 82:183-189.<br />
<br/><br />
Ancsin, J.B. & Kisilevsky, R. (1999). The heparin/heparin sulfate-binding site on apo-serum amyloid A. <i>Journal of Biological Chemistry.</i> Vol. 274, 11:7172-7181.<br />
</p><br />
<br/><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td align="left"><br />
<a name="Part2"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Two: Effector System</p></a><br />
<br/><br />
<br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Ideally, our effector system should only begin to produce the effectors once a threshold density of <i>E.coli</i> cells is reached at the plaque site. Since the system is in a prokaryotic chassis, signal transduction via the VLA-4/VCAM binding is not a realistic approach. Instead, we choose to employ the highly characterized LuxI/LuxR quorum sensing system in bacteria. Briefly, the effector proteins to be released at the site of plaque are placed under the control of pLux promoter, activated by a threshold concentration of AHL. AHL is constitutively produced by our cells and it will reach the threshold concentration once a sufficient amount of E.coli cells is bound to the plaque. <br />
</p><br />
<br/><br />
<center><img src="https://static.igem.org/mediawiki/2009/5/55/QueensEffectors.jpg"></center><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In terms of treatment options we explored a number of routes; however, the following three proved to be the most feasible and advantageous choices. While we are not medical professionals and have not addressed the concept of dosage, the effectors we have chosen are such that mild overdoses would not be of great concern (i.e. beneficial effects far outweigh negative effects). We have also chosen these effectors for having minimal side effects. <br />
</p><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<b> 1. Serum Amyloid A</b><br />
</p><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
SAA converts cholesterol stored in plaques into a form more accessible by High Density Lipoprotein (HDL), which is the body’s natural mechanism for returning cholesterol to the liver for packaging, metabolism and/or excretion. It is our hope that this effector, when released specifically at the site of atherosclerosis, will induce plaque regression. <br />
</p><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
However, the clinical effectiveness of SAA is still in doubt for several reasons:<br />
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
* Not all atherosclerotic plaques are associated with cholesterol.<br />
* The body can naturally produce SAA at an astoundingly high rate, given the correct signals.<br />
* We are unsure if the molecule will reach the area of the plaque it is effective in.<br />
* We are unsure if the body produces enough HDL to clear atherosclerotic plaques.<br />
</pre></p><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<b> 2. Heme Oxygenase 1 (HO-1)</b><br />
</p><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
The HO-1 effector system involves the production of heme and HO-1. HO-1 catalyzes the degradation of heme into carbon monoxide (CO), biliverdin (BV), and free iron (Fe++), all of which have therapeutically beneficial effects on atherosclerotic plaques. CO acts as a local vasodilator, which may minimize chances of plaque rupture, as well as retard plaque growth. BV inhibits the proliferation of vascular smooth muscle cells, which has been shown to lead to stenosis of blood vessels. Fe++ induces the production of ferritin, which has an antioxidant effect that may protect the surrounding tissues from free radical attack and reduce the chance of plaque rupture. <br />
</p><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<b> 3. Atrial Natriuretic Peptide (ANP)</b><br />
</p><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
ANP activates membrane-bound guanylate cyclase (GCA), which increases the level of intracellular cGMP, a signalling molecule that mediates vasodilation. The effect of ANP is similar to that of carbon monoxide. cGMP also possess an anti-clotting effect, although it is unlikely that we can take advantage of this since platelets do not have membrane-bound guanylate cyclase.<br />
</p><br />
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</td><br />
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<a name="Part3"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Three: Cleavage and Termination</p></a><br />
<br/><br />
<br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
After our chassis has been bound to the site of the plaque for a period of time, it should begin to produce DNases and proteases. The DNases will function to sheer the bacterial genome, making it unable to proliferate in the blood; this functions as a safe-guard against bacteremia. The proteases serve to detach the chassis from the plaque site, so as to avoid a build-up of dead cell debris in an already inflamed area.<br />
</p><br />
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<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Please note that Results are also available in PDF Format. <br />
Please click on the document you wish to view.<br />
</pre></p></td><br />
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<a href="https://static.igem.org/mediawiki/2009/f/fc/QueensResultsJamesBogdanBryantHarry.pdf"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">Laboratory One: Harry, Bogdan, James, Bryant</p></a><br />
<a href="https://static.igem.org/mediawiki/2009/2/26/QueensResultsKateMike.pdf"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">Laboratory Two: Kate, Mike</p></a><br />
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<td align="left"><p style="font-size:175%;font-family:corbel;color:#172C4E;font-weight:bold"><i>Results<i><br />
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The following summarizes the results of the various aspects of this year's QGEM Project. <br />
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<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part One: SAA<br />
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<a href="#Part2"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Two: Binding Construct<br />
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Part Three: Heme and HO-1<br />
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<a href="#Part4"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Four: Future Directions<br />
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<a name="Part1"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part One: SAA</p></a><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Serum amyloid A (SAA) featured prominently on our list of possible effectors to release at the site of atherosclerotic plaques due to research done at Queen’s University by Tam et al. (2005). This research showed that treatment of SAA caused macrophages to reverse and prevent esterification of cholesterol, thereby allowing it to be exported out of plaques by high-density lipoprotein (HDL). In order to get the SAA to be picked up by the macrophages we needed to produce large quantities of the molecule, and then secrete it extracellularly. In order to secrete the protein we decided on using the twin-arginine translocase (TAT) system, which can transport fully folded proteins outside the cell (Sargent et al., 2006) and is found in the majority of prokaryotes. We then fused the TAT signal sequence to the front of our SAA sequence in order to produce a construct that should be able produce and secrete the protein, allowing it to be taken up by macrophages.<br />
</p><br />
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<center><img src="https://static.igem.org/mediawiki/2009/c/cb/QueensFigure1.png"></center><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<i>Fig. 1</i> SAA expression and secretion by <i>E. coli</i> cells. Cultures of <i>E. coli</i> cells containing either the SAA construct or control plasmid were spun down at 0, 4, and 12 hours after seeding. The culture medium and whole cell lysates were analyzed by SDS-PAGE and Western blot analysis with a polyclonal antibody recognizing whole SAA protein. <br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Our <i>E. coli</i> cells transformed with SAA construct did not appear to express or secrete SAA into the growth medium even after growing for 12 hours in optimum condition. This might be due to defective ligation of the Ptet-RBS fragment to the SAA-encoding gene. We plan to sequence the construct in the future. <br />
</p><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
References:<br />
<br/><br />
Benditt, E.P., Hoffman, J.S. & Eriksen, N. (1982). SAA, an apoprotein of HDL: its structure and function. <i> New York Academy of Sciences. </i> 82:183-189.<br />
<br/><br />
Ancsin, J.B. & Kisilevsky, R. (1999). The heparin/heparin sulfate-binding site on apo-serum amyloid A. <i>Journal of Biological Chemistry.</i> Vol. 274, 11:7172-7181.<br />
</p><br />
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<a name="Part2"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Two: Effector System</p></a><br />
<br/><br />
<br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Ideally, our effector system should only begin to produce the effectors once a threshold density of <i>E.coli</i> cells is reached at the plaque site. Since the system is in a prokaryotic chassis, signal transduction via the VLA-4/VCAM binding is not a realistic approach. Instead, we choose to employ the highly characterized LuxI/LuxR quorum sensing system in bacteria. Briefly, the effector proteins to be released at the site of plaque are placed under the control of pLux promoter, activated by a threshold concentration of AHL. AHL is constitutively produced by our cells and it will reach the threshold concentration once a sufficient amount of E.coli cells is bound to the plaque. <br />
</p><br />
<br/><br />
<center><img src="https://static.igem.org/mediawiki/2009/5/55/QueensEffectors.jpg"></center><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In terms of treatment options we explored a number of routes; however, the following three proved to be the most feasible and advantageous choices. While we are not medical professionals and have not addressed the concept of dosage, the effectors we have chosen are such that mild overdoses would not be of great concern (i.e. beneficial effects far outweigh negative effects). We have also chosen these effectors for having minimal side effects. <br />
</p><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<b> 1. Serum Amyloid A</b><br />
</p><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
SAA converts cholesterol stored in plaques into a form more accessible by High Density Lipoprotein (HDL), which is the body’s natural mechanism for returning cholesterol to the liver for packaging, metabolism and/or excretion. It is our hope that this effector, when released specifically at the site of atherosclerosis, will induce plaque regression. <br />
</p><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
However, the clinical effectiveness of SAA is still in doubt for several reasons:<br />
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
* Not all atherosclerotic plaques are associated with cholesterol.<br />
* The body can naturally produce SAA at an astoundingly high rate, given the correct signals.<br />
* We are unsure if the molecule will reach the area of the plaque it is effective in.<br />
* We are unsure if the body produces enough HDL to clear atherosclerotic plaques.<br />
</pre></p><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<b> 2. Heme Oxygenase 1 (HO-1)</b><br />
</p><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
The HO-1 effector system involves the production of heme and HO-1. HO-1 catalyzes the degradation of heme into carbon monoxide (CO), biliverdin (BV), and free iron (Fe++), all of which have therapeutically beneficial effects on atherosclerotic plaques. CO acts as a local vasodilator, which may minimize chances of plaque rupture, as well as retard plaque growth. BV inhibits the proliferation of vascular smooth muscle cells, which has been shown to lead to stenosis of blood vessels. Fe++ induces the production of ferritin, which has an antioxidant effect that may protect the surrounding tissues from free radical attack and reduce the chance of plaque rupture. <br />
</p><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<b> 3. Atrial Natriuretic Peptide (ANP)</b><br />
</p><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
ANP activates membrane-bound guanylate cyclase (GCA), which increases the level of intracellular cGMP, a signalling molecule that mediates vasodilation. The effect of ANP is similar to that of carbon monoxide. cGMP also possess an anti-clotting effect, although it is unlikely that we can take advantage of this since platelets do not have membrane-bound guanylate cyclase.<br />
</p><br />
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<a name="Part3"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Three: Cleavage and Termination</p></a><br />
<br/><br />
<br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
After our chassis has been bound to the site of the plaque for a period of time, it should begin to produce DNases and proteases. The DNases will function to sheer the bacterial genome, making it unable to proliferate in the blood; this functions as a safe-guard against bacteremia. The proteases serve to detach the chassis from the plaque site, so as to avoid a build-up of dead cell debris in an already inflamed area.<br />
</p><br />
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<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Please note that Results are also available in PDF Format. <br />
Please click on the document you wish to view.<br />
</pre></p></td><br />
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<a href="https://static.igem.org/mediawiki/2009/f/fc/QueensResultsJamesBogdanBryantHarry.pdf"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">Laboratory One: Harry, Bogdan, James, Bryant</p></a><br />
<a href="https://static.igem.org/mediawiki/2009/2/26/QueensResultsKateMike.pdf"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">Laboratory Two: Kate, Mike</p></a><br />
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<div></div>ParthivAhttp://2009.igem.org/File:QueensResultsJamesBogdanBryantHarry.pdfFile:QueensResultsJamesBogdanBryantHarry.pdf2009-10-20T23:56:05Z<p>ParthivA: </p>
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<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Nickname: francium phosphide (Fr<sub>3</sub>P) / fatal attrACTION</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Program: B.Sc. BioChemical Engineering 2011</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">QGEM Position: Student Volunteer/Webmaster</P><br />
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About: I am currently entering my third year in BioChemical Engineering at Queen's University. I garnered an interest in iGEM due to my fascination of the field of biofuels, especially the use of engineered algal strains for biodiesel production. As a volunteer member of the QGEM, my focus is mostly on the technological aspects of iGEM: maintaining the Wiki, compiling data, and animations. Outside of QGEM I'll be working on the Queen's BAJA SAE car, as well as getting some of the paperwork filed for a new chemical engineering design team a few of my friends and I are developing. In my free time I'll be playing my guitar & Halo 2, surfing the net, and of course doing a metric tonne of homework.</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Michael Freeman</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Nickname: The Naked Sasquatch / Microbicurious</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Program: B.Sc. BioChemical Engineering 2010</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">QGEM Position: Student Employee</P><br />
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About: I am in my fourth year at Queen's after transferring degree programs from chemistry to biochemical engineering. iGEM is a competition that grabbed my interest immediately because of the unbelievable possibilities synthetic biology makes available. Solving problems in general is incredibly interesting to me, and solving them in unconventional or obscure ways is even more exciting. I came into iGEM with a few project ideas like including engineered microbes in specific waste water treatment processes, creating a more conclusive indicator test for aggressive prostate cancers, and creating some microbial suspension which could be ingested and used to break down cellulose in the human stomach, a hugely abundant source of food and energy. For iGEM this year I was a 'wet labber' responsible for creating, engineering and testing our Heme/Heme-Oxygenase system which would be used as one of our plaque degrading effectors. In order to keep from going nuts from talking with bacteria all day I bar tend at night, I compete in triathlons and varsity rowing for Queen's, I am deeply in love with traveling and hosting travelers, and I have just entered the wonderful (and expensive) world of skydiving! Ever touched a cloud? This guy has.</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Jonas Elliott Gerson</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Nickname: Sex Pili</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Program:</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">QGEM Position:</P><br />
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About: More to come later</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">James MacLeod</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Nickname: Bond</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Program: B.Sc.H. Biology</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">QGEM Position: Co-Founder</P><br />
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About: I am currently in my third year at Queen's University pursuing a Bachelor's of Science (BScH). I was born in Britain, lived in the Middle East for a period, and now reside in Canada. I love the skiing that Canada has to offer, and I'm very excited that Queen's will be participating in the 2009 Jamboree and am looking forward to meeting teams from across the world! </p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Bogdan Momciu</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Nickname: The Count </p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Program: B.Sc.H. Life Sciences SSP 2010</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">QGEM Position: Student Employee</P><br />
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About: I am 4th year student in the Queen's University Life Sciences program with a focus towards research and biomedical applications. Once I heard about iGEM I was greatly enthusiastic about the idea due to the sheer number of innovations this kind of cooperative initiative could end up producing. During this past summer I worked on the design and synthesis of our SAA and Endothelial Cell Binding Constructs as well as testing and troubleshooting.<br />
In my spare time I like to play a variety of sports including hockey, soccer, and volleyball. </p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Chris Palmer</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Nickname: FRECsecutioner</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Program: B.Sc. Engineering Chemistry 2012</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">QGEM Position: Student Volunteer</P><br />
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About: I am a second year student in the Engineering Chemistry program at Queen's university, but I have always had a strong interest in microbiology and biochemistry as well. As a result, I was intrigued by the opportunities presented by the iGEM competition. As soon as I heard that Queen's was forming an iGEM team, I knew that I wanted to be a part of it and help get it off the ground in its first year. I have thoroughly enjoyed my time this summer working with everyone else on the team, and I look forward to meeting people and hearing about everyone's exciting projects at the jamboree!</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Bryant Shum</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Nickname: Blossom</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Program: Life Science SSP 2011</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">QGEM Position: Student Employee</P><br />
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About: I am currently a third year undergraduate student at Queen's, with a concentration in Life Science. Given the nature of my studies, I have always been fascinated by the sheer variety of phenotypes that can arise from a simple genetic code; therefore, the chance to play "Lego" with these complex molecules of life was an irresistible opportunity for me. Specifically, my role on the team this year was to help construct the genetic components required for plaque binding, as well as to conduct background research on the pathogenesis of atherosclerosis. And, so far, this has proven to be a challenging but unforgettable project.</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Kate Turner</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Nickname: Chicken Joe / The Octagon</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Program: B.Sc. BioChemical Engineering 2010</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">QGEM Position: Student Employee</P><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
About: I am in my fourth year of a chemical engineering degree at Queen’s, focusing on the biomedical stream. I spent last summer working as a field engineer in the Alberta oil patch, but this year I wanted to broaden my horizons and focus on biotechnology or biomedical research. I was especially interested in iGEM due to the wide-ranging applications of synthetic biology – from smog cleanup to combating tumours to degrading plastics – and now I hope to continue in this field after graduation. During the summer I worked in one of the labs testing the heme/HO-1 effector system and researching the amelioration of atherosclerotic plaques by CO, HO-1, and biliverdin. Outside of QGEM, I spent my summer camping, BBQing, and reading like a fiend.</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Christopher Yan</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Nickname: YanTASTIC</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Program: B.Sc.H. Life Sciences 2012</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">QGEM Position: Student Employee</P><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
About: I am a second year Life Sciences student at Queen's University. This is my first time participating in an event of such grand scale and am very much looking forward to the Jamboree.</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Harry Zhou</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Nickname: Bubbles</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Program: B.Sc.H. Biochemistry 2011</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">QGEM Position: Co-Founder</P><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
About: I am a third-year Biochemistry student at Queen’s University. Starting in high school, I had a strong interest in biotechnology that has the potential of benefiting health care and environment. When I was in grade 12, a professor at University of Calgary introduced me to synthetic biology and the iGEM competition and I volunteered for the UCalgary team in 2007. I thoroughly enjoyed the experience and was fascinated by the iGEM program, which provides a platform for students to tackle important problems concerning the environment, health care, and new technology. This was why I decided to put together a multidisciplinary team of undergraduate students at Queen’s to participate in this year’s iGEM competition. I had tons of fun this summer planning and carrying out experiments for the project and working with students from other discipline. In my spare time, I like to play sports, guitar, and read. </p><br />
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<td align="left"><p style="font-size:175%;font-family:corbel;color:#172C4E;font-weight:bold"><i>The Faculty Advisors</i></p></td><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Ian D. Chin-Sang, Ph.D.</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Faculty: Arts & Science</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Department: Biology</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Position: Associate Professor and CCS/NCIC Research Scientist</P><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Research: Molecular Genetics of <i>C. elegans</i> Development</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Peter A. Greer, Ph.D.</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Faculty: Arts & Science</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Department: BioChemistry</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Position: Professor of Biochemistry and Pathology & Molecular Medicine</P><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Research: Cancer Signal Transduction</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">David P. Lebrun, MD</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Faculty: Medicine</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Department: Pathology and Molecular Medicine</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Position: Associate Professor of Pathology & Molecular Medicine</P><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Research: Molecular Mechanisms of Leukemia & Clinicopathological Correlations in Malignant Lymphoma</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Nancy Martin, Ph.D.</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Faculty: Arts & Science</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Department: Microbiology and Immunology</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Position: Associate Professor</P><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Research: Sensing and Adaptation to Environmental Changes in <i>Salmonella typhimurium</i></p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Ronald J. Neufeld, Ph.D.</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Faculty: Applied Science</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Department: Chemical Engineering</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Position: Professor</P><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Research: Bioencapsulation & Bioactives Processing and Controlled Release</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Waheed Sangrar, Ph.D.</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Faculty: Medicine</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Department: Pathology and Molecular Medicine</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Position: Assistant Professor</P><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Research: Breast Cancer</p><br />
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<p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Virginia K. Walker, Ph.D.</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Faculty: Arts & Science</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Department: Biology</p><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">Position: Professor</P><br />
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Research: Genetics and Molecular Biology of Resistance</p><br />
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<p style="font-size:110%; font-family:corbel;color:#172C4E"><br />
Last Updated: October 19, 2009 by Fr<subs>3</subs>P</p></center><br />
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</html></div>ParthivAhttp://2009.igem.org/Team:Queens/ProjectTeam:Queens/Project2009-10-19T21:59:25Z<p>ParthivA: </p>
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The QGEM project centres on the treatment of atherosclerosis by targeted drug delivery from <i>E.coli</i>. The project is broken down into three major components. The first, and the main, component is the cell membrane protein that allows <i>E.coli</i> to bind to the site of atherosclerotic plaque. The second component is the inducible effector system that produces factors to treat the plaque. The last component is the terminator system that detaches <i>E.coli</i> from the plaque and inhibits proliferation of <i>E.coli</i> in the blood stream.<br />
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Part One: Binding System<br />
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Part Two: Effector System<br />
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<a name="Part1"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part One: Binding System</p></a><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In order to target <i>E.coli</i> cells to atherosclerotic plaques, we selected the VLA-4/VCAM-1 binding system. VCAM-1 (vascular cell adhesion molecule-1) is a specific marker that is commonly found at sites of damaged endothelium, such as the site of an atherosclerotic plaque. VLA-4 (Integrin α4β1 or very late antigen-4) is normally expressed on leukocyte membranes, and it directs the leukocytes to damaged sites in the vascular system. Studies have shown that the ITGA-4 chain of VLA-4 antigen can sufficient bind to VCAM-1.<br />
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Our goal is to express a fragment of ITGA-4 on the surface of the E.coli plasma membrane. To do this, we have modified the attachment system previously explored by NYMU-Taipei team 2008. Below is a schematic diagram of the binding construct. <br />
</p><br />
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Pconst – RBS – LppOmpA – linker - TEV cleavage site X2 – linker – VLA-4 – STOP <br />
</pre><br />
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The Lpp (Lipoprotein signal peptide) and OmpA (Outer membrane protein A) fusion results in presentation of the protein at the outer membrane of E.coli. Two cleavage sites for Tobacco-Etch Virus (TEV) protease are inserted between two linker regions. The TEV protease is a part of the terminator system that detaches E.coli from the endothelial cells. The length of the linker sequence between TEV cut site and VLA-4 can be adjusted in order to optimize binding efficiency.<br />
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<a name="Part2"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Two: Effector System</p></a><br />
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Ideally, our effector system should only begin to produce the effectors once a threshold density of <i>E.coli</i> cells is reached at the plaque site. Since the system is in a prokaryotic chassis, signal transduction via the VLA-4/VCAM binding is not a realistic approach. Instead, we choose to employ the highly characterized LuxI/LuxR quorum sensing system in bacteria. Briefly, the effector proteins to be released at the site of plaque are placed under the control of pLux promoter, activated by a threshold concentration of AHL. AHL is constitutively produced by our cells and it will reach the threshold concentration once a sufficient amount of E.coli cells is bound to the plaque. <br />
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In terms of treatment options we explored a number of routes; however, the following three proved to be the most feasible and advantageous choices. While we are not medical professionals and have not addressed the concept of dosage, the effectors we have chosen are such that mild overdoses would not be of great concern (i.e. beneficial effects far outweigh negative effects). We have also chosen these effectors for having minimal side effects. <br />
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<b> 1. Serum Amyloid A</b><br />
</p><br />
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SAA converts cholesterol stored in plaques into a form more accessible by High Density Lipoprotein (HDL), which is the body’s natural mechanism for returning cholesterol to the liver for packaging, metabolism and/or excretion. It is our hope that this effector, when released specifically at the site of atherosclerosis, will induce plaque regression. <br />
</p><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
However, the clinical effectiveness of SAA is still in doubt for several reasons:<br />
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
* Not all atherosclerotic plaques are associated with cholesterol.<br />
* The body can naturally produce SAA at an astoundingly high rate, given the correct signals.<br />
* We are unsure if the molecule will reach the area of the plaque it is effective in.<br />
* We are unsure if the body produces enough HDL to clear atherosclerotic plaques.<br />
</pre></p><br />
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<b> 2. Heme Oxygenase 1 (HO-1)</b><br />
</p><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
The HO-1 effector system involves the production of heme and HO-1. HO-1 catalyzes the degradation of heme into carbon monoxide (CO), biliverdin (BV), and free iron (Fe++), all of which have therapeutically beneficial effects on atherosclerotic plaques. CO acts as a local vasodilator, which may minimize chances of plaque rupture, as well as retard plaque growth. BV inhibits the proliferation of vascular smooth muscle cells, which has been shown to lead to stenosis of blood vessels. Fe++ induces the production of ferritin, which has an antioxidant effect that may protect the surrounding tissues from free radical attack and reduce the chance of plaque rupture. <br />
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<b> 3. Atrial Natriuretic Peptide (ANP)</b><br />
</p><br />
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ANP activates membrane-bound guanylate cyclase (GCA), which increases the level of intracellular cGMP, a signalling molecule that mediates vasodilation. The effect of ANP is similar to that of carbon monoxide. cGMP also possess an anti-clotting effect, although it is unlikely that we can take advantage of this since platelets do not have membrane-bound guanylate cyclase.<br />
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<a name="Part3"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Three: Cleavage and Termination</p></a><br />
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After our chassis has been bound to the site of the plaque for a period of time, it should begin to produce DNases and proteases. The DNases will function to sheer the bacterial genome, making it unable to proliferate in the blood; this functions as a safe-guard against bacteremia. The proteases serve to detach the chassis from the plaque site, so as to avoid a build-up of dead cell debris in an already inflamed area.<br />
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The QGEM project centres on the treatment of atherosclerosis by targeted drug delivery from <i>E.coli</i>. The project is broken down into three major components. The first, and the main, component is the cell membrane protein that allows <i>E.coli</i> to bind to the site of atherosclerotic plaque. The second component is the inducible effector system that produces factors to treat the plaque. The last component is the terminator system that detaches <i>E.coli</i> from the plaque and inhibits proliferation of <i>E.coli</i> in the blood stream.<br />
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Part One: Binding System<br />
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<a href="#Part2"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Two: Effector System<br />
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<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Three: Cleavage and Termination<br />
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<a name="Part1"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part One: Binding System</p></a><br />
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<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In order to target <i>E.coli</i> cells to atherosclerotic plaques, we selected the VLA-4/VCAM-1 binding system. VCAM-1 (vascular cell adhesion molecule-1) is a specific marker that is commonly found at sites of damaged endothelium, such as the site of an atherosclerotic plaque. VLA-4 (Integrin α4β1 or very late antigen-4) is normally expressed on leukocyte membranes, and it directs the leukocytes to damaged sites in the vascular system. Studies have shown that the ITGA-4 chain of VLA-4 antigen can sufficient bind to VCAM-1.<br />
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<center><img src="https://static.igem.org/mediawiki/2009/1/18/QueensBindingMechanism.jpg"></center><br />
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Our goal is to express a fragment of ITGA-4 on the surface of the E.coli plasma membrane. To do this, we have modified the attachment system previously explored by NYMU-Taipei team 2008. Below is a schematic diagram of the binding construct. <br />
</p><br />
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Pconst – RBS – LppOmpA – linker - TEV cleavage site X2 – linker – VLA-4 – STOP <br />
</pre><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
The Lpp (Lipoprotein signal peptide) and OmpA (Outer membrane protein A) fusion results in presentation of the protein at the outer membrane of E.coli. Two cleavage sites for Tobacco-Etch Virus (TEV) protease are inserted between two linker regions. The TEV protease is a part of the terminator system that detaches E.coli from the endothelial cells. The length of the linker sequence between TEV cut site and VLA-4 can be adjusted in order to optimize binding efficiency.<br />
</p><br />
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</td><br />
</tr><br />
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<tr><br />
<td align="left"><br />
<a name="Part2"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Two: Effector System</p></a><br />
<br/><br />
<br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Ideally, our effector system should only begin to produce the effectors once a threshold density of <i>E.coli</i> cells is reached at the plaque site. Since the system is in a prokaryotic chassis, signal transduction via the VLA-4/VCAM binding is not a realistic approach. Instead, we choose to employ the highly characterized LuxI/LuxR quorum sensing system in bacteria. Briefly, the effector proteins to be released at the site of plaque are placed under the control of pLux promoter, activated by a threshold concentration of AHL. AHL is constitutively produced by our cells and it will reach the threshold concentration once a sufficient amount of E.coli cells is bound to the plaque. <br />
</p><br />
<br/><br />
<center><img src="https://static.igem.org/mediawiki/2009/5/55/QueensEffectors.jpg"></center><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In terms of treatment options we explored a number of routes; however, the following three proved to be the most feasible and advantageous choices. While we are not medical professionals and have not addressed the concept of dosage, the effectors we have chosen are such that mild overdoses would not be of great concern (i.e. beneficial effects far outweigh negative effects). We have also chosen these effectors for having minimal side effects. <br />
</p><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<b> 1. Serum Amyloid A</b><br />
</p><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
SAA converts cholesterol stored in plaques into a form more accessible by High Density Lipoprotein (HDL), which is the body’s natural mechanism for returning cholesterol to the liver for packaging, metabolism and/or excretion. It is our hope that this effector, when released specifically at the site of atherosclerosis, will induce plaque regression. <br />
</p><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
However, the clinical effectiveness of SAA is still in doubt for several reasons:<br />
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
* Not all atherosclerotic plaques are associated with cholesterol.<br />
* The body can naturally produce SAA at an astoundingly high rate, given the correct signals.<br />
* We are unsure if the molecule will reach the area of the plaque it is effective in.<br />
* We are unsure if the body produces enough HDL to clear atherosclerotic plaques.<br />
</pre></p><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<b> 2. Heme Oxygenase 1 (HO-1)</b><br />
</p><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
The HO-1 effector system involves the production of heme and HO-1. HO-1 catalyzes the degradation of heme into carbon monoxide (CO), biliverdin (BV), and free iron (Fe++), all of which have therapeutically beneficial effects on atherosclerotic plaques. CO acts as a local vasodilator, which may minimize chances of plaque rupture, as well as retard plaque growth. BV inhibits the proliferation of vascular smooth muscle cells, which has been shown to lead to stenosis of blood vessels. Fe++ induces the production of ferritin, which has an antioxidant effect that may protect the surrounding tissues from free radical attack and reduce the chance of plaque rupture. <br />
</p><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<b> 3. Atrial Natriuretic Peptide (ANP)</b><br />
</p><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
ANP activates membrane-bound guanylate cyclase (GCA), which increases the level of intracellular cGMP, a signalling molecule that mediates vasodilation. The effect of ANP is similar to that of carbon monoxide. cGMP also possess an anti-clotting effect, although it is unlikely that we can take advantage of this since platelets do not have membrane-bound guanylate cyclase.<br />
</p><br />
<br/><br />
</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td align="left"><br />
<a name="Part3"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Three: Cleavage and Termination</p></a><br />
<br/><br />
<br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
After our chassis has been bound to the site of the plaque for a period of time, it should begin to produce DNases and proteases. The DNases will function to sheer the bacterial genome, making it unable to proliferate in the blood; this functions as a safe-guard against bacteremia. The proteases serve to detach the chassis from the plaque site, so as to avoid a build-up of dead cell debris in an already inflamed area.<br />
</p><br />
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</td><br />
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<table style="background-color:#922334; position:relative; overflow:auto; left:200px; top:-215px; width:750px"><br />
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<td align="left"><p style="font-size:175%;font-family:corbel;color:#172C4E;font-weight:bold"><i>Overview<i><br />
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The QGEM project centres on the treatment of atherosclerosis by targeted drug delivery from <i>E.coli</i>. The project is broken down into three major components. The first, and the main, component is the cell membrane protein that allows <i>E.coli</i> to bind to the site of atherosclerotic plaque. The second component is the inducible effector system that produces factors to treat the plaque. The last component is the terminator system that detaches <i>E.coli</i> from the plaque and inhibits proliferation of <i>E.coli</i> in the blood stream.<br />
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<a href="#Part1"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part One: Binding System<br />
</p></a><br />
<a href="#Part2"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Two: Effector System<br />
</p></a><br />
<a href="#Part3"><br />
<p style="font-size=150%;font-family:Corbel;color:#172C4E;font-weight:bold"><br />
Part Three: Cleavage and Termination<br />
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<a name="Part1"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part One: Binding System</p></a><br />
<br/><br />
<br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In order to target <i>E.coli</i> cells to atherosclerotic plaques, we selected the VLA-4/VCAM-1 binding system. VCAM-1 (vascular cell adhesion molecule-1) is a specific marker that is commonly found at sites of damaged endothelium, such as the site of an atherosclerotic plaque. VLA-4 (Integrin α4β1 or very late antigen-4) is normally expressed on leukocyte membranes, and it directs the leukocytes to damaged sites in the vascular system. Studies have shown that the ITGA-4 chain of VLA-4 antigen can sufficient bind to VCAM-1.<br />
</p><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Our goal is to express a fragment of ITGA-4 on the surface of the E.coli plasma membrane. To do this, we have modified the attachment system previously explored by NYMU-Taipei team 2008. Below is a schematic diagram of the binding construct. <br />
</p><br />
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Pconst – RBS – LppOmpA – linker - TEV cleavage site X2 – linker – VLA-4 – STOP <br />
</pre><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
The Lpp (Lipoprotein signal peptide) and OmpA (Outer membrane protein A) fusion results in presentation of the protein at the outer membrane of E.coli. Two cleavage sites for Tobacco-Etch Virus (TEV) protease are inserted between two linker regions. The TEV protease is a part of the terminator system that detaches E.coli from the endothelial cells. The length of the linker sequence between TEV cut site and VLA-4 can be adjusted in order to optimize binding efficiency.<br />
</p><br />
<br/><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td align="left"><br />
<a name="Part2"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Two: Effector System</p></a><br />
<br/><br />
<br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
Ideally, our effector system should only begin to produce the effectors once a threshold density of <i>E.coli</i> cells is reached at the plaque site. Since the system is in a prokaryotic chassis, signal transduction via the VLA-4/VCAM binding is not a realistic approach. Instead, we choose to employ the highly characterized LuxI/LuxR quorum sensing system in bacteria. Briefly, the effector proteins to be released at the site of plaque are placed under the control of pLux promoter, activated by a threshold concentration of AHL. AHL is constitutively produced by our cells and it will reach the threshold concentration once a sufficient amount of E.coli cells is bound to the plaque. <br />
</p><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
In terms of treatment options we explored a number of routes; however, the following three proved to be the most feasible and advantageous choices. While we are not medical professionals and have not addressed the concept of dosage, the effectors we have chosen are such that mild overdoses would not be of great concern (i.e. beneficial effects far outweigh negative effects). We have also chosen these effectors for having minimal side effects. <br />
</p><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<b> 1. Serum Amyloid A</b><br />
</p><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
SAA converts cholesterol stored in plaques into a form more accessible by High Density Lipoprotein (HDL), which is the body’s natural mechanism for returning cholesterol to the liver for packaging, metabolism and/or excretion. It is our hope that this effector, when released specifically at the site of atherosclerosis, will induce plaque regression. <br />
</p><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
However, the clinical effectiveness of SAA is still in doubt for several reasons:<br />
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
* Not all atherosclerotic plaques are associated with cholesterol.<br />
* The body can naturally produce SAA at an astoundingly high rate, given the correct signals.<br />
* We are unsure if the molecule will reach the area of the plaque it is effective in.<br />
* We are unsure if the body produces enough HDL to clear atherosclerotic plaques.<br />
</pre></p><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<b> 2. Heme Oxygenase 1 (HO-1)</b><br />
</p><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
The HO-1 effector system involves the production of heme and HO-1. HO-1 catalyzes the degradation of heme into carbon monoxide (CO), biliverdin (BV), and free iron (Fe++), all of which have therapeutically beneficial effects on atherosclerotic plaques. CO acts as a local vasodilator, which may minimize chances of plaque rupture, as well as retard plaque growth. BV inhibits the proliferation of vascular smooth muscle cells, which has been shown to lead to stenosis of blood vessels. Fe++ induces the production of ferritin, which has an antioxidant effect that may protect the surrounding tissues from free radical attack and reduce the chance of plaque rupture. <br />
</p><br />
<br/><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
<b> 3. Atrial Natriuretic Peptide (ANP)</b><br />
</p><br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
ANP activates membrane-bound guanylate cyclase (GCA), which increases the level of intracellular cGMP, a signalling molecule that mediates vasodilation. The effect of ANP is similar to that of carbon monoxide. cGMP also possess an anti-clotting effect, although it is unlikely that we can take advantage of this since platelets do not have membrane-bound guanylate cyclase.<br />
</p><br />
<br/><br />
</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td align="left"><br />
<a name="Part3"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Part Three: Cleavage and Termination</p></a><br />
<br/><br />
<br />
<p style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"><br />
After our chassis has been bound to the site of the plaque for a period of time, it should begin to produce DNases and proteases. The DNases will function to sheer the bacterial genome, making it unable to proliferate in the blood; this functions as a safe-guard against bacteremia. The proteases serve to detach the chassis from the plaque site, so as to avoid a build-up of dead cell debris in an already inflamed area.<br />
</p><br />
<br/><br />
</td><br />
</tr><br />
<br />
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<br />
</table><br />
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Last Updated: October 16, 2009 by Fr<sub>3</sub>P</center></font><br />
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