http://2014.igem.org/wiki/index.php?title=Special:Contributions/Maexlich&feed=atom&limit=50&target=Maexlich&year=&month=2014.igem.org - User contributions [en]2024-03-29T15:19:02ZFrom 2014.igem.orgMediaWiki 1.16.5http://2014.igem.org/Team:Heidelberg/pages/SponsoringTeam:Heidelberg/pages/Sponsoring2015-07-10T04:14:53Z<p>Maexlich: </p>
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<div class="col-md-7 col-sm-12 col-xs-12"><h1>Platinum Sponsor</div><br />
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<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"> <br />
<h4>Klaus Tschira Foundation</h4><br />
In 1995, Klaus Tschira, physicist and co-founder of the software company SAP, founded the Klaus Tschira Stiftung (KTS). With the intention to place strong emphasis on natural sciences in the society, the commitment of the KTS ranges from kindergarten to schools, universities and research institutions all over Germany. The support is differentiated in three main fields: natural sciences for children and young people, research and science communication. The foundation is located at the Villa Bosch in Heidelberg, the former residence of Nobel Prize laureate for chemistry Carl Bosch.<br />
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<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img src="/wiki/images/2/20/Klaus-Tschira-Stiftung_Logo.png" class="img-responsive" alt="Klaus Tschira Foundation Logo"><br />
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<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"> <br />
<h4>Dietmar Hopp Foundation</h4><br />
Dietmar Hopp founded his foundation in 1995 to enable the implementation of charitable projects. His foundation encourages projects in the area sports, medicine, social support and education. Thus the Hopp foundation supports medical research projects on the highest level including cancer research and pediatrics. In the area of education the foundation facilitates institutions and projects for young people- from kindergarten to university. The Hopp foundation also supports our project and our team of young scientists! <br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img style="margin-top:10px;" src="/wiki/images/8/8a/Dietmar_hopp_stiftung.png" class="img-responsive" alt="Dietmar Hopp Foundation"><br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"> <br />
<h4>Helmholtz-Initiative Synthetic Biology </h4><br />
The Helmholtz Association is Germany’s largest scientific organization. The Association brings together 18 scientific-technical and biological-medical research centres and follows the tradition of the great natural scientist Hermann von Helmholtz (1821-1894). The Helmholtz Association is dedicated to pursuing the long-term research goals of state and society, and to maintaining and improving the livelihoods of the population. In order to do this, the Helmholtz Association carries out top-level research to identify and explore the major challenges facing society, science and the economy. Its work is divided into six strategic research fields: Energy; Earth and Environment; Health; Key Technologies; Structure of Matter; and Aeronautics, Space and Transport. The Helmholtz Initiative on Synthetic Biology has been supporting the emerging field of synthetic biology since September 2012.<br />
</div><br />
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<div class="col-lg-8 col-lg-offset-2 col-md-offset-2 col-md-8 col-sm-offset-1 col-sm-10 col-xs-12"><br />
<img style="margin-top:20px;" src="/wiki/images/1/17/Helmholtz_logo.jpg" class="img-responsive" alt="Helmholtz Association Logo"><br />
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</div><br />
</div><br />
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<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"> <br />
<h4>CellNetworks</h4><br />
CellNetworks is an interdisciplinary research cluster centered around Heidelberg´s life sciences. The Heidelberg life science community features an unparalleled number of internationally well-noted institutions. Since 2006 CellNetworks is part of the German Universities Excellence Initiative. CellNetworks assembles excellent groups focusing on research in molecular life science and computational sciences, chemistry and physics to tackle fundamental questions in cell biology, its network structure and architecture, dynamics and regulation.<br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img src="/wiki/images/a/a9/CellNetWorks_logo.jpg" class="img-responsive" alt="Klaus Tschira Foundation Logo"><br />
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<div class="col-md-7 col-sm-12 col-xs-12"><h1>Gold Sponsors</div><br />
<br />
</div><br />
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<div class="col-md-7 col-sm-12 col-xs-12"> <br />
<h4>geneious</h4><br />
Geneious is a DNA, RNA and protein sequence alignment, assembly and analysis software platform, integrating bioinformatic and molecular biology tools into a simple interface. Geneious provides us their software which helped us design primers and plan our cloning.<br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img src="/wiki/images/6/63/Heidelberg_Geneious_Logo.png" class="img-responsive" alt="Helmholtz Association Logo"><br />
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</div><br />
</div><br />
</br><br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"> <br />
<h4>Lange + Pflanz</h4><br />
Lange + Pflanz advertising agency Gmbh has been a specialist for Marketing and Design since 1992. Our focus covers the fields of financial , corporate and strategic communication. As an owner-operated communication company we offer individual and enduring communication solutions for variable media platforms.<br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img src="/wiki/images/6/6c/LangePflanz_Logo.png" class="img-responsive" alt="Lange & Pflanz Logo"><br />
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<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"><h1>Silver Sponsors</div><br />
<br />
</div><br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"> <br />
<h4>Eppendorf</h4><br />
Eppendorf is a company, which products used in all types of life science research and clinical testing settings—from basic laboratory applications to highly specialized cell and molecular biology applications. They are highly regarded for their quality design and performance—beginning with extensive research and development, adding state-of-the-art technology and ending with strict quality-controlled manufacturing are what make our products stand out from the rest. It is what has made us a brand you have been able to rely on for over 60 years.<br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img src="/wiki/images/f/f4/Eppendorf-Logo.png" class="img-responsive" alt="Eppendorf Logo"><br />
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</div><br />
</div><br />
<br /><br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"> <br />
<h4>GATC</h4><br />
GATC is a leading service provider of DNA sequencing. Head quartered in Constance, Germany, GATC serves over 10,000 customers in 40 countries around the globe and have subsidiary companies in Great Britain, France and Sweden. GATC provides multi platform strategies, using all leading sequencing technologies on the market, such as Applied Biosystems ABI 3730xl for Sanger sequencing, Illumina’s HiSeq 2000 and Roche Diagnostics GS FLX+ System for Next Generation sequencing as well as the new single molecule sequencer PacBio RS from Pacific Biosciences.<br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img src="/wiki/images/9/97/GATC_logo.gif" class="img-responsive" alt="Helmholtz Association Logo"><br />
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</div><br />
</div><br />
</br><br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"> <br />
<h4>Chroma</h4><br />
Chroma Technology Corp. is a manufacturer of interference filters for the ultra-violet, visible and near-infrared portions of the spectrum, including bandpass, multiple bandpass, and long and short pass filters, as well as beamsplitters, dichroic mirrors and laser rejection filters. We specialize in precision spectral control, including extra high signal-to-noise ratio filters and those with rapid cut-on and cut-off profiles. The manufacturing process involves precisely depositing, in a vacuum, extremely thin layers of two or more materials on a glass or similarly transparent substrate.<br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img src="/wiki/images/1/1f/Chroma_LOGO.JPG" class="img-responsive" alt="Chroma Logo"><br />
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<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"> <br />
<h4>Carl Roth GmbH & Co</h4><br />
Carl Roth is a private German enterprise with tradition based in Karlsruhe Rheinhafen. It was founded in 1879 with the establishment of a grocer's-paint supplier's-chemist's-store at Karlsruhe. As supplier for lab ware, life sciences and chemicals, they are your partner for best performance reflected in their basic values quality, logistics, fair rates, competent customer advice and service.<br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img src="/wiki/images/8/88/Roth_logo.jpg" class="img-responsive" alt="Roth Logo"><br />
</div><br />
</div><br />
</div><br />
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<div class="col-md-7 col-sm-12 col-xs-12"><h1>Academic Sponsors</div><br />
</div><br />
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<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"> <br />
<h4>Heidelberg University</h4><br />
Heidelberg University, founded in 1386, is Germany’s oldest university and one of the strongest research institutions in Europe. The successes in both rounds of the Excellence Initiative and in international rankings prove its leading role in the scientific community. Heidelberg University is a comprehensive university with the full spectrum of subjects including medicine. It aims to strengthen the individual disciplines, to further interdisciplinary cooperation and to make research results usable for society and industry. Heidelberg also draws its strength from its cooperation with local non-university research institutions and is tied into a worldwide network of research and teaching collaborations.<br />
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<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img src="/wiki/images/0/07/University-of-Heidelberg_logo.jpg" class="img-responsive" alt="Helmholtz Association Logo"><br />
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<h4>German Research Center (DKFZ)</h4><br />
The DKFZ (Deutsches Krebsforschungszentrum) is the largest biomedical research institute in Germany and a member of the Helmholtz Association of National Research Centers. In over 90 divisions and research groups, our more than 3,000 employees, of which more than 1,000 are scientists, are investigating the mechanisms of cancer, are identifying cancer risk factors and are trying to find strategies to prevent people from getting cancer .They are developing novel approaches to make tumor diagnosis more precise and treatment of cancer patients more successful.<br />
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<img src="/wiki/images/0/01/Dkfz_logo.jpg" class="img-responsive" alt="Helmholtz Association Logo"><br />
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<h4>BioQuant</h4><br />
The BioQuant, the Center for “Quantitative Analysis of Molecular and Cellular Biosystems” at Heidelberg University was established in 2007 as an interdisciplinary University research center that is solely dedicated to research and training in systems biology. BioQuants’s objective is to function as a platform for the development and constant refinement of mathematical models of complex biological systems as well as the swift validation of scientific hypotheses via experimental data. Currently, up to 40 University and non-University research groups (DKFZ, EMBL, the Heidelberg Institute for Theoretical Studies (HITS, formerly the European Media Lab), and the MPI for Medical Research) are affiliated with BioQuant. These research groups are instrumental in implementing numerous national and international systems biology funding initiatives.<br />
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<img src="/wiki/images/0/09/BioQuant_Logo.png" class="img-responsive" alt="BioQuant Logo"><br />
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</html></div>Maexlichhttp://2014.igem.org/Team:Heidelberg/pages/SponsoringTeam:Heidelberg/pages/Sponsoring2015-07-10T04:13:04Z<p>Maexlich: </p>
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<div class="col-md-7 col-sm-12 col-xs-12"><h1>Platinum Sponsor</div><br />
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<div class="col-md-7 col-sm-12 col-xs-12"> <br />
<h4>Klaus Tschira Foundation</h4><br />
In 1995, Klaus Tschira, physicist and co-founder of the software company SAP, founded the Klaus Tschira Stiftung (KTS). With the intention to place strong emphasis on natural sciences in the society, the commitment of the KTS ranges from kindergarten to schools, universities and research institutions all over Germany. The support is differentiated in three main fields: natural sciences for children and young people, research and science communication. The foundation is located at the Villa Bosch in Heidelberg, the former residence of Nobel Prize laureate for chemistry Carl Bosch.<br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img src="/wiki/images/2/20/Klaus-Tschira-Stiftung_Logo.png" class="img-responsive" alt="Klaus Tschira Foundation Logo"><br />
</div><br />
</div><br />
</div><br />
</br><br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"> <br />
<h4>Dietmar Hopp Foundation</h4><br />
Dietmar Hopp founded his foundation in 1995 to enable the implementation of charitable projects. His foundation encourages projects in the area sports, medicine, social support and education. Thus the Hopp foundation supports medical research projects on the highest level including cancer research and pediatrics. In the area of education the foundation facilitates institutions and projects for young people- from kindergarten to university. The Hopp foundation also supports our project and our team of young scientists! <br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img style="margin-top:10px;" src="/wiki/images/8/8a/Dietmar_hopp_stiftung.png" class="img-responsive" alt="Dietmar Hopp Foundation"><br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"> <br />
<h4>Helmholtz-Initiative Synthetic Biology </h4><br />
The Helmholtz Association is Germany’s largest scientific organization. The Association brings together 18 scientific-technical and biological-medical research centres and follows the tradition of the great natural scientist Hermann von Helmholtz (1821-1894). The Helmholtz Association is dedicated to pursuing the long-term research goals of state and society, and to maintaining and improving the livelihoods of the population. In order to do this, the Helmholtz Association carries out top-level research to identify and explore the major challenges facing society, science and the economy. Its work is divided into six strategic research fields: Energy; Earth and Environment; Health; Key Technologies; Structure of Matter; and Aeronautics, Space and Transport. The Helmholtz Initiative on Synthetic Biology has been supporting the emerging field of synthetic biology since September 2012.<br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img style="margin-top:20px;" src="/wiki/images/1/17/Helmholtz_logo.jpg" class="img-responsive" alt="Helmholtz Association Logo"><br />
</div><br />
</div><br />
</div><br />
</br><br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"> <br />
<h4>CellNetworks</h4><br />
CellNetworks is an interdisciplinary research cluster centered around Heidelberg´s life sciences. The Heidelberg life science community features an unparalleled number of internationally well-noted institutions. Since 2006 CellNetworks is part of the German Universities Excellence Initiative. CellNetworks assembles excellent groups focusing on research in molecular life science and computational sciences, chemistry and physics to tackle fundamental questions in cell biology, its network structure and architecture, dynamics and regulation.<br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img src="/wiki/images/a/a9/CellNetWorks_logo.jpg" class="img-responsive" alt="Klaus Tschira Foundation Logo"><br />
</div><br />
</div><br />
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<div class="col-md-7 col-sm-12 col-xs-12"><h1>Gold Sponsors</div><br />
<br />
</div><br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"> <br />
<h4>geneious</h4><br />
Geneious is a DNA, RNA and protein sequence alignment, assembly and analysis software platform, integrating bioinformatic and molecular biology tools into a simple interface. Geneious provides us their software which helped us design primers and plan our cloning.<br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img src="/wiki/images/6/63/Heidelberg_Geneious_Logo.png" class="img-responsive" alt="Helmholtz Association Logo"><br />
</div><br />
</div><br />
</div><br />
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<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"> <br />
<h4>Lange + Pflanz</h4><br />
Lange + Pflanz advertising agency Gmbh has been a specialist for Marketing and Design since 1992. Our focus covers the fields of financial , corporate and strategic communication. As an owner-operated communication company we offer individual and enduring communication solutions for variable media platforms.<br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img src="/wiki/images/6/6c/LangePflanz_Logo.png" class="img-responsive" alt="Lange & Pflanz Logo"><br />
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<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"><h1>Silver Sponsors</div><br />
<br />
</div><br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"> <br />
<h4>Eppendorf</h4><br />
Eppendorf is a company, which products used in all types of life science research and clinical testing settings—from basic laboratory applications to highly specialized cell and molecular biology applications. They are highly regarded for their quality design and performance—beginning with extensive research and development, adding state-of-the-art technology and ending with strict quality-controlled manufacturing are what make our products stand out from the rest. It is what has made us a brand you have been able to rely on for over 60 years.<br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img src="/wiki/images/f/f4/Eppendorf-Logo.png" class="img-responsive" alt="Eppendorf Logo"><br />
</div><br />
</div><br />
</div><br />
<br /><br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"> <br />
<h4>GATC</h4><br />
GATC is a leading service provider of DNA sequencing. Head quartered in Constance, Germany, GATC serves over 10,000 customers in 40 countries around the globe and have subsidiary companies in Great Britain, France and Sweden. GATC provides multi platform strategies, using all leading sequencing technologies on the market, such as Applied Biosystems ABI 3730xl for Sanger sequencing, Illumina’s HiSeq 2000 and Roche Diagnostics GS FLX+ System for Next Generation sequencing as well as the new single molecule sequencer PacBio RS from Pacific Biosciences.<br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img src="/wiki/images/9/97/GATC_logo.gif" class="img-responsive" alt="Helmholtz Association Logo"><br />
</div><br />
</div><br />
</div><br />
</br><br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"> <br />
<h4>Chroma</h4><br />
Chroma Technology Corp. is a manufacturer of interference filters for the ultra-violet, visible and near-infrared portions of the spectrum, including bandpass, multiple bandpass, and long and short pass filters, as well as beamsplitters, dichroic mirrors and laser rejection filters. We specialize in precision spectral control, including extra high signal-to-noise ratio filters and those with rapid cut-on and cut-off profiles. The manufacturing process involves precisely depositing, in a vacuum, extremely thin layers of two or more materials on a glass or similarly transparent substrate.<br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img src="/wiki/images/1/1f/Chroma_LOGO.JPG" class="img-responsive" alt="Chroma Logo"><br />
</div><br />
</div><br />
</div><br />
</br><br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"> <br />
<h4>Carl Roth GmbH & Co</h4><br />
Carl Roth is a private German enterprise with tradition based in Karlsruhe Rheinhafen. It was founded in 1879 with the establishment of a grocer's-paint supplier's-chemist's-store at Karlsruhe. As supplier for lab ware, life sciences and chemicals, they are your partner for best performance reflected in their basic values quality, logistics, fair rates, competent customer advice and service.<br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img src="/wiki/images/8/88/Roth_logo.jpg" class="img-responsive" alt="Roth Logo"><br />
</div><br />
</div><br />
</div><br />
</br><br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"><h1>Academic Sponsors</div><br />
</div><br />
<br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"> <br />
<h4>Heidelberg University</h4><br />
Heidelberg University, founded in 1386, is Germany’s oldest university and one of the strongest research institutions in Europe. The successes in both rounds of the Excellence Initiative and in international rankings prove its leading role in the scientific community. Heidelberg University is a comprehensive university with the full spectrum of subjects including medicine. It aims to strengthen the individual disciplines, to further interdisciplinary cooperation and to make research results usable for society and industry. Heidelberg also draws its strength from its cooperation with local non-university research institutions and is tied into a worldwide network of research and teaching collaborations.<br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img src="/wiki/images/0/07/University-of-Heidelberg_logo.jpg" class="img-responsive" alt="Helmholtz Association Logo"><br />
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<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"><br />
<h4>German Research Center (DKFZ)</h4><br />
The DKFZ (Deutsches Krebsforschungszentrum) is the largest biomedical research institute in Germany and a member of the Helmholtz Association of National Research Centers. In over 90 divisions and research groups, our more than 3,000 employees, of which more than 1,000 are scientists, are investigating the mechanisms of cancer, are identifying cancer risk factors and are trying to find strategies to prevent people from getting cancer .They are developing novel approaches to make tumor diagnosis more precise and treatment of cancer patients more successful.<br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img src="/wiki/images/0/01/Dkfz_logo.jpg" class="img-responsive" alt="Helmholtz Association Logo"><br />
</div><br />
</div><br />
</div><br />
</br><br />
<br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"><br />
<h4>BioQuant</h4><br />
The BioQuant, the Center for “Quantitative Analysis of Molecular and Cellular Biosystems” at Heidelberg University was established in 2007 as an interdisciplinary University research center that is solely dedicated to research and training in systems biology. BioQuants’s objective is to function as a platform for the development and constant refinement of mathematical models of complex biological systems as well as the swift validation of scientific hypotheses via experimental data. Currently, up to 40 University and non-University research groups (DKFZ, EMBL, the Heidelberg Institute for Theoretical Studies (HITS, formerly the European Media Lab), and the MPI for Medical Research) are affiliated with BioQuant. These research groups are instrumental in implementing numerous national and international systems biology funding initiatives.<br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img src="/wiki/images/0/09/BioQuant_Logo.png" class="img-responsive" alt="BioQuant Logo"><br />
</div><br />
</div><br />
</div><br />
<br />
</html></div>Maexlichhttp://2014.igem.org/Team:Heidelberg/pages/SponsoringTeam:Heidelberg/pages/Sponsoring2015-07-10T04:09:30Z<p>Maexlich: </p>
<hr />
<div><html><br />
<br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"><h1>Platinum Sponsor</div><br />
<br />
</div><br />
<br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"> <br />
<h4>Klaus Tschira Foundation</h4><br />
In 1995, Klaus Tschira, physicist and co-founder of the software company SAP, founded the Klaus Tschira Stiftung (KTS). With the intention to place strong emphasis on natural sciences in the society, the commitment of the KTS ranges from kindergarten to schools, universities and research institutions all over Germany. The support is differentiated in three main fields: natural sciences for children and young people, research and science communication. The foundation is located at the Villa Bosch in Heidelberg, the former residence of Nobel Prize laureate for chemistry Carl Bosch.<br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img src="/wiki/images/2/20/Klaus-Tschira-Stiftung_Logo.png" class="img-responsive" alt="Klaus Tschira Foundation Logo"><br />
</div><br />
</div><br />
</div><br />
</br><br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"> <br />
<h4>Dietmar Hopp Foundation</h4><br />
Dietmar Hopp founded his foundation in 1995 to enable the implementation of charitable projects. His foundation encourages projects in the area sports, medicine, social support and education. Thus the Hopp foundation supports medical research projects on the highest level including cancer research and pediatrics. In the area of education the foundation facilitates institutions and projects for young people- from kindergarten to university. The Hopp foundation also supports our project and our team of young scientists! <br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img src="/wiki/images/8/8a/Dietmar_hopp_stiftung.png" class="img-responsive" alt="Dietmar Hopp Foundation"><br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"> <br />
<h4>Helmholtz-Initiative Synthetic Biology </h4><br />
The Helmholtz Association is Germany’s largest scientific organization. The Association brings together 18 scientific-technical and biological-medical research centres and follows the tradition of the great natural scientist Hermann von Helmholtz (1821-1894). The Helmholtz Association is dedicated to pursuing the long-term research goals of state and society, and to maintaining and improving the livelihoods of the population. In order to do this, the Helmholtz Association carries out top-level research to identify and explore the major challenges facing society, science and the economy. Its work is divided into six strategic research fields: Energy; Earth and Environment; Health; Key Technologies; Structure of Matter; and Aeronautics, Space and Transport. The Helmholtz Initiative on Synthetic Biology has been supporting the emerging field of synthetic biology since September 2012.<br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img src="/wiki/images/1/17/Helmholtz_logo.jpg" class="img-responsive" alt="Helmholtz Association Logo"><br />
</div><br />
</div><br />
</div><br />
</br><br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"> <br />
<h4>CellNetworks</h4><br />
CellNetworks is an interdisciplinary research cluster centered around Heidelberg´s life sciences. The Heidelberg life science community features an unparalleled number of internationally well-noted institutions. Since 2006 CellNetworks is part of the German Universities Excellence Initiative. CellNetworks assembles excellent groups focusing on research in molecular life science and computational sciences, chemistry and physics to tackle fundamental questions in cell biology, its network structure and architecture, dynamics and regulation.<br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img src="/wiki/images/a/a9/CellNetWorks_logo.jpg" class="img-responsive" alt="Klaus Tschira Foundation Logo"><br />
</div><br />
</div><br />
</div><br />
</br><br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"><h1>Gold Sponsors</div><br />
<br />
</div><br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"> <br />
<h4>geneious</h4><br />
Geneious is a DNA, RNA and protein sequence alignment, assembly and analysis software platform, integrating bioinformatic and molecular biology tools into a simple interface. Geneious provides us their software which helped us design primers and plan our cloning.<br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img src="/wiki/images/6/63/Heidelberg_Geneious_Logo.png" class="img-responsive" alt="Helmholtz Association Logo"><br />
</div><br />
</div><br />
</div><br />
</br><br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"> <br />
<h4>Lange + Pflanz</h4><br />
Lange + Pflanz advertising agency Gmbh has been a specialist for Marketing and Design since 1992. Our focus covers the fields of financial , corporate and strategic communication. As an owner-operated communication company we offer individual and enduring communication solutions for variable media platforms.<br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img src="/wiki/images/6/6c/LangePflanz_Logo.png" class="img-responsive" alt="Lange & Pflanz Logo"><br />
</div><br />
</div><br />
</div><br />
</br><br />
<br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"><h1>Silver Sponsors</div><br />
<br />
</div><br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"> <br />
<h4>Eppendorf</h4><br />
Eppendorf is a company, which products used in all types of life science research and clinical testing settings—from basic laboratory applications to highly specialized cell and molecular biology applications. They are highly regarded for their quality design and performance—beginning with extensive research and development, adding state-of-the-art technology and ending with strict quality-controlled manufacturing are what make our products stand out from the rest. It is what has made us a brand you have been able to rely on for over 60 years.<br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img src="/wiki/images/f/f4/Eppendorf-Logo.png" class="img-responsive" alt="Eppendorf Logo"><br />
</div><br />
</div><br />
</div><br />
<br /><br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"> <br />
<h4>GATC</h4><br />
GATC is a leading service provider of DNA sequencing. Head quartered in Constance, Germany, GATC serves over 10,000 customers in 40 countries around the globe and have subsidiary companies in Great Britain, France and Sweden. GATC provides multi platform strategies, using all leading sequencing technologies on the market, such as Applied Biosystems ABI 3730xl for Sanger sequencing, Illumina’s HiSeq 2000 and Roche Diagnostics GS FLX+ System for Next Generation sequencing as well as the new single molecule sequencer PacBio RS from Pacific Biosciences.<br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img src="/wiki/images/9/97/GATC_logo.gif" class="img-responsive" alt="Helmholtz Association Logo"><br />
</div><br />
</div><br />
</div><br />
</br><br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"> <br />
<h4>Chroma</h4><br />
Chroma Technology Corp. is a manufacturer of interference filters for the ultra-violet, visible and near-infrared portions of the spectrum, including bandpass, multiple bandpass, and long and short pass filters, as well as beamsplitters, dichroic mirrors and laser rejection filters. We specialize in precision spectral control, including extra high signal-to-noise ratio filters and those with rapid cut-on and cut-off profiles. The manufacturing process involves precisely depositing, in a vacuum, extremely thin layers of two or more materials on a glass or similarly transparent substrate.<br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img src="/wiki/images/1/1f/Chroma_LOGO.JPG" class="img-responsive" alt="Chroma Logo"><br />
</div><br />
</div><br />
</div><br />
</br><br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"> <br />
<h4>Carl Roth GmbH & Co</h4><br />
Carl Roth is a private German enterprise with tradition based in Karlsruhe Rheinhafen. It was founded in 1879 with the establishment of a grocer's-paint supplier's-chemist's-store at Karlsruhe. As supplier for lab ware, life sciences and chemicals, they are your partner for best performance reflected in their basic values quality, logistics, fair rates, competent customer advice and service.<br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img src="/wiki/images/8/88/Roth_logo.jpg" class="img-responsive" alt="Roth Logo"><br />
</div><br />
</div><br />
</div><br />
</br><br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"><h1>Academic Sponsors</div><br />
</div><br />
<br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"> <br />
<h4>Heidelberg University</h4><br />
Heidelberg University, founded in 1386, is Germany’s oldest university and one of the strongest research institutions in Europe. The successes in both rounds of the Excellence Initiative and in international rankings prove its leading role in the scientific community. Heidelberg University is a comprehensive university with the full spectrum of subjects including medicine. It aims to strengthen the individual disciplines, to further interdisciplinary cooperation and to make research results usable for society and industry. Heidelberg also draws its strength from its cooperation with local non-university research institutions and is tied into a worldwide network of research and teaching collaborations.<br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img src="/wiki/images/0/07/University-of-Heidelberg_logo.jpg" class="img-responsive" alt="Helmholtz Association Logo"><br />
</div><br />
</div><br />
</div><br />
</br><br />
<br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"><br />
<h4>German Research Center (DKFZ)</h4><br />
The DKFZ (Deutsches Krebsforschungszentrum) is the largest biomedical research institute in Germany and a member of the Helmholtz Association of National Research Centers. In over 90 divisions and research groups, our more than 3,000 employees, of which more than 1,000 are scientists, are investigating the mechanisms of cancer, are identifying cancer risk factors and are trying to find strategies to prevent people from getting cancer .They are developing novel approaches to make tumor diagnosis more precise and treatment of cancer patients more successful.<br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img src="/wiki/images/0/01/Dkfz_logo.jpg" class="img-responsive" alt="Helmholtz Association Logo"><br />
</div><br />
</div><br />
</div><br />
</br><br />
<br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"><br />
<h4>BioQuant</h4><br />
The BioQuant, the Center for “Quantitative Analysis of Molecular and Cellular Biosystems” at Heidelberg University was established in 2007 as an interdisciplinary University research center that is solely dedicated to research and training in systems biology. BioQuants’s objective is to function as a platform for the development and constant refinement of mathematical models of complex biological systems as well as the swift validation of scientific hypotheses via experimental data. Currently, up to 40 University and non-University research groups (DKFZ, EMBL, the Heidelberg Institute for Theoretical Studies (HITS, formerly the European Media Lab), and the MPI for Medical Research) are affiliated with BioQuant. These research groups are instrumental in implementing numerous national and international systems biology funding initiatives.<br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img src="/wiki/images/0/09/BioQuant_Logo.png" class="img-responsive" alt="BioQuant Logo"><br />
</div><br />
</div><br />
</div><br />
<br />
</html></div>Maexlichhttp://2014.igem.org/File:Dietmar_hopp_stiftung.pngFile:Dietmar hopp stiftung.png2015-07-10T04:07:50Z<p>Maexlich: </p>
<hr />
<div></div>Maexlichhttp://2014.igem.org/Team:Heidelberg/pages/SponsoringTeam:Heidelberg/pages/Sponsoring2015-07-10T04:02:09Z<p>Maexlich: </p>
<hr />
<div><html><br />
<br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"><h1>Platinum Sponsor</div><br />
<br />
</div><br />
<br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"> <br />
<h4>Klaus Tschira Foundation</h4><br />
In 1995, Klaus Tschira, physicist and co-founder of the software company SAP, founded the Klaus Tschira Stiftung (KTS). With the intention to place strong emphasis on natural sciences in the society, the commitment of the KTS ranges from kindergarten to schools, universities and research institutions all over Germany. The support is differentiated in three main fields: natural sciences for children and young people, research and science communication. The foundation is located at the Villa Bosch in Heidelberg, the former residence of Nobel Prize laureate for chemistry Carl Bosch.<br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img src="/wiki/images/2/20/Klaus-Tschira-Stiftung_Logo.png" class="img-responsive" alt="Klaus Tschira Foundation Logo"><br />
</div><br />
</div><br />
</div><br />
</br><br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"> <br />
<h4>Dietmar Hopp Foundation</h4><br />
Dietmar Hopp founded his foundation in 1995 to enable the implementation of charitable projects. His foundation encourages projects in the area sports, medicine, social support and education. Thus the Hopp foundation supports medical research projects on the highest level including cancer research and pediatrics. In the area of education the foundation facilitates institutions and projects for young people- from kindergarten to university. The Hopp foundation also supports our project and our team of young scientists! <br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img src="/wiki/images/a/a9/CellNetWorks_logo.jpg" class="img-responsive" alt="Klaus Tschira Foundation Logo"><br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"> <br />
<h4>Helmholtz-Initiative Synthetic Biology </h4><br />
The Helmholtz Association is Germany’s largest scientific organization. The Association brings together 18 scientific-technical and biological-medical research centres and follows the tradition of the great natural scientist Hermann von Helmholtz (1821-1894). The Helmholtz Association is dedicated to pursuing the long-term research goals of state and society, and to maintaining and improving the livelihoods of the population. In order to do this, the Helmholtz Association carries out top-level research to identify and explore the major challenges facing society, science and the economy. Its work is divided into six strategic research fields: Energy; Earth and Environment; Health; Key Technologies; Structure of Matter; and Aeronautics, Space and Transport. The Helmholtz Initiative on Synthetic Biology has been supporting the emerging field of synthetic biology since September 2012.<br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img src="/wiki/images/1/17/Helmholtz_logo.jpg" class="img-responsive" alt="Helmholtz Association Logo"><br />
</div><br />
</div><br />
</div><br />
</br><br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"> <br />
<h4>CellNetworks</h4><br />
CellNetworks is an interdisciplinary research cluster centered around Heidelberg´s life sciences. The Heidelberg life science community features an unparalleled number of internationally well-noted institutions. Since 2006 CellNetworks is part of the German Universities Excellence Initiative. CellNetworks assembles excellent groups focusing on research in molecular life science and computational sciences, chemistry and physics to tackle fundamental questions in cell biology, its network structure and architecture, dynamics and regulation.<br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img src="/wiki/images/a/a9/CellNetWorks_logo.jpg" class="img-responsive" alt="Klaus Tschira Foundation Logo"><br />
</div><br />
</div><br />
</div><br />
</br><br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"><h1>Gold Sponsors</div><br />
<br />
</div><br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"> <br />
<h4>geneious</h4><br />
Geneious is a DNA, RNA and protein sequence alignment, assembly and analysis software platform, integrating bioinformatic and molecular biology tools into a simple interface. Geneious provides us their software which helped us design primers and plan our cloning.<br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img src="/wiki/images/6/63/Heidelberg_Geneious_Logo.png" class="img-responsive" alt="Helmholtz Association Logo"><br />
</div><br />
</div><br />
</div><br />
</br><br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"> <br />
<h4>Lange + Pflanz</h4><br />
Lange + Pflanz advertising agency Gmbh has been a specialist for Marketing and Design since 1992. Our focus covers the fields of financial , corporate and strategic communication. As an owner-operated communication company we offer individual and enduring communication solutions for variable media platforms.<br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img src="/wiki/images/6/6c/LangePflanz_Logo.png" class="img-responsive" alt="Lange & Pflanz Logo"><br />
</div><br />
</div><br />
</div><br />
</br><br />
<br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"><h1>Silver Sponsors</div><br />
<br />
</div><br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"> <br />
<h4>Eppendorf</h4><br />
Eppendorf is a company, which products used in all types of life science research and clinical testing settings—from basic laboratory applications to highly specialized cell and molecular biology applications. They are highly regarded for their quality design and performance—beginning with extensive research and development, adding state-of-the-art technology and ending with strict quality-controlled manufacturing are what make our products stand out from the rest. It is what has made us a brand you have been able to rely on for over 60 years.<br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img src="/wiki/images/f/f4/Eppendorf-Logo.png" class="img-responsive" alt="Eppendorf Logo"><br />
</div><br />
</div><br />
</div><br />
<br /><br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"> <br />
<h4>GATC</h4><br />
GATC is a leading service provider of DNA sequencing. Head quartered in Constance, Germany, GATC serves over 10,000 customers in 40 countries around the globe and have subsidiary companies in Great Britain, France and Sweden. GATC provides multi platform strategies, using all leading sequencing technologies on the market, such as Applied Biosystems ABI 3730xl for Sanger sequencing, Illumina’s HiSeq 2000 and Roche Diagnostics GS FLX+ System for Next Generation sequencing as well as the new single molecule sequencer PacBio RS from Pacific Biosciences.<br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img src="/wiki/images/9/97/GATC_logo.gif" class="img-responsive" alt="Helmholtz Association Logo"><br />
</div><br />
</div><br />
</div><br />
</br><br />
<div class="row"><br />
<div class="col-md-7 col-sm-12 col-xs-12"> <br />
<h4>Chroma</h4><br />
Chroma Technology Corp. is a manufacturer of interference filters for the ultra-violet, visible and near-infrared portions of the spectrum, including bandpass, multiple bandpass, and long and short pass filters, as well as beamsplitters, dichroic mirrors and laser rejection filters. We specialize in precision spectral control, including extra high signal-to-noise ratio filters and those with rapid cut-on and cut-off profiles. The manufacturing process involves precisely depositing, in a vacuum, extremely thin layers of two or more materials on a glass or similarly transparent substrate.<br />
</div><br />
<div class="col-md-5 col-sm-12 col-xs-12"><br />
<div class="col-lg-6 col-lg-offset-3 col-md-offset-2 col-md-8 col-sm-offset-3 col-sm-6 col-xs-12"><br />
<img src="/wiki/images/1/1f/Chroma_LOGO.JPG" class="img-responsive" alt="Chroma Logo"><br />
</div><br />
</div><br />
</div><br />
</br><br />
<div class="row"><br />
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<h4>Carl Roth GmbH & Co</h4><br />
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</html></div>Maexlichhttp://2014.igem.org/Team:Heidelberg/Project/Linker_ScreeningTeam:Heidelberg/Project/Linker Screening2014-12-08T16:58:48Z<p>Maexlich: </p>
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{{:Team:Heidelberg/templates/mathjax}}</div>Maexlichhttp://2014.igem.org/Team:Heidelberg/pages/Linker_ScreeningTeam:Heidelberg/pages/Linker Screening2014-12-08T16:56:53Z<p>Maexlich: </p>
<hr />
<div><br/><br />
We developed a modeling approach to design linkers that can circularize proteins and that may confer some rigidity in particular after heat shock. The general concept of the approach can be found in the [https://2014.igem.org/Team:Heidelberg/Software Software], and the detailed theoretical development can be found in the software as well as in the [https://2014.igem.org/Team:Heidelberg/Modeling Modeling] sections. <br />
The experimental work presented in this section has two main goals. The first one was testing the validity of the idea: Can rigid linkers with angles indeed provide heat stability to a protein, and can it do this better than flexible linkers? And the second one was to measure this stabilization depending on the properties of the linkers to calibrate the [https://2014.igem.org/Team:Heidelberg/Software/Linker_Software Linker Software]. As already seen in the software part, these properties are the total length of the linker, the angles between consecutive alpha helices, the regions that the linker passes and the distance from the proteins surface. As we had no a priori knowledge on the contribution of those different properties for the heat stability of protein, we performed an extensive linker screening on the lambda phage lysozyme. The tested linkers were predicted to belong to different groups by the software: Very good, good, average, bad and too short. By measuring their activity after heatshock, we could transform this qualitative classification into a quantitative one.<br />
<br />
=Introduction=<br />
<br />
{{:Team:Heidelberg/templates/image-quarter| align=right| caption=figure 1) Lambda Lysozyme| descr=Secondary structure of lambda lysozyme. The distance between the two ends is 27.3 Ångström.| file=lysozyme-pdb.png}}<br />
Designing linkers for circularization is a non trivial task: Circularization is a narrow path between gaining heat-stability and loosing function due to deformation. <br />
The choice of the protein to perform the linker screen was constrained by different requirements. First, it needed to be easily and fast expressed in ''E.coli''. We needed to be able to measure its functionality without purification and in an easy, fast, cheap and reliable way. It needed to be known for having a certain stability at high temperature with, at the same time, a loss of functionality so that the heat stabilization could be tested. Finally, the major constrain for the choice was the structure of the protein.<br />
<br />
First, we needed a crystal structure of the complete protein at high resolution. Second, the ends should be separated by a distance of 15 to 30 Ångström, so that the rigid linkers containing alpha helices would be relevant. On the other hand, the linker should not pass over the active site of the protein. Furthermore it should be easy to obtain. <br />
<br />
Lysozymes are well characterized enzymes that are able to digest the peptidoglycans that form the bacterial wall. This process is performed by many different species for different applications, including antibacterial defence by plants and animals [[#References|[1]]] or bacterial penetration by viruses [[#References|[2]]]. On top, lysozyme is applied in different fields of biotechnology and medicine. It is notably one of the most important proteins in food preservation and is produced in the 100 tons scale a year. <br />
We anticipated that the lysozyme of the bacteriophage lambda could reasonably fulfill the requirements for our linker screen. <br />
<br />
Its crystal structure is known [[#References|[2]]] and the ends are 27.3 Angströms apart (figure.1). It is easy to obtain: one can clone it from the bacteriophage lambda genome, and express it in ''E.coli''. The fact that the overexpression of the enzyme eventually lyse the cells was not considered as a problem as long as enough enzyme would be produced. Finally substrates to measure the enzymatic activity are commercially available.<br />
<br />
=Main results=<br />
{{:Team:Heidelberg/templates/image-half|<br />
caption=Figure 5) Lysozyme assay|<br />
align=right |<br />
descr=Lysozyme assay with linker sho2 with a heat shock span between 45.8°C and 56.5°C. You can see very clearly a difference between the temperatures.|<br />
file=plot_of_E_axis2014_10_08.png}} <br />
We could clone and express the lysozyme of bacteriophage lambda and its fusion to different linkers (table 1: Linker and their amino acid sequence) chosen to calibrate our linker software. We used lyophilized ''Micrococcus lysodeikticus'' as substrate to measure lysozyme activity and we established a heat shock assay to measure the heat-stability of the enzyme. We observed that while lysozyme shows a clear activity at 37°C, this activity is gradually lost when the enzyme is pretreated at higher temperatures up to 57°C. Thanks to our measurement of the kinetics of substrate processing and to our model on enzymatic activity with product inhibition (see our model in section [https://2014.igem.org/Team:Heidelberg/Modeling/Linker_Modeling Linker-Modeling]), we could extract the activity of the enzyme after different heat shock temperatures and with different linkers. This quantitative information was then used to calculate the weighting constants of the different linker features. <br />
The following figure (figure 5) shows the substrate processing measured from the decrease of optical density of the ''M. lysodeikticus'' for the different temperatures of heat shock and the different linkers.<br />
<br />
<br/><br />
<br />
{| class="table table-hover"<br />
|+ '''table 1''': Linker and their amino acid sequence. Green: attachment sequences to prevent the flexible regions from being perturbed; Blue: angle; Purple: extein.<br />
! Predicted linker quality <br />
! Amino acid sequence<br />
|- <br />
|<br />
|<br />
|-<br />
|'''Very good linkers'''<br />
|<br />
|-<br />
| sgt2<br />
| <span style="color:#3ADF00;">GG</span>AEAAAK<span style="color:#00BFFF;">AAAHPEA</span>AEAAAK<span style="color:#A901DB;">RGTCWE</span><br />
|-<br />
| rigid<br />
| <span style="color:#3ADF00;">GG</span>AEAAAKEAAAKAA<span style="color:#3ADF00;">P</span><span style="color:#A901DB;">RGKCWE</span><br />
|-<br />
| <br />
| <br />
|-<br />
| '''Average linkers'''<br />
|<br />
|-<br />
| may1<br />
| <span style="color:#3ADF00;">GG</span>AEAAAKEAAAKA<span style="color:#00BFFF;">AAAHPEA</span>AEAAAKEAAAKA<span style="color:#00BFFF;">KTA</span>AEAAAKEAAAKA<span style="color:#A901DB;">RGTCWE</span><br />
|-<br />
| ord1<br />
| <span style="color:#3ADF00;">GG</span>AEAAAKEAAAK<span style="color:#00BFFF;">ATGDLA</span>AEAAAKAA<span style="color:#A901DB;">RGTCWE</span><br />
|-<br />
| ord3<br />
| <span style="color:#3ADF00;">GG</span>AEAAAKEAAAK<span style="color:#00BFFF;">ASLPAA</span>AEAAAKEAAAK<span style="color:#A901DB;">RGTCWE</span><br />
|-<br />
|<br />
| <br />
|-<br />
| '''Short linkers'''<br />
| <br />
|-<br />
| sho1<br />
| <span style="color:#3ADF00;">GG</span><span style="color:#A901DB;">RGTCWE</span><br />
|-<br />
| sho2<br />
| <span style="color:#3ADF00;">GG</span>AEAAAK<span style="color:#A901DB;">RGTCWE</span><br />
|-<br />
| '''Flexible linker'''<br />
| <br />
|-<br />
| flex<br />
| GGSGGGSG<span style="color:#A901DB;">RGKCWE</span><br />
|}<br />
<br />
=Experimental procedure=<br />
<br />
==Cloning of the expression constructs==<br />
<br />
The construct to express linear lambda lysozyme [http://parts.igem.org/Part:BBa_K1362011 BBa_K1362011] was assembled by [https://2014.igem.org/Team:Heidelberg/Notebook/Methods#CPEC CPEC] from PCR products of [http://parts.igem.org/Part:BBa_K1362093 pSBX1K30] and phage lambda genome. The RBS [http://parts.igem.org/Part:BBa_K1362090 BBa_K1362090] was added by primer overhangs.<br />
In order to create the construct to express linear lambda lysozyme with His6-tag and exteins, primers to linearize [http://parts.igem.org/Part:BBa_K1362011 BBa_K1362011] between the lambda lysozyme coding sequence and stop codon and add BsaI sites at the ends were designed. An insert coding for the sequence GSHHHHHHSGRGKCWE (His6-tag + exteins) with overhangs corresponding to the overhangs caused by BsaI restriction of the vector PCR product was designed and ordered as DNA-oligos. The insert was annealed and inserted into the purified PCR product of [http://parts.igem.org/Part:BBa_K1362011 BBa_K1362011] by [https://2014.igem.org/Team:Heidelberg/Notebook/Methods#Golden_Gate_Assembly Golden Gate assembly].<br />
<br />
The constructs to express circular lambda lysozyme with a rigid linker [http://parts.igem.org/Part:BBa_K1362013 (BBa_K1362013)], a His6 linker [http://parts.igem.org/Part:BBa_K1362012 (BBa_K1362012)] and all other linkers in shown in table 1 are based on the [http://parts.igem.org/Part:BBa_K1362000 NpuDnaE intein RFC(105) circularization construct]. These cloning steps were carried out according to our new RFC[105].<br />
<br />
For [http://parts.igem.org/Part:BBa_K1362013 BBa_K1362013] and [http://parts.igem.org/Part:BBa_K1362012 BBa_K1362012], the coding sequence of lambda lysozyme without start and stop codon was aquired from genomic lambda DNA by PCR. Overhangs containing a BsaI site, the RFC[105] E standard overhang, the coding sequences of the extein CWE (forward) and the corresponding linker sequence, RGK, the RFC[105] B standard overhang, a BsaI site (reverse) were added by primers. The PCR products were purified and inserted each into the [http://parts.igem.org/Part:BBa_K1362000 NpuDnaE intein RFC(105) circularization construct] by Golden Gate assembly.<br />
<br />
For the constructs with the linkers sgt2, may1, ord1, ord3, sho1, sho2 and flex, a cloning intermediate containing both lambda lysozyme and the selection marker ccdB instead of the mRFP selection marker was created by Golden Gate assembly of [http://parts.igem.org/Part:BBa_K1362000 NpuDnaE intein RFC(i) circularization construct] and two inserts with subsequent [https://2014.igem.org/Team:Heidelberg/Notebook/Methods#Religation_after_Golden_gate_Assembly religation]. The first insert was created by a PCR of the coding sequence of lambda lysozyme without start and stop codon aquired from genomic lambda DNA. Overhangs containing a BsaI site, the RFC[105] E standard overhang, the coding sequences of the extein CWE (forward) and a BsaI site (reverse) were added by primers. The ccdB insert was created by PCR of the ccdB selection marker from pDONR. Overhangs containing a BsaI site, the last 4 nucleotides of lambda lysozyme (forward) and the RFC[105] B standard overhang and a BsaI site (reverse) were added by primers.<br />
Inserts coding for the linker sequences with overhangs corresponding to the overhangs caused by BsaI restriction of the cloning intermediate were ordered as DNA-oligos. The linker inserts were annealed and inserted to the cloning intermediate by Golden gate assembly. <br />
<br />
==Expression and detection of circular lysozyme variants==<br />
<br />
{{:Team:Heidelberg/templates/image-quarter| align=right| caption=Figure 5) Measurement of substrate degradation after recovery| descr=All samples are active and not degraded. | file=plot_of_12_axis2014_09_29.png}}<br />
{{:Team:Heidelberg/templates/image-quarter| align=right| caption=Figure 4) Measurement of substrate degradation before recovery| descr= There is a difference between the activity of the lysozyme samples. | file=plot_of_12_axis2014_09_28.png}}<br />
With a Coomassie Gel and a Western Blot we tested the expressed construct on circularization. To do a Western Blot we built a circular form of lambda lysozyme with a His-tag as linker structure and a linear control including a His-tag, too. Circular proteins run faster in a gel than proteins of the same length because of their structure [[#References|[3]]]. So you can see a shift on the gel between the linear and the circular form of a protein (figure 2). But what you could see on the Western Blot is that the product of the splicing reaction is not entirely circular. So in samples of circular lambda lysozyme there is still linear lysozyme.<br />
<br />
Because of the high expression level, tested with a Coomassie Gel (figure 3), we did not purify the lysozyme constructs and used non-purified protein mix. On the gel in figure 3 one can see that there is a difference between the expression level of the different constructs. But all constructs were expressed enough to measure their activity. <br />
To make sure our lysozyme samples were not degraded by proteases we measures samples two times, direct and after 20 hours at 37°C. But the activity persisted on the same level (figure 4 and 5).<br />
<br />
The linker screening was performed with ten different linkers. We wanted to show that different linkers have different effects on the thermostability and function of a protein, as claimed by our [https://2014.igem.org/Team:Heidelberg/Modeling/Linker_Modeling Linker-Modeling]. The linker software calculated linkers classified according to fitness as great, bad, short and proper linker. The length of a linker had a significant effect on folding structure of proteins.<br />
<br />
<div><br />
{{:Team:Heidelberg/templates/image-half| align=right| caption=Figure 3) Coomassie Gel of the linker constructs| descr=The expression levels of the linker constructs are different. In all lanes you can see intein fragments as result of the trans-splicing reaction.| file=62.png}}<br />
{{:Team:Heidelberg/templates/image-half| align=right| caption=Figure 2) Western Blot to prove circularization| descr=To test if lambda lysozyme is circular after the splicing reaction of the intein Npu Dna E we did a Western Blot with Penta·His Antibody. There is a clear shift between linear and circular samples.| file=36_20140923_lysozyme.jpg}}<br />
<div class="clearfix"></div><br />
</div><br />
<br />
==Development of assay to measure lysozyme activity==<br />
<br />
To test and quantify the differences in thermostability of the linear and several circular forms with different linkers of lambda lysozyme we needed an assay that is simple and reproducible enough. To have a comparison and a great check on functionality we tested two different methods to measure lysozyme activity and established the range of temperature to test the heat stability of the enzyme.<br />
Lysozyme of the bacteriophage lambda like the other lysozymes cleaves glycosidic bonds between 1,4-beta-linkages between N-acetylmuramic acid (NAM) and N-acetyl-D-glucosamine residues (NAG) in peptidoglycan and between N-acetyl-D-glucosamine residues in. As a really small protein built of only 158 amino acids its mechanism is different from other lysozymes: the hydroxyl function OH on the C6 of the muramic acid is the nucleophile on cleavage of the muropeptide instead of the more common water molecule. So the protein is a transglycosidase, not a hydrolase [[#Reference|[4]]]. <br />
The scientific background of the first assay was the capacity of lambda lysozyme to degrade the cell wall of ''Micrococcus lysodeikticus'', a gram-positive bacterium often used as a lysozyme substrate [[#Reference|[5]]]. The rate of lysis of ''Micrococcus lysodeikticus'' was measured by a change of OD of the substrate solution at 600 nm. Using 96-well plates we have the possibility to test on several constructs within one assay.<br />
<br />
The basis of the second assay was the labeling of peptidoglycan with fluorescein isothiocyanate (FITC), an amine-reactive derivative of fluorescein dye. When the FITC-labeled substrate was subjected to the enzyme digestion, an increase of fluorescence intensity and a decrease of fluorescence polarization value should be observable. The labeling of peptidoglycane was done by ourself [[#Reference|[6]]].<br />
<br />
Performing both assays we realized in an early state that the first assay is less error-prone than the fluorescence intensity measuring assay. Both assays were measured in the plate reader Tecan Infinite F200 PRO. Even after several washing steps we could not exactly identify how effective the labeling was. Furthermore we had problems with the adjustment of the wavelength and the gain.<br />
The assay measuring the degradation of the substrate had disadvantages, too. Because of the substrate ''M. lysodeicticus'' there were unknown values we could not predict. <br />
<br />
The degradation of the substrate and associated with it the decrease of the OD was measured every 2 minutes for two or three hours to get all kinetic information and recognize a potential refolding.<br />
At the beginning of the assay setup we measured the behavior of lambda lysozyme after a heat shock at temperatures up to 70°C until 90°C. Because of special characteristics of lambda lysozyme such as non-enzymatic behaviour around 70°C [[#Reference|[7]]] we did not enhance the measurement due to higher temperatures.<br />
For the linker screening the samples were heated for 1 minute between 37°C and 57°C. <br />
<br />
Focus of the assay is to test the heated protein on stability not on functionality. If the lysozyme still degrades the substrate and retains function it shows that the protein is still folded correctly. We measured the activity and associated the heat-stability of lambda lysosyme after a heat shock to see behavior of refolding. So the first minutes after heating are very important to see potential refolding. So the speed the assay were performed is critical, too.<br />
<br />
=Materials and Methods=<br />
<div>{{:Team:Heidelberg/templates/image-half| align=right|caption=Figure 6) Composition of the final assay.|descr=| file=Aufbau_Assay.png}}<br />
Bacteria were transformed with lambda lysozyme constructs, grown to an OD of 0.6 and induced with 1 mM IPTG. After 4 hours of expression, the samples were diluted to the same OD of 2.0 and centrifuged down at 2,850 rpm. The pellet was resuspended in 10 mM potassium phosphate buffer with a pH of 6.24 and sonicated for two minutes on ice. After centrifugation a second time at 2,850 rpm, the supernatant containing the lambda lysozyme was kept.<br />
For substrate preparation, the lyophilized cells of ''M.lysodeikticus'' (Sigma Aldrich), were resuspended in ultrapure water. The supernantant with the protein mix was transfered and aliquoted for biological replicates and to prepare dilution series. <br />
Subsequently the samples were transfered into the thermocycler to do a one minute heat-shock in a temperature span between 45 and 55°C. <br />
After mixing enzyme and substrate the OD was measured every two minutes in a plate reader over 100 minutes at 37°C (figure 6). <br />
The expression of the lysozyme samples for the assay using FITC-labeled peptidoglycan was carried out in the same as we did it in the first assay. We resuspended the pellet in PBS. The protocol of labeling of peptidoglycan with fluorescein isothiocyanate (FITC) you can find in the Materials and Methods. <br />
<div class="clearfix"><br />
</div><br />
</div><br />
<br />
=Results=<br />
<br />
We have established a protocol with which one is able to make a quantitative analysis on the effect of different linkers heat stability of lysozyme. This method can not only be performed in large scale, but is also resistant to various influences, like different expression levels of the constructs. With this assay in short time sufficient data of high quality can be produced, so that an extensive enzyme modeling approach can be made for their evaluation. <br />
<br />
For the evaluation the data of the degradation curves from the assays were analyzed for the change in activity after different heatshocks. This was done applying different models to the data and analyzing it by use of Profile Likelihood Estimation, like we described in our [[Team:Heidelberg/Modeling/Enzyme_Modeling | enzyme modeling]] part. To each degradation curve by itself we did not apply a complex model, but the advantage lies in modeling the data in total. The complexity lies in the combination of the different model parameters. The models were always fitted to the data of multiple experiments at once, leading to an increased reliability than fitting single data.<br />
<br />
We could show, that the data was well described by applying product inhibited Michaelis Menten kinetics. Further on we found out, that the model's quality did not increase significantly by letting the enzyme kinetics ($K_M, K_I$) parameters change over temperature. This was done by making identifiability analysis of model parameters. We showed, that when not restraining $K_M$ and $K_I$ for different temperatures none of the important parameters was identifiable anymore. For better readability we introduced parameters representing the loss in activity after heat shock. These were called activities at a temperature and were built as the ratio between $V^T_{Max}$ and $V^{37°C}_{Max}$. These parameters were the most interesting ones for us. When each lysozyme was modeled with constant $K_M$ and $K_I$ for different temperatures nearly the activities were identifiable within 95% confidence level. For some very good datasets even the kinetic parameters $K_M$ and $K_I$ could be identified on a lower confidence level. Therefore we concluded that the change in these parameters for the different temperatures was minor and could be neglected in the further evaluation.<br />
<br />
We showed that the different linker constructs show different changes in activity after heat shock, figure 7. <br />
<br />
{{:Team:Heidelberg/templates/image-full|<br />
caption=Figure 7, Activity after heatshock|<br />
align=right |<br />
descr=Ratio of activity after a heatshock of a certain temperature. The single points come from different technical and biological replicates. For clarification a simple spline was fitted to the data. The fits produced 95% confidence intervals, that would exceed the scale. But as on can see that mostly the different replicates don't scatter too much, these errors are not too relevant. A clear difference can be seen for ord1- and ord3-linker in comparison to the flexible linker and the linear lysozyme|<br />
file=resultsofscreening_new.png}} <br />
<br />
For further clarification we have calculated the average activity between 45 and 50°C, because there the biggest change in activity could be observed for the different constructs. Furthermore the values of the activities vary a lot, what can especially be seen at circsho1 linker. This averaging was made for clarification and for obtaining a final ranking of the linkers to feed back to the linker software (see table 2)<br />
<br />
<br />
{| class="table table-hover"<br />
|+ '''table 2''': Ranking of different linkers on enhancement of thermostability and their classification<br />
! Linker<br />
! classification<br />
! average activity from 45°C to 50°C<br />
|- <br />
|<br />
|<br />
|<br />
|-<br />
| ord3<br />
| Average<br />
| 1.390<br />
|-<br />
| ord1<br />
| Average<br />
| 0.956<br />
|-<br />
| rigid<br />
| Very good<br />
| 0.945<br />
|-<br />
| may1<br />
| Average<br />
| 0.749<br />
|-<br />
| sgt2<br />
| Very good<br />
| 0.748<br />
|-<br />
| sho1<br />
| bad<br />
| 0.709<br />
|-<br />
| linlys<br />
| linear<br />
| 0.704<br />
|-<br />
| flex<br />
| Flexible<br />
| 0.685<br />
|-<br />
| sho2<br />
| bad<br />
| 0.574<br />
|-<br />
|}<br />
<br />
=Discussion=<br />
<br />
Our Linker screening was the first time that rigid linkers with angles were tested on their effect on thermostability. Further on we could extract the relevant data to calibrate [[Team:Heidelberg/Software/Linker_Software | CRAUT]] software with the results. Still important enzymatic parameters could not be identified reliably. Due to the lack of time the amount of replicates was restricted. Mainly the time between adding the enzyme to the substrate and the first measurement influences the quality of the data. Further refinement there could lead to better identifiability of the parameters. The errors on the parameters obtained from the fitting would need to be reduced. On the other hand one is tightly limited in the initial substrate concentration in a range until 0.7 mg/ml, which makes it difficult to identify all the parameters. Further on the linkers could be tested with different proteins to make predictions more independent of the protein.<br />
<br />
=The long way to setup the assay=<br />
<br />
Enzyme kinetics and protein interactions are very complex fields of modeling and biochemistry. For the present assay, there were many different dimensions that has to be tested and optimized: concentration of enzymes, of substrates, temperature for the enzyme-substrate reaction, heat shock temperature and length, as well as length of recovery after heat shock. So we had to test many factors, solve many problems and find new ways to establish our linker screening to a standardized and ideal method for testing the effect of different linkers on proteins. The following part details the establishment of our assay.<br />
The main issues were finding the correct temperatures, the correct lengthes of heatshock and the right concentrations.<br />
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caption=21th of August| file=plot_of_9_axis2014_08_21.png}}<br />
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caption=20th of August| file=plot_of_A_axis2014_08_20.png}}<br />
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caption= 19th of August| file=plot_of_A_axis2014_08_19.png}}<br />
<br />
====14th of August====<br />
In the first assay we used HEWL (ordered from Sigma Aldrich) and ''E.coli'' lysate without lysozyme to see how lysozyme behaves in general and how the assay works in total. Furthermore we wanted to see a difference between HEWL containing samples and ''E.coli'' lysate. All samples were heated from 70°C to 90°C in the cycler.<br />
The results of this first test were curious. It seems that even without lysozyme and without ''E.coli'' lysate, the substrate is degraded.<br />
<br />
====17th of August====<br />
Repeating the first assay we could improve the results and saw first temperature dependence. This assay did not give great results, too. But we figured out that the concentration of HEWL was too high, which might explain the behavior of the samples, because at the higher temperatures we observed big coagulation. Furthermore we mentioned that we are too slow preparing and performing the assay, as most of the substrate is already degraded, when we had the first measurement. The most information about the enzyme kinetics can only be seen in the degradation of the ''Micrococcus lysodeicticus'' in the very first minutes. <br />
<br />
====19th of August====<br />
After using HEWL in the last assays we now tested a linear lambda lysozyme construct we had cloned and expressed the weeks before. The samples were heated at a different temperature span between 50°C and 70°C. This was the first time we really could see different curve progressions after a heat-shock at different temperatures. We changed our method, so that we were faster in the platereader after adding the lysozyme. The sobering message: The activity grows with higher temperature.<br />
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caption=26th of August| file=plot_of_6_axis2014_08_26.png}}<br />
<br />
====20th of August====<br />
To explain the behavior of the lysozyme around 70°C we saw in the last assay we measured the degradation of substrate after a heat-shock in a temperature span between 70°C and 95°C. It seems that lysozyme has his highest activity around 70°C. Later we detected that lysozymes has a non-enzymatic activity [[#References|[7]]], which explains the increase of activity around 70°C. Controls containing no lysozyme did not show any activity. All graphs showed "shoulders" and not a clean exponentiell curve. Troubleshooting possibilities like salt, measuring temperature had to be checked before performing the next assay.<br />
<br />
====21st of August====<br />
To find out the best concentration of the lysate we measured a dilution series. High concentrated samples were coagulating, protein lysate less diluted did not showe this effect. It was the first time we saw a significant activity of lysozyme at 37°C. The 1 to 10 dilution is seems to be the appropriate dilution, because activity was high enough.<br />
<br />
====22nd of August====<br />
Testing lower temperatures we decided to do a heat shock at temperatures between 30°C and 50°C. The activity of lambda lysozyme decreases until 50°C, the maximum activity samples showed that were heated at lower temperatures. <br />
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caption=30th of August| file=plot_of_lys1to1045diff2014_08_30.png}}<br />
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caption=30th of August| file=plot_of_plate_before_lyse.png}}<br />
<br />
====26th of August====<br />
Next to enzyme dilutions we tested substrate dilutions to. All constructs were kept on 37°C, even during the measurement the plate reader had this temperatur. We found out the perfect concentration for the substrate, next assays were done with 0.5 % (w/v). With lower concentrations the errors were large. <br />
<br />
====30th of August====<br />
Next to the speed the accuracy of pipetting is significant, too. In this assay we payed heed to exact pipetting. Furthermore we measured many replicates on one well to check on correctness of pipetting. The differences between the replicates were low.<br />
<br />
====5th of September====<br />
The cloning of the circular form of lambda lysozyme was finished, so we tested this construct the first time to see, if the circular form of lysozyme is still active. Because it was the first assay with circular constructs, the rigid-linker construct and the His-linker construct, we did not heated them. All samples stayed on 37°C.<br />
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caption= 10th of September (1)| file=plot_of_A_axis2014_09_10.png}}<br />
</div><br />
<br />
====9th of September====<br />
Aim of this assay was to see differences in the temperature stability of the different lysozyme constructs, linear and circular, after a heat shock of half of an hour between 35°C and 55°C. The circular lysozyme constructs are clearly working and the probes with the rigid linker work better than the construct including a His-tag. Before declaring a difference between circular and linear constructs we needed more results for great statistics.<br />
<br />
====10th of September (1)====<br />
Next to the first assay we tested a second assay based on an increase of fluorescence intensity. We performed the assay with many different dilutions of substrate and of lysozymes, testing the fluorescein labeled peptidoglycane.To proof the labeling we added the supernatants of the washing steps to see whether they are still fluorescing or not. The fluorescence absolutely doesn't behave as we expected. We don't see any enlargement of the fluorescence. The different curves have huge variations between them.<br />
<div><br />
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caption= 10th of September (2)| file=resultsplotlamlys2014_09_10.png}}<br />
</div><br />
<br />
====10th of September (2)====<br />
To have a higher statistical coverage we reproduced the last assays. The temperature span of the heat shock was 37°C until 54°C. From this assay on, we could test and evaluate different linkertypes properly, as we have introduced a fitting method, showing the activity of the lysozymes as the exponent of the exponential decay. As we always had different expression levels, we introduced a normalization of the activity to the activity at 37°C.<br />
There seems to exist some offset activity, the lysozymes always keep.<br />
In the plots above you can see, how important a normalization is, as the activity plots sometimes are too crowded. But on the other hand, these plots can be misleading, as they overestimate, when there is low activity already.<br />
<br />
====11th of September (1)====<br />
To have a comparison between biological replicates we repeated the last assay under the same conditions. The results are similar, our assay indeed is reproducable with different biological samples.<br />
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caption= 12th of September| file=resultsplotlamlys2014_09_12.png}}<br />
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caption= 11th of September| file=resultsplotlamlysnormed2014_09_11_1.png}}{{:Team:Heidelberg/templates/image-quarter| align=right| descr=|<br />
caption= 11th of September| file=resultsplotlamlys2014_09_11_1.png}}<br />
</div><br />
<br />
====11th of September (2)====<br />
To find a temperature span where we can finally screen our linkers we did the last assay at higher temperatures up to 70°C. <br />
<br />
====12th of September====<br />
In this assay we increased the temperature level of the heat shock, again. Aim of the last and following assays is to have data for the complete span up to 95°C. One can see, that while linear lysozyme has an activity maximum at 75°C circular lysozyme has not.<br />
<br />
====14th of September====<br />
The samples were heat shocked for half of an hour at 78°C up to 95°C. <br />
At least at a heat shock of 95°C all the samples loose activity, but somehow are always clearly distinguishable from the controls.<br />
<br />
====15th of September====<br />
In the last assays we recognized that the temperature span between 45°C and 55°C is interesting one for us related to a difference between the linear lysozyme and the lysozyme with the rigid linker in loosing the conformation from 37°C. So we repeated the assay in this span. There can be seen huge differences in the temperature behaviour of the different lysozymes after normalization. But it is not clear, whether they come from differences in expression level, or not.<br />
<div><br />
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caption= 15th of September| file=resultsplotlamlys2014_09_15.png}}<br />
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caption= 15th of September| file=resultsplotlamlysnormed2014_09_15.png}}{{:Team:Heidelberg/templates/image-quarter| align=right| descr=|<br />
caption= 14th of September| file=resultsplotlamlys2014_09_14.png}}<br />
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caption= 14th of September| file=resultsplotlamlys2014_09_14.png}}<br />
</div><br />
<br />
====19th of September====<br />
On this assay OD calibration was made. Also we tried to have same "standard" activity at 37°C for all constructs, so that normalization isn't that grave anymore. The OD can be calculated to ''M. lysodeicticus'' concentration in the linear range until dilution of substrate of 0.65 mg/ml with 1.160 ml/mg + 0.13. So we can extend our range to 0.66 mg/ml, so that we have more time where we can measure. <br />
<br />
====28th of September====<br />
To test the recovery of the lysozyme constructs after heat-shock we did two assays with the same heated samples. One part we let recover for 20h at 37°C. The temperature span is from 40.3°C-54 °C with a reference temperature at 37°C. The heat shock was 30 minutes. The substrate concentration was after calculation of the last assay 0.66 mg/ml. We had two aims: Is there protease activity and is there some effect of heatshock.<br />
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caption=29th of September| file=plot_of_12_axis2014_09_29.png}}<br />
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caption= 28th of September| file=plot_of_12_axis2014_09_28.png}}<br />
<br />
====29th of September====<br />
This assay was made after a recovery time of the lysozyme samples of 20h. The samples recovered at 37°C over night. It was measured at 37°C in the platereader as well. This is the continuation of the test of 29th of september. The rest of the samples that could recover over night was put on a plate. We could not see any recovery from the heatshock and protease activity. The activity levels for both lysozyme constructs were nearly the same before and after the recovery time.<br />
<br />
====30th of September====<br />
<br />
<div><br />
We want to find the appropriate length of heat shock for our assay to see the effect of different heatshock lengths and figure out whether there is a difference between the different lysozyme constructs. The samples were heated for 1 and for 7 minutes. For such short heatshocks in the temperatures where normally the activity is lost, most of the activitiy for all of the constructs remained still. For the ridid linker activity even increased and had a maximum activity at 46 degrees. Unfortunately the errors are enormous as we only had duplicates on the wellplate. The 1 minute heatsock seems the range where we could observe differences between the different linkers.<br />
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caption= 1st of October| file=resultsplotlamlys2014_10_01.png}}<br />
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caption=4th of October| file=plot_of_11_axis2014_10_01_4.png}}<br />
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caption= 4th of October| file=resultsplotlamlysnormed2014_10_01_4.png}}<br />
</div><br />
<br />
====1st of October====<br />
The samples were heated for 14 and for 21 minutes. With this longer heatshocks we observe the same behaviour as with the 30 minutes, so no clear tendencies can be seen. So we decided to perform all following assays of the linker screening with a heat shock time of 1 minute. The last preperations to set up the final assay were finished.<br />
<br />
====4th of October====<br />
The final protocol you can find in in Materials and Methods or in parts of the text above!<br />
<br />
=References=<br />
[1] Callewaert, L. & Michiels, C. W. Lysozymes in the animal kingdom. J. Biosci. 35, 127–160 (2010).<br />
<br />
[2] Evrard, C., Fastrez, J. & Declercq, J. P. Crystal structure of the lysozyme from bacteriophage lambda and its relationship with V and C-type lysozymes. J. Mol. Biol. 276, 151–64 (1998).<br />
<br />
[3] Iwai, H., Lingel, a & Pluckthun, a. Cyclic green fluorescent protein produced in vivo using an artificially split PI-PfuI intein from Pyrococcus furiosus. J. Biol. Chem. 276, 16548–54 (2001).<br />
<br />
[4] Callewaert, L. et al. Purification of Ivy, a lysozyme inhibitor from Escherichia coli, and characterisation of its specificity for various lysozymes. Enzyme Microb. Technol. 37, 205–211 (2005).<br />
<br />
[5] May, R. Turbidimetric Determination of Lysozyme with Micrococcus Cells : Reexamination of Reaction Conditions. 85, 77–85 (1983).<br />
<br />
[6] Maeda, H. A New Lysozyme Assay Based on Fluorescence Polarization or Fluorescence Intensity Utilizing a Fluorescent Peptidoglycan Substrate 1. 1191, 1185–1191 (1980).<br />
<br />
[7] Du, K., Porsch, P., Mahn, A., Brinkmann, O. & Gie, W. The non-enzymatic microbicidal activity of lysozymes. 449, 93–100 (1999).</div>Maexlichhttp://2014.igem.org/Team:Heidelberg/ProjectTeam:Heidelberg/Project2014-10-18T04:00:07Z<p>Maexlich: </p>
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<h4><img id="dropdownImg" src="/wiki/images/7/70/Heidelberg_Abstract-dropdown.png"/>&nbsp;Project overview</h4><br />
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<!--<div class="col-lg-9"><br />
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<div id="abstract-content" class="col-lg-12" ><br />
<p>Proteins are the functional basis of all biological processes and being able to control and improve their functions through design and engineering is one of the fundamental goals of Synthetic Biology. Protein splicing, a process in which a catalytically active internal polypeptide termed INTEIN excises itself from a precursor protein, has been used by our team to alter the functionalities of proteins in various ways post-translationally. <br />
<br></br><br />
We have developed a comprehensive <a href="/Team:Heidelberg/Project/Toolbox">toolbox</a> compromising five sets of <a href="/Team:Heidelberg/Parts">standardized parts</a> that provide standard mechanisms for specific modifications of proteins: <br />
(1) A <a href="/Team:Heidelberg/Project/Toolbox#Purification">purification</a> standard to faciliate purification of recombinant proteins. <br />
(2) A construct that allows for post-translational <a href="/Team:Heidelberg/Project/Toolbox#Fusion_and_Tagging">fusion</a> of protein domains to produce synthetic proteins<br />
(3) A standard that allows for switching proteins <a href="/Team:Heidelberg/Project/Toolbox#On_Off">on and off</a>.<br />
(4) One to <a href="/Team:Heidelberg/Project/Toolbox#Oligomerization">oligomerize</a> protein monomers.<br />
And (5) perhaps the most intriguing standard construct of this toolbox that was built to produce heat stable proteins, which can be achieved by <a href="/Team:Heidelberg/Toolbox/Circularization">circularization</a>.<br />
In order to offer a comfortable way to apply any of the above mechanisms to a protein and a biological system we designed a <a href="/Team:Heidelberg/Toolbox_Guide">toolbox guide</a> that provides step by step instructions for cloning based on the intein standard parts described in our <a href="/Team:Heidelberg/Parts/RFC">RFC</a>.<br />
<br></br><br />
Besides achieving a solid and broad foundation for future intein applications in synthetic biology, we aimed on deeply exploring one of the functions provided in our toolbox: circularization of proteins. In head to tail circularized peptides the terminal amino acids are joined together just like in the rest of the chain, forming a circular structure. Such peptides have been discovered in all kingdoms of life during the past years and they are unified by an extreme stability towards high temperatures, proteases and changes in pH.<br />
Synthetically connecting a protein's termini without disrupting its 3D structure and function is, however, a delicate task which has so far been accomplished only for relatively small proteins whose ends lie close to each other. With <a href="/Team:Heidelberg/Modeling/Linker_Modeling">CRAUT</a> we have brought into existence a powerful open-source software to predict an optimal rigid linker to support stabilizing circularization of a protein preserving its 3D structure and function. <br />
In order to provide us - and future iGEM teams - with a standard way to conduct the needed consuming modelling calculations we deployed a distributed computing platform we call <a href="/Team:Heidelberg/Human_Practice/igemathome">iGEM@home</a> that we also developed into a powerful science communication platform.<br />
As an evaluation of the linker software we screened linkers generated by the software by circularizing <a href="/Team:Heidelberg/Project/Linker_Screening">Lambda Lysozyme</a> and experimentally evaluating the heat stability of the products.<br />
<br></br><br />
Based on the calibrated software, we constructed linkers to circularize the 871 a.a. long <a href="/Team:Heidelberg/Project/PCR2.0">methyltransferase Dnmt1</a> and provide data suggesting that circular DNMT1 is more functional than its linear counterpart at high temperatures. Our results have strong implications for developing an innovative PCR-based technique that could revolutionize epigenetic studies and cancer research by maintaining the methylation pattern of the DNA template during amplification.<br />
Besides a protein with potential medical (lysozyme) and one with biotechnological (DNMT1) applications we chose the hemicellulose <a href="/Team:Heidelberg/Project/Xylanase">Xylanase</a> as a third target for circularization, that has applications in large scale production in paper and food industry.<br />
<br></br><br />
As an additional feature we decided to add another dimension to intein mediated modifications of our toolbox by developing optically induced small inteins that are photocaged by an <a href=”/Team:Heidelberg/Project/LOV”>As LOV2</a> domain. While sterically hampering intein dimerization in the dark, the LOV domain opens up when exposed to blue light, releasing the intein to proceed the splicing reaction.<br />
<br></br><br />
Setting new standards also for wiki documentation we created and developed a new way of documenting collaborative lab work by bringing the <a href="/Team:Heidelberg/Notebook">MidnightDoc</a> to life.<br />
<br></br><br />
Finally we complemented our already elaborate <a href="/Team:Heidelberg/Human_Practice">Human Practice</a> activities with two events on education and religion, philosophy and ethics in synthetic biology.<br />
<br />
<br />
<br />
</p><br />
</div><br />
</div><br />
<div class="graphicalAbstract"><br />
<div id="redOverlay"></div><br />
<div style="z-index:3;position:relative; height:100%;"><br />
<div id="linker-links"><br />
<a href="https://2014.igem.org/Team:Heidelberg/Software/igemathome" class="block box" id="calibrated"><br />
<img style="height:60px;display: inline-block;" src="/wiki/images/e/ea/Heidelberg_Project_Computer.png"><br />
<span style="position: relative;top: 10px;display: inline-block;"><br />
calibrated<br><br />
<span class="red-text">in silico</span></span><br />
</a><br />
<a href="https://2014.igem.org/Team:Heidelberg/Project/Linker_Screening" class="block box" id="screened"><br />
<span style="position: relative;top:10px;display: inline-block;"><br />
screened <span class="red-text">in vitro</span><br><br />
with lysozyme</span><br />
<img style="height:60px; display:inline-block;" src="/wiki/images/d/df/Heidelberg_Lysozyme.png" /><br />
</a><br />
<a href="/Team:Heidelberg/Software/Linker_Software" class="block box" style="bottom:0; left:-20px; position:absolute;width:170px;"><br />
<img src="/wiki/images/4/42/Craut_small.png" alt="..." style="width:100%;"/><br />
<span class="red-text" >circularize</span> it<br><br />
with calculated linkers<br />
</a><br />
</div><br />
<div class="ringbox"><br />
<img src="/wiki/images/0/0d/Heidelberg_Firering_red.png" id="ring-background" /><br />
<div id="circ-box" class="descr-box"><br />
<a href="/Team:Heidelberg/Toolbox/Circularization"><br />
<img src="/wiki/images/5/58/Heidelberg_Toolbox_Circularization.png" id="circ-icon" class="toolbox-icon toolbox-icon-scale"/><br />
</a><br />
<h3>CIRCULARIZATION</h3><br />
<div><span>Create a linker with our crowd computing software and make your protein heat stable</span></div><br />
</div><br />
<div id="oligo-box" class="descr-box"><a href="https://2014.igem.org/Team:Heidelberg/Project/Toolbox#Oligomerization"><img src="/wiki/images/4/40/Oligomerization.png" id="oligo-icon" class="toolbox-icon toolbox-icon-scale"/></a><h3>OLIGOMERIZATION</h3><div><span>Fuse multiple Proteins or Domains using Inteins</span></div><br />
</div><br />
<div id="fusion-box" class="descr-box"><a href="https://2014.igem.org/Team:Heidelberg/Project/Toolbox#Fusion_and_Tagging"><img src="/wiki/images/8/87/Heidelberg_Toolbox_Fusion.png" id="fusion-icon" class="toolbox-icon toolbox-icon-scale"/></a><h3>FUSION</h3><div><span>Fuse two Proteins or Domains together using Inteins</span></div><br />
</div><br />
<div id="onoff-box" class="descr-box"><a href="https://2014.igem.org/Team:Heidelberg/Project/Toolbox#On_Off"><img src="/wiki/images/c/c2/Heidelberg_Toolbox_On-Off.png" id="onoff-icon" class="toolbox-icon toolbox-icon-scale"/></a><h3>ON/OFF</h3><div><span>Activate or deactivate Proteins using Inteins</span></div></div><br />
<div id="purification-box" class="descr-box"><a href="https://2014.igem.org/Team:Heidelberg/Project/Toolbox#Purification"><img src="/wiki/images/0/04/Heidelberg_Toolbox_Purification.png" id="purification-icon" class="toolbox-icon toolbox-icon-scale"/></a><div><h3>PURIFICATION</h3></div><span>Placeholder</span></div><br />
<div id="toolbox-text"><br />
the intein<br><br />
<span><a class="scheisslinkbleibweiss" href="/Team:Heidelberg/Project/Toolbox">toolbox</a><span><br />
</div><br />
</div><br />
<a href="https://2014.igem.org/Team:Heidelberg/Toolbox/Induction" class="block" id="lightning"><br />
<img class="toolbox-icon-scale" style="height:110px;display: inline-block;" src="/wiki/images/8/83/Heidelberg_Project_Lightning.png"><span style="position: relative;top: 20px;display: inline-block;">&nbsp;inducible via<br><br />
<span style="font-weight:bold;position: relative;left: -18px;" class="red-text">light induction</span></span><br />
</a><br />
<a href="https://2014.igem.org/Team:Heidelberg/Project/PCR_2.0" class="block box" style="width:250px;" id="dnmt1-box"><br />
<img id="dnmt1-img" src="/wiki/images/a/a6/Heidelberg_Project_Dnmt1.png"><br />
<span style="position:relative; z-index:5;" class="block"><br />
<span style="font-size:1.5em;"><br />
<span class="red-text">Heat-stable</span><br> circular <br><span style="font-weight:bold;">DNA-<br/>Methyltransferase</span><br />
</span><br><br />
<span style="font-weight:bold;font-size: 2.5em; text-align:right;line-height: 35px;"><br />
<span class="red-text">PCR 2.0</span><br />
</span><br />
</span><br />
</a><br />
<a href="/Team:Heidelberg/Toolbox_Guide" class="block" id="toolbox"><br />
<span style="position:relative; display: inline-block;height:110px; width:110px; vertical-align: middle;"><br />
<img style="height:100%; position:absolute; top:0; left:0;" id="toolbox-img" src="/wiki/images/2/24/Heidelberg_Project_Toolbox_guide.png" /><br />
<img style="height:100%; position:absolute; top:0; left:0; display:none;" id="toolbox-img-hover" src="/wiki/images/4/4c/Heidelberg_Toolbox_guide_highlighted.png" /><br />
</span><br />
<span style="position: relative;top: 20px;display: inline-block; vertical-align:middle;"><br />
<span style="font-weight:bold;" class="red-text">modify your protein</span><br/><br />
using the toolbox guide<br />
</span><br />
</a><br />
<div class="clearfix"></div><br />
</div><br />
</div><br />
</div><br />
<div class="container" id="Achievements"><br />
<ul class="nav nav-tabs" role="tablist"><br />
<li class="active" style="font-size:30px"><a href="#Achievements-tab" role="tab" data-toggle="tab"><img src="https://static.igem.org/mediawiki/2014/2/22/Heidelberg_Achievements_red.png" height="50px" alt="Button"> Achievements</a></li><br />
<li style="font-size:30px"><a href="#Medal_Criteria-tab" role="tab" data-toggle="tab"> <img src="https://static.igem.org/mediawiki/2014/3/3f/Heidelberg_Gold_red.png" height="50px" alt="Button"> Medal Criteria</a></li><br />
</ul><br />
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</div><br />
<br />
<div class="col-md-9 col-xs-12 vcenter-cell"><br />
<div class="col-xs-9"><br />
<p style="font-weight:light; font-size:25px">Establishing protein circularization as a NEW BIOENGINEERING TOOL in synthetic biology.</p> <br />
<p style="margin-left:50px; font-size:20px">Contributing to iGEM with a new foundational advance!</p><br />
</div><br />
<br />
<div class="col-xs-3"><br />
<img src="/wiki/images/7/7d/Check_new.png" style="height:100px; margin-right:15px;"><br />
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</div><br />
</div><br />
<div class="linie"></div><br />
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<img src="https://static.igem.org/mediawiki/2014/1/11/Achievement_1.png" class="img-responsive" style="margin-left:15px;"><br />
</div><br />
<br />
<div class="col-md-9 col-xs-12 vcenter-cell"><br />
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<p style="font-weight:light; font-size:25px">Providing a NEW COMPREHENSIVE TOOLBOX based on inteins for modifying proteins post-translationally.</p> <br />
<p style="margin-left:50px; font-size:20px">Sending 67 Biobricks to Registry of Biological parts!</p><br />
</div><br />
<br />
<div class="col-xs-3"><br />
<img src="/wiki/images/7/7d/Check_new.png" style="height:100px; margin-right:15px;"><br />
</div><br />
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</div><br />
<div class="linie"></div><br />
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<img src="https://static.igem.org/mediawiki/2014/f/f9/Achievement_2.png" class="img-responsive" style="margin-left:15px;"> <br />
</div><br />
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<div class="col-md-9 col-xs-12 vcenter-cell"><br />
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<p style="font-weight:light; font-size:25px">Development of a NEW STANDARD to make the use of inteins easy and modular.</p> <br />
<p style="margin-left:50px; font-size:20px">Establishment of a new <a href="https://2014.igem.org/Team:Heidelberg/Parts/RFC">RFC</a>!</p><br />
</div><br />
<br />
<div class="col-xs-3"><br />
<img src="/wiki/images/7/7d/Check_new.png" style="height:100px; margin-right:15px;"><br />
</div><br />
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</div><br />
</div><br />
<div class="linie"></div><br />
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<div class="row vcenter-table" style="margin:0;"><br />
<div class="col-md-3 col-xs-12 vcenter-cell"><br />
<img src="https://static.igem.org/mediawiki/2014/1/1b/Achievements_toolbox.png" class="img-responsive" style="margin-left:15px;"> <br />
</div><br />
<br />
<div class="col-md-9 col-xs-12 vcenter-cell"><br />
<div class="col-xs-9"><br />
<p style="font-weight:light; font-size:25px">Showing that the toolbox WORKS: proteins are circularized and split fluorescent proteins are reconstituted.</p> <br />
<p style="margin-left:50px; font-size:20px">Making Gels, Western Blots, Fluorescence-based Assays and Mass spectrometry to prove it! </p><br />
</div><br />
<br />
<div class="col-xs-3"><br />
<img src="/wiki/images/7/7d/Check_new.png" style="height:100px; margin-right:15px;"><br />
</div><br />
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</div><br />
<div class="linie"></div><br />
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<img src="https://static.igem.org/mediawiki/2014/2/26/Achievements_4.png" class="img-responsive" style="margin-left:15px;"> <br />
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<div class="col-md-9 col-xs-12 vcenter-cell"><br />
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<p style="font-weight:light; font-size:25px">Creating circular DNMT1 and showing that it is ACTIVE.</p> <br />
<p style="margin-left:50px; font-size:20px">For the first time achieving the circularization of a large protein!</p><br />
<p style="margin-left:50px; font-size:20px">Circularizing LYSOZYME and XYLANASE, two very important proteins for<br />
research and industry!</p><br />
</div><br />
<br />
<div class="col-xs-3"><br />
<img src="/wiki/images/7/7d/Check_new.png" style="height:100px; margin-right:15px;"><br />
</div><br />
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</div><br />
</div><br />
<div class="linie"></div><br />
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<br />
<div class="col-md-9 col-xs-12 vcenter-cell"><br />
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<p style="font-weight:light; font-size:25px">Developing a NEW SOFTWARE to calculate customized linkers to circularize proteins.</p> <br />
<p style="margin-left:50px; font-size:20px">Making CRAUT open-source for the scientific community.</p><br />
</div><br />
<br />
<div class="col-xs-3"><br />
<img src="/wiki/images/7/7d/Check_new.png" style="height:100px; margin-right:15px;"><br />
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<br />
</div><br />
</div><br />
<div class="linie"></div><br />
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<div class="col-md-3 col-xs-12 vcenter-cell"><br />
<img src="https://static.igem.org/mediawiki/2014/e/e5/Heidelberg_Frontpage_igemathome.png" class="img-responsive" style="margin-left:15px;"> <br />
</div><br />
<br />
<div class="col-md-9 col-xs-12 vcenter-cell"><br />
<div class="col-xs-9"><br />
<p style="font-weight:light; font-size:25px"> Establishing a distributed computing platform called <a href="https://2014.igem.org/Team:Heidelberg/Software/igemathome">iGEM@home</a>.</p> <br />
<p style="margin-left:50px; font-size:20px">Using this platform as an entirely new way to reach out to the world with synthetic biology concepts!</p><br />
</div><br />
<br />
<br />
<div class="col-xs-3"><br />
<img src="/wiki/images/7/7d/Check_new.png" style="height:100px; margin-right:15px;"><br />
</div><br />
</div><br />
</div><br />
<div class="linie"></div><br />
<div class="row" style="margin:0;"><br />
<div class="col-md-3 col-xs-12 vcenter-cell"><br />
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</div><br />
<br />
<div class="col-md-9 col-xs-12 vcenter-cell"><br />
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<p style="font-weight:light; font-size:25px">Creating a NEW SOFTWARE to display the notebook on the wiki.</p> <br />
<p style="margin-left:50px; font-size:20px">Distributing MidNightDOC to the iGEM community to help future teams organize their protocols!</p><br />
</div><br />
<br />
<br />
<div class="col-xs-3"><br />
<img src="/wiki/images/7/7d/Check_new.png" style="height:100px; margin-right:15px;"><br />
</div><br />
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</div><br />
</div><br />
<br />
<div class="tab-pane fade" style="color:white;" id="Medal_Criteria-tab"><br />
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<div class="col-md-12 col-sm-12 col-xs-12" style="margin-top: 20px"><br />
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<div class="row "> <br />
<div class="col-md-3 col-xs-12"><br />
<img class="medal" src="https://static.igem.org/mediawiki/2014/6/63/Heidelberg_Bronze.png"><br />
</div><br />
<br />
<div class="col-md-9 col-xs-12"><br />
<h1>Bronze</h1><br />
<br><br />
<ul><br />
<li>Please find a comprehensive compilation of <a href="https://2014.igem.org/Team:Heidelberg/Team/Sponsoring">sponsors</a>, partners and scientific contributors on our <a href="https://2014.igem.org/Team:Heidelberg/Team/Attributions">acknowledgements page</a>. </li><br />
<br/><br />
<li>We also encourage you to take notice of the projects “Photo-intein” and “Mito-intein” by <a href="https://2014.igem.org/Team:Queens_Canada/Project">iGEM team Queens from Canada</a> that may supply you with complementary information and tools for the use of inteins in synthetic biology!</li><br />
<br/><br />
<li>A list of links to more than 60 parts in the registry submitted by our team (being or not being part of the new intein toolbox) can be found <a href="https://2014.igem.org/Team:Heidelberg/Parts#allParts">here</a>.</li><br />
</ul><br />
</div><br />
</div><br />
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<br />
<div class="col-md-9 col-xs-12"><br />
<h1>Silver</h1><br />
<br><br />
<ul><br />
<li>We experimentally validated that our biobricks <a href="http://parts.igem.org/Part:BBa_K1362000">BBa_K1362000</a>, <a href="http://parts.igem.org/Part:BBa_K1362100">BBa_K1362100 </a>and <a href="http://parts.igem.org/Part:BBa_K1362101">BBa_K1362101</a> work as expected. For more information on the <a href="https://2014.igem.org/Team:Heidelberg/Parts#Favorite Parts">parts</a> please visit the corresponding main pages in the parts registry or explore their involvement in our subprojects.</li><br />
<br/><br />
<li>Religious perceptions of synthetic biology have been part of several surveys during the past ten years of iGEM and Human Practices projects. Since religious groups cover the majority of worlds population, deliver moral values and wield power at the same time, we decided to dedicate a whole event on the topic of religion, philosophy and ethics regarding synthetic biology. Please find an <a href="https://2014.igem.org/Team:Heidelberg/Human_Practice/Ethics">evaluation of our event</a> on the corresponding Human Practices pages. <!--In order to reassure ourselves about the acceptability of our project and synthetic biology in general, we also used this opportunity to build up on the work of the iGEM Team Heidelberg 2013 and conducted a survey that addresses basic questions regarding the public reflection of our work.--><br />
</li><br />
</ul><br />
</div><br />
</div><br />
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<br />
<div class="col-md-9 col-xs-12"><br />
<h1>Gold</h1><br />
<ul><br />
<li>We improved the function of the <b>already existing</b> biobrick part <a href="http://parts.igem.org/Part:BBa_K117505">BBa_K1175005 </a>by optimizing and resubmitting the corresponding sequence of B. subtilis xylanase to the registry (Part:<a href="http://parts.igem.org/Part:BBa_K1362020"> BBa_K1362020</a>). In addition, we submitted a new part for expression of <a href="http://parts.igem.org/Part:BBa_K1362022">circularized xylanase </a>(BBa_K1362022) that might be used in future applications with need for refined enzyme stability.</li><br />
<br/><br />
<li>Despite the fact that we focused on building a set of powerful soft- and wetware tools to help future iGEM-teams developing and realizing projects in synthetic biology, we are happy to announce that we were also able to help out several team during the course of our project, aspecially with sending <a href="https://2014.igem.org/Team:Heidelberg/Parts#Backbones"> our expression vectors</a>. Read more abou in in our <a href="https://2014.igem.org/Team:Heidelberg/Team/Collaborations">Collaborations</a>.</li><br />
<br/><br />
<li>In the style of of the new iGEM community labs track that involves science amateurs “beyond the accolades of scientific publishing and economic reward”, we sought for a new way to involve laymen in actual science and build a strong community of well informed supporters and communicators of synthetic biology at the same time. Now we proudly present the crowd sourcing and communication platform <a href="https://2014.igem.org/Team:Heidelberg/Human_Practice/igemathome">iGEM@home</a>.</li><br />
</ul><br />
</div><br />
</div><br />
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<div class="col-lg-offset-1 col-lg-11"><br />
<h4><img id="dropdownImg" src="/wiki/images/7/70/Heidelberg_Abstract-dropdown.png"/>&nbsp;Project overview</h4><br />
</div><br />
<!--<div class="col-lg-9"><br />
<span>Click to read the project overview</span><br />
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<div id="abstract-content" class="col-lg-12" ><br />
<p>Proteins are the functional basis of all biological processes and being able to control and improve their functions through design and engineering is one of the fundamental goals of Synthetic Biology. Protein splicing, a process in which a catalytically active internal polypeptide termed INTEIN excises itself from a precursor protein, has been used by our team to alter the functionalities of proteins in various ways post-translationally. <br />
<br></br><br />
We have developed a comprehensive <a href="/Team:Heidelberg/Project/Toolbox">toolbox</a> compromising five sets of <a href="/Team:Heidelberg/Parts">standardized parts</a> that provide standard mechanisms for specific modifications of proteins: <br />
(1) A <a href="/Team:Heidelberg/Project/Toolbox#Purification">purification</a> standard to faciliate purification of recombinant proteins. <br />
(2) A construct that allows for post-translational <a href="/Team:Heidelberg/Project/Toolbox#Fusion_and_Tagging">fusion</a> of protein domains to produce synthetic proteins<br />
(3) A standard that allows for switching proteins <a href="/Team:Heidelberg/Project/Toolbox#On_Off">on and off</a>.<br />
(4) One to <a href="/Team:Heidelberg/Project/Toolbox#Oligomerization">oligomerize</a> protein monomers.<br />
And (5) perhaps the most intriguing standard construct of this toolbox that was built to produce heat stable proteins, which can be achieved by <a href="/Team:Heidelberg/Toolbox/Circularization">circularization</a>.<br />
In order to offer a comfortable way to apply any of the above mechanisms to a protein and a biological system we designed a <a href="/Team:Heidelberg/Toolbox_Guide">toolbox guide</a> that provides step by step instructions for cloning based on the intein standard parts described in our <a href="/Team:Heidelberg/Parts/RFC">RFC</a>.<br />
<br></br><br />
Besides achieving a solid and broad foundation for future intein applications in synthetic biology, we aimed on deeply exploring one of the functions provided in our toolbox: circularization of proteins. In head to tail circularized peptides the terminal amino acids are joined together just like in the rest of the chain, forming a circular structure. Such peptides have been discovered in all kingdoms of life during the past years and they are unified by an extreme stability towards high temperatures, proteases and changes in pH.<br />
Synthetically connecting a protein's termini without disrupting its 3D structure and function is, however, a delicate task which has so far been accomplished only for relatively small proteins whose ends lie close to each other. With <a href="/Team:Heidelberg/Modeling/Linker_Modeling">CRAUT</a> we have brought into existence a powerful open-source software to predict an optimal rigid linker to support stabilizing circularization of a protein preserving its 3D structure and function. <br />
In order to provide us - and future iGEM teams - with a standard way to conduct the needed consuming modelling calculations we deployed a distributed computing platform we call <a href="/Team:Heidelberg/Human_Practice/igemathome">iGEM@home</a> that we also developed into a powerful science communication platform.<br />
As an evaluation of the linker software we screened linkers generated by the software by circularizing <a href="/Team:Heidelberg/Project/Linker_Screening">Lambda Lysozyme</a> and experimentally evaluating the heat stability of the products.<br />
<br></br><br />
Based on the calibrated software, we constructed linkers to circularize the 871 a.a. long <a href="/Team:Heidelberg/Project/PCR2.0">methyltransferase Dnmt1</a> and provide data suggesting that circular DNMT1 is more functional than its linear counterpart at high temperatures. Our results have strong implications for developing an innovative PCR-based technique that could revolutionize epigenetic studies and cancer research by maintaining the methylation pattern of the DNA template during amplification.<br />
Besides a protein with potential medical (lysozyme) and one with biotechnological (DNMT1) applications we chose the hemicellulose <a href="/Team:Heidelberg/Project/Xylanase">Xylanase</a> as a third target for circularization, that has applications in large scale production in paper and food industry.<br />
<br></br><br />
As an additional feature we decided to add another dimension to intein mediated modifications of our toolbox by developing optically induced small inteins that are photocaged by an <a href=”/Team:Heidelberg/Project/LOV”>As LOV2</a> domain. While sterically hampering intein dimerization in the dark, the LOV domain opens up when exposed to blue light, releasing the intein to proceed the splicing reaction.<br />
<br></br><br />
Setting new standards also for wiki documentation we created and developed a new way of documenting collaborative lab work by bringing the <a href="/Team:Heidelberg/Notebook">MidnightDoc</a> to life.<br />
<br></br><br />
Finally we complemented our already elaborate Human Practice activities with two events on education and religion, philosophy and ethics in synthetic biology.<br />
<br />
<br />
<br />
</p><br />
</div><br />
</div><br />
<div class="graphicalAbstract"><br />
<div id="redOverlay"></div><br />
<div style="z-index:3;position:relative; height:100%;"><br />
<div id="linker-links"><br />
<a href="https://2014.igem.org/Team:Heidelberg/Software/igemathome" class="block box" id="calibrated"><br />
<img style="height:60px;display: inline-block;" src="/wiki/images/e/ea/Heidelberg_Project_Computer.png"><br />
<span style="position: relative;top: 10px;display: inline-block;"><br />
calibrated<br><br />
<span class="red-text">in silico</span></span><br />
</a><br />
<a href="https://2014.igem.org/Team:Heidelberg/Project/Linker_Screening" class="block box" id="screened"><br />
<span style="position: relative;top:10px;display: inline-block;"><br />
screened <span class="red-text">in vitro</span><br><br />
with lysozyme</span><br />
<img style="height:60px; display:inline-block;" src="/wiki/images/d/df/Heidelberg_Lysozyme.png" /><br />
</a><br />
<a href="/Team:Heidelberg/Software/Linker_Software" class="block box" style="bottom:0; left:-20px; position:absolute;width:170px;"><br />
<img src="/wiki/images/4/42/Craut_small.png" alt="..." style="width:100%;"/><br />
<span class="red-text" >circularize</span> it<br><br />
with calculated linkers<br />
</a><br />
</div><br />
<div class="ringbox"><br />
<img src="/wiki/images/0/0d/Heidelberg_Firering_red.png" id="ring-background" /><br />
<div id="circ-box" class="descr-box"><br />
<a href="/Team:Heidelberg/Toolbox/Circularization"><br />
<img src="/wiki/images/5/58/Heidelberg_Toolbox_Circularization.png" id="circ-icon" class="toolbox-icon toolbox-icon-scale"/><br />
</a><br />
<h3>CIRCULARIZATION</h3><br />
<div><span>Create a linker with our crowd computing software and make your protein heat stable</span></div><br />
</div><br />
<div id="oligo-box" class="descr-box"><a href="https://2014.igem.org/Team:Heidelberg/Project/Toolbox#Oligomerization"><img src="/wiki/images/4/40/Oligomerization.png" id="oligo-icon" class="toolbox-icon toolbox-icon-scale"/></a><h3>OLIGOMERIZATION</h3><div><span>Fuse multiple Proteins or Domains using Inteins</span></div><br />
</div><br />
<div id="fusion-box" class="descr-box"><a href="https://2014.igem.org/Team:Heidelberg/Project/Toolbox#Fusion_and_Tagging"><img src="/wiki/images/8/87/Heidelberg_Toolbox_Fusion.png" id="fusion-icon" class="toolbox-icon toolbox-icon-scale"/></a><h3>FUSION</h3><div><span>Fuse two Proteins or Domains together using Inteins</span></div><br />
</div><br />
<div id="onoff-box" class="descr-box"><a href="https://2014.igem.org/Team:Heidelberg/Project/Toolbox#On_Off"><img src="/wiki/images/c/c2/Heidelberg_Toolbox_On-Off.png" id="onoff-icon" class="toolbox-icon toolbox-icon-scale"/></a><h3>ON/OFF</h3><div><span>Activate or deactivate Proteins using Inteins</span></div></div><br />
<div id="purification-box" class="descr-box"><a href="https://2014.igem.org/Team:Heidelberg/Project/Toolbox#Purification"><img src="/wiki/images/0/04/Heidelberg_Toolbox_Purification.png" id="purification-icon" class="toolbox-icon toolbox-icon-scale"/></a><div><h3>PURIFICATION</h3></div><span>Placeholder</span></div><br />
<div id="toolbox-text"><br />
the intein<br><br />
<span><a class="scheisslinkbleibweiss" href="/Team:Heidelberg/Project/Toolbox">toolbox</a><span><br />
</div><br />
</div><br />
<a href="https://2014.igem.org/Team:Heidelberg/Toolbox/Induction" class="block" id="lightning"><br />
<img class="toolbox-icon-scale" style="height:110px;display: inline-block;" src="/wiki/images/8/83/Heidelberg_Project_Lightning.png"><span style="position: relative;top: 20px;display: inline-block;">&nbsp;inducible via<br><br />
<span style="font-weight:bold;position: relative;left: -18px;" class="red-text">light induction</span></span><br />
</a><br />
<a href="https://2014.igem.org/Team:Heidelberg/Project/PCR_2.0" class="block box" style="width:250px;" id="dnmt1-box"><br />
<img id="dnmt1-img" src="/wiki/images/a/a6/Heidelberg_Project_Dnmt1.png"><br />
<span style="position:relative; z-index:5;" class="block"><br />
<span style="font-size:1.5em;"><br />
<span class="red-text">Heat-stable</span><br> circular <br><span style="font-weight:bold;">DNA-<br/>Methyltransferase</span><br />
</span><br><br />
<span style="font-weight:bold;font-size: 2.5em; text-align:right;line-height: 35px;"><br />
<span class="red-text">PCR 2.0</span><br />
</span><br />
</span><br />
</a><br />
<a href="/Team:Heidelberg/Toolbox_Guide" class="block" id="toolbox"><br />
<span style="position:relative; display: inline-block;height:110px; width:110px; vertical-align: middle;"><br />
<img style="height:100%; position:absolute; top:0; left:0;" id="toolbox-img" src="/wiki/images/2/24/Heidelberg_Project_Toolbox_guide.png" /><br />
<img style="height:100%; position:absolute; top:0; left:0; display:none;" id="toolbox-img-hover" src="/wiki/images/4/4c/Heidelberg_Toolbox_guide_highlighted.png" /><br />
</span><br />
<span style="position: relative;top: 20px;display: inline-block; vertical-align:middle;"><br />
<span style="font-weight:bold;" class="red-text">modify your protein</span><br/><br />
using the toolbox guide<br />
</span><br />
</a><br />
<div class="clearfix"></div><br />
</div><br />
</div><br />
</div><br />
<div class="container" id="Achievements"><br />
<ul class="nav nav-tabs" role="tablist"><br />
<li class="active" style="font-size:30px"><a href="#Achievements-tab" role="tab" data-toggle="tab"><img src="https://static.igem.org/mediawiki/2014/2/22/Heidelberg_Achievements_red.png" height="50px" alt="Button"> Achievements</a></li><br />
<li style="font-size:30px"><a href="#Medal_Criteria-tab" role="tab" data-toggle="tab"> <img src="https://static.igem.org/mediawiki/2014/3/3f/Heidelberg_Gold_red.png" height="50px" alt="Button"> Medal Criteria</a></li><br />
</ul><br />
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<br />
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</div><br />
<div class="row" style="margin:0;"><br />
<div class="col-md-3 col-xs-12 vcenter-cell"><br />
<img src="https://static.igem.org/mediawiki/2014/a/ae/Achievements_3.png" class="img-responsive" style="margin-left:15px;"><br />
</div><br />
<br />
<div class="col-md-9 col-xs-12 vcenter-cell"><br />
<div class="col-xs-9"><br />
<p style="font-weight:light; font-size:25px">Establishing protein circularization as a NEW BIOENGINEERING TOOL in synthetic biology.</p> <br />
<p style="margin-left:50px; font-size:20px">Contributing to iGEM with a new foundational advance!</p><br />
</div><br />
<br />
<div class="col-xs-3"><br />
<img src="/wiki/images/7/7d/Check_new.png" style="height:100px; margin-right:15px;"><br />
</div><br />
<br />
</div><br />
</div><br />
<div class="linie"></div><br />
<br />
<div class="row" style="margin:0;"><br />
<div class="col-md-3 col-xs-12 vcenter-cell"><br />
<img src="https://static.igem.org/mediawiki/2014/1/11/Achievement_1.png" class="img-responsive" style="margin-left:15px;"><br />
</div><br />
<br />
<div class="col-md-9 col-xs-12 vcenter-cell"><br />
<div class="col-xs-9"><br />
<p style="font-weight:light; font-size:25px">Providing a NEW COMPREHENSIVE TOOLBOX based on inteins for modifying proteins post-translationally.</p> <br />
<p style="margin-left:50px; font-size:20px">Sending 67 Biobricks to Registry of Biological parts!</p><br />
</div><br />
<br />
<div class="col-xs-3"><br />
<img src="/wiki/images/7/7d/Check_new.png" style="height:100px; margin-right:15px;"><br />
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<br />
<div class="col-md-9 col-xs-12 vcenter-cell"><br />
<div class="col-xs-9"><br />
<p style="font-weight:light; font-size:25px">Development of a NEW STANDARD to make the use of inteins easy and modular.</p> <br />
<p style="margin-left:50px; font-size:20px">Establishment of a new <a href="https://2014.igem.org/Team:Heidelberg/Parts/RFC">RFC</a>!</p><br />
</div><br />
<br />
<div class="col-xs-3"><br />
<img src="/wiki/images/7/7d/Check_new.png" style="height:100px; margin-right:15px;"><br />
</div><br />
<br />
</div><br />
</div><br />
<div class="linie"></div><br />
<br />
<br />
<div class="row vcenter-table" style="margin:0;"><br />
<div class="col-md-3 col-xs-12 vcenter-cell"><br />
<img src="https://static.igem.org/mediawiki/2014/1/1b/Achievements_toolbox.png" class="img-responsive" style="margin-left:15px;"> <br />
</div><br />
<br />
<div class="col-md-9 col-xs-12 vcenter-cell"><br />
<div class="col-xs-9"><br />
<p style="font-weight:light; font-size:25px">Showing that the toolbox WORKS: proteins are circularized and split fluorescent proteins are reconstituted.</p> <br />
<p style="margin-left:50px; font-size:20px">Making Gels, Western Blots, Fluorescence-based Assays and Mass spectrometry to prove it! </p><br />
</div><br />
<br />
<div class="col-xs-3"><br />
<img src="/wiki/images/7/7d/Check_new.png" style="height:100px; margin-right:15px;"><br />
</div><br />
<br />
</div><br />
</div><br />
<div class="linie"></div><br />
<br />
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<div class="col-md-3 col-xs-12 vcenter-cell"><br />
<img src="https://static.igem.org/mediawiki/2014/2/26/Achievements_4.png" class="img-responsive" style="margin-left:15px;"> <br />
</div><br />
<br />
<div class="col-md-9 col-xs-12 vcenter-cell"><br />
<div class="col-xs-9"><br />
<p style="font-weight:light; font-size:25px">Creating circular DNMT1 and showing that it is ACTIVE.</p> <br />
<p style="margin-left:50px; font-size:20px">For the first time achieving the circularization of a large protein!</p><br />
<p style="margin-left:50px; font-size:20px">Circularizing LYSOZYME and XYLANASE, two very important proteins for<br />
research and industry!</p><br />
</div><br />
<br />
<div class="col-xs-3"><br />
<img src="/wiki/images/7/7d/Check_new.png" style="height:100px; margin-right:15px;"><br />
</div><br />
<br />
</div><br />
</div><br />
<div class="linie"></div><br />
<br />
<div class="row vcenter-table" style="margin:0;"><br />
<div class="col-md-3 col-xs-12 vcenter-cell"><br />
<img src="https://static.igem.org/mediawiki/2014/5/5a/Achievements_craut.png" class="img-responsive" style="margin-left:15px;"> <br />
</div><br />
<br />
<div class="col-md-9 col-xs-12 vcenter-cell"><br />
<div class="col-xs-9"><br />
<p style="font-weight:light; font-size:25px">Developing a NEW SOFTWARE to calculate customized linkers to circularize proteins.</p> <br />
<p style="margin-left:50px; font-size:20px">Making CRAUT open-source for the scientific community.</p><br />
</div><br />
<br />
<div class="col-xs-3"><br />
<img src="/wiki/images/7/7d/Check_new.png" style="height:100px; margin-right:15px;"><br />
</div><br />
<br />
</div><br />
</div><br />
<div class="linie"></div><br />
<br />
<div class="row vcenter-table" style="margin:0;"><br />
<div class="col-md-3 col-xs-12 vcenter-cell"><br />
<img src="https://static.igem.org/mediawiki/2014/e/e5/Heidelberg_Frontpage_igemathome.png" class="img-responsive" style="margin-left:15px;"> <br />
</div><br />
<br />
<div class="col-md-9 col-xs-12 vcenter-cell"><br />
<div class="col-xs-9"><br />
<p style="font-weight:light; font-size:25px"> Establishing a distributed computing platform called <a href="https://2014.igem.org/Team:Heidelberg/Software/igemathome">iGEM@home</a>.</p> <br />
<p style="margin-left:50px; font-size:20px">Using this platform as an entirely new way to reach out to the world with synthetic biology concepts!</p><br />
</div><br />
<br />
<br />
<div class="col-xs-3"><br />
<img src="/wiki/images/7/7d/Check_new.png" style="height:100px; margin-right:15px;"><br />
</div><br />
</div><br />
</div><br />
<div class="linie"></div><br />
<div class="row" style="margin:0;"><br />
<div class="col-md-3 col-xs-12 vcenter-cell"><br />
<img src="https://static.igem.org/mediawiki/2014/1/18/Achievements_md.png" class="img-responsive" style="margin-left:15px;"> <br />
</div><br />
<br />
<div class="col-md-9 col-xs-12 vcenter-cell"><br />
<div class="col-xs-9"><br />
<p style="font-weight:light; font-size:25px">Creating a NEW SOFTWARE to display the notebook on the wiki.</p> <br />
<p style="margin-left:50px; font-size:20px">Distributing MidNightDOC to the iGEM community to help future teams organize their protocols!</p><br />
</div><br />
<br />
<br />
<div class="col-xs-3"><br />
<img src="/wiki/images/7/7d/Check_new.png" style="height:100px; margin-right:15px;"><br />
</div><br />
<br />
</div><br />
</div><br />
</div><br />
<br />
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<br />
<div class="col-md-12 col-sm-12 col-xs-12" style="margin-top: 20px"><br />
</div><br />
<div class="row "> <br />
<div class="col-md-3 col-xs-12"><br />
<img class="medal" src="https://static.igem.org/mediawiki/2014/6/63/Heidelberg_Bronze.png"><br />
</div><br />
<br />
<div class="col-md-9 col-xs-12"><br />
<h1>Bronze</h1><br />
<br><br />
<ul><br />
<li>Please find a comprehensive compilation of <a href="https://2014.igem.org/Team:Heidelberg/Team/Sponsoring">sponsors</a>, partners and scientific contributors on our <a href="https://2014.igem.org/Team:Heidelberg/Team/Attributions">acknowledgements page</a>. </li><br />
<br/><br />
<li>We also encourage you to take notice of the projects “Photo-intein” and “Mito-intein” by <a href="https://2014.igem.org/Team:Queens_Canada/Project">iGEM team Queens from Canada</a> that may supply you with complementary information and tools for the use of inteins in synthetic biology!</li><br />
<br/><br />
<li>A list of links to more than 60 parts in the registry submitted by our team (being or not being part of the new intein toolbox) can be found <a href="https://2014.igem.org/Team:Heidelberg/Parts#allParts">here</a>.</li><br />
</ul><br />
</div><br />
</div><br />
<br />
<div class="row "><br />
<div class="col-md-3 col-xs-12"><br />
<img class="medal" src="https://static.igem.org/mediawiki/2014/b/bf/Heidelberg_Silver.png"><br />
</div><br />
<br />
<div class="col-md-9 col-xs-12"><br />
<h1>Silver</h1><br />
<br><br />
<ul><br />
<li>We experimentally validated that our biobricks <a href="http://parts.igem.org/Part:BBa_K1362000">BBa_K1362000</a>, <a href="http://parts.igem.org/Part:BBa_K1362100">BBa_K1362100 </a>and <a href="http://parts.igem.org/Part:BBa_K1362101">BBa_K1362101</a> work as expected. For more information on the <a href="https://2014.igem.org/Team:Heidelberg/Parts#Favorite Parts">parts</a> please visit the corresponding main pages in the parts registry or explore their involvement in our subprojects.</li><br />
<br/><br />
<li>Religious perceptions of synthetic biology have been part of several surveys during the past ten years of iGEM and Human Practices projects. Since religious groups cover the majority of worlds population, deliver moral values and wield power at the same time, we decided to dedicate a whole event on the topic of religion, philosophy and ethics regarding synthetic biology. Please find an <a href="https://2014.igem.org/Team:Heidelberg/Human_Practice/Ethics">evaluation of our event</a> on the corresponding Human Practices pages. <!--In order to reassure ourselves about the acceptability of our project and synthetic biology in general, we also used this opportunity to build up on the work of the iGEM Team Heidelberg 2013 and conducted a survey that addresses basic questions regarding the public reflection of our work.--><br />
</li><br />
</ul><br />
</div><br />
</div><br />
<br />
<div class="row"><br />
<div class="col-md-3 col-xs-12"><br />
<img class="medal" src="https://static.igem.org/mediawiki/2014/0/0a/Heidelberg_Gold.png"><br />
</div><br />
<br />
<div class="col-md-9 col-xs-12"><br />
<h1>Gold</h1><br />
<ul><br />
<li>We improved the function of the <b>already existing</b> biobrick part <a href="http://parts.igem.org/Part:BBa_K117505">BBa_K1175005 </a>by optimizing and resubmitting the corresponding sequence of B. subtilis xylanase to the registry (Part:<a href="http://parts.igem.org/Part:BBa_K1362020"> BBa_K1362020</a>). In addition, we submitted a new part for expression of <a href="http://parts.igem.org/Part:BBa_K1362022">circularized xylanase </a>(BBa_K1362022) that might be used in future applications with need for refined enzyme stability.</li><br />
<br/><br />
<li>Despite the fact that we focused on building a set of powerful soft- and wetware tools to help future iGEM-teams developing and realizing projects in synthetic biology, we are happy to announce that we were also able to help out several team during the course of our project, aspecially with sending <a href="https://2014.igem.org/Team:Heidelberg/Parts#Backbones"> our expression vectors</a>. Read more abou in in our <a href="https://2014.igem.org/Team:Heidelberg/Team/Collaborations">Collaborations</a>.</li><br />
<br/><br />
<li>In the style of of the new iGEM community labs track that involves science amateurs “beyond the accolades of scientific publishing and economic reward”, we sought for a new way to involve laymen in actual science and build a strong community of well informed supporters and communicators of synthetic biology at the same time. Now we proudly present the crowd sourcing and communication platform <a href="https://2014.igem.org/Team:Heidelberg/Human_Practice/igemathome">iGEM@home</a>.</li><br />
</ul><br />
</div><br />
</div><br />
<br />
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<h4><img id="dropdownImg" src="/wiki/images/7/70/Heidelberg_Abstract-dropdown.png"/>&nbsp;Project overview</h4><br />
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<div id="abstract-content" class="col-lg-12" ><br />
<p>Proteins are the functional basis of all biological processes and being able to control and improve their functions through design and engineering is one of the fundamental goals of Synthetic Biology. Protein splicing, a process in which a catalytically active internal polypeptide termed INTEIN excises itself from a precursor protein, has been used by our team to alter the functionalities of proteins in various ways post-translationally. <br />
<br></br><br />
We have developed a comprehensive <a href="/Team:Heidelberg/Project/Toolbox">toolbox</a> compromising five sets of <a href="/Team:Heidelberg/Parts">standardized parts</a> that provide standard mechanisms for specific modifications of proteins: <br />
(1) A <a href="/Team:Heidelberg/Project/Toolbox#Purification">purification</a> standard to faciliate purification of recombinant proteins. <br />
(2) A construct that allows for post-translational <a href="/Team:Heidelberg/Project/Toolbox#Fusion_and_Tagging">fusion</a> of protein domains to produce synthetic proteins<br />
(3) A standard that allows for switching proteins <a href="/Team:Heidelberg/Project/Toolbox#On_Off">on and off</a>.<br />
(4) One to <a href="/Team:Heidelberg/Project/Toolbox#Oligomerization">oligomerize</a> protein monomers.<br />
And (5) perhaps the most intriguing standard construct of this toolbox that was built to produce heat stable proteins, which can be achieved by <a href="/Team:Heidelberg/Toolbox/Circularization">circularization</a>.<br />
In order to offer a comfortable way to apply any of the above mechanisms to a protein and a biological system we designed a <a href="/Team:Heidelberg/Toolbox_Guide">toolbox guide</a> that provides step by step instructions for cloning based on the intein standard parts described in our <a href="/Team:Heidelberg/Parts/RFC">RFC</a>.<br />
<br></br><br />
Besides achieving a solid and broad foundation for future intein applications in synthetic biology, we aimed on deeply exploring one of the functions provided in our toolbox: circularization of proteins. In head to tail circularized peptides the terminal amino acids are joined together just like in the rest of the chain, forming a circular structure. Such peptides have been discovered in all kingdoms of life during the past years and they are unified by an extreme stability towards high temperatures, proteases and changes in pH.<br />
Synthetically connecting a protein's termini without disrupting its 3D structure and function is, however, a delicate task which has so far been accomplished only for relatively small proteins whose ends lie close to each other. With <a href="/Team:Heidelberg/Modeling/Linker_Modeling">CRAUT</a> we have brought into existence a powerful open-source software to predict an optimal rigid linker to support stabilizing circularization of a protein preserving its 3D structure and function. <br />
In order to provide us - and future iGEM teams - with a standard way to conduct the needed consuming modelling calculations we deployed a distributed computing platform we call <a href="/Team:Heidelberg/Human_Practice/igemathome">iGEM@home</a> that we also developed into a powerful science communication platform.<br />
As an evaluation of the linker software we screened linkers generated by the software by circularizing <a href="/Team:Heidelberg/Project/Linker_Screening">Lambda Lysozyme</a> and experimentally evaluating the heat stability of the products.<br />
<br></br><br />
Based on the calibrated software, we constructed linkers to circularize the 871 a.a. long <a href="/Team:Heidelberg/Project/PCR2.0">methyltransferase Dnmt1</a> and provide data suggesting that circular DNMT1 is more functional than its linear counterpart at high temperatures. Our results have strong implications for developing an innovative PCR-based technique that could revolutionize epigenetic studies and cancer research by maintaining the methylation pattern of the DNA template during amplification.<br />
Besides a protein with potential medical (lysozyme) and one with biotechnological (DNMT1) applications we chose the hemicellulose <a href="/Team:Heidelberg/Project/Xylanase">Xylanase</a> as a third target for circularization, that has applications in large scale production in paper and food industry.<br />
<br></br><br />
As an additional feature we decided to add another dimension to intein mediated modifications of our toolbox by developing optically induced small inteins that are photocaged by an <a href=”/Team:Heidelberg/Project/LOV”>As LOV2</a> domain. While sterically hampering intein dimerization in the dark, the LOV domain opens up when exposed to blue light, releasing the intein to proceed the splicing reaction.<br />
<br></br><br />
Setting new standards also for wiki documentation we created and developed a new way of documenting collaborative lab work by bringing the MidnightDoc to life.<br />
<br></br><br />
Finally we complemented our already elaborate Human Practice activities with two events on education and religion, philosophy and ethics in synthetic biology.<br />
<br />
<br />
<br />
</p><br />
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<div class="graphicalAbstract"><br />
<div id="redOverlay"></div><br />
<div style="z-index:3;position:relative; height:100%;"><br />
<div id="linker-links"><br />
<a href="https://2014.igem.org/Team:Heidelberg/Software/igemathome" class="block box" id="calibrated"><br />
<img style="height:60px;display: inline-block;" src="/wiki/images/e/ea/Heidelberg_Project_Computer.png"><br />
<span style="position: relative;top: 10px;display: inline-block;"><br />
calibrated<br><br />
<span class="red-text">in silico</span></span><br />
</a><br />
<a href="https://2014.igem.org/Team:Heidelberg/Project/Linker_Screening" class="block box" id="screened"><br />
<span style="position: relative;top:10px;display: inline-block;"><br />
screened <span class="red-text">in vitro</span><br><br />
with lysozyme</span><br />
<img style="height:60px; display:inline-block;" src="/wiki/images/d/df/Heidelberg_Lysozyme.png" /><br />
</a><br />
<a href="/Team:Heidelberg/Software/Linker_Software" class="block box" style="bottom:0; left:-20px; position:absolute;width:170px;"><br />
<img src="/wiki/images/4/42/Craut_small.png" alt="..." style="width:100%;"/><br />
<span class="red-text" >circularize</span> it<br><br />
with calculated linkers<br />
</a><br />
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<div class="ringbox"><br />
<img src="/wiki/images/0/0d/Heidelberg_Firering_red.png" id="ring-background" /><br />
<div id="circ-box" class="descr-box"><br />
<a href="/Team:Heidelberg/Toolbox/Circularization"><br />
<img src="/wiki/images/5/58/Heidelberg_Toolbox_Circularization.png" id="circ-icon" class="toolbox-icon toolbox-icon-scale"/><br />
</a><br />
<h3>CIRCULARIZATION</h3><br />
<div><span>Create a linker with our crowd computing software and make your protein heat stable</span></div><br />
</div><br />
<div id="oligo-box" class="descr-box"><a href="https://2014.igem.org/Team:Heidelberg/Project/Toolbox#Oligomerization"><img src="/wiki/images/4/40/Oligomerization.png" id="oligo-icon" class="toolbox-icon toolbox-icon-scale"/></a><h3>OLIGOMERIZATION</h3><div><span>Fuse multiple Proteins or Domains using Inteins</span></div><br />
</div><br />
<div id="fusion-box" class="descr-box"><a href="https://2014.igem.org/Team:Heidelberg/Project/Toolbox#Fusion_and_Tagging"><img src="/wiki/images/8/87/Heidelberg_Toolbox_Fusion.png" id="fusion-icon" class="toolbox-icon toolbox-icon-scale"/></a><h3>FUSION</h3><div><span>Fuse two Proteins or Domains together using Inteins</span></div><br />
</div><br />
<div id="onoff-box" class="descr-box"><a href="https://2014.igem.org/Team:Heidelberg/Project/Toolbox#On_Off"><img src="/wiki/images/c/c2/Heidelberg_Toolbox_On-Off.png" id="onoff-icon" class="toolbox-icon toolbox-icon-scale"/></a><h3>ON/OFF</h3><div><span>Activate or deactivate Proteins using Inteins</span></div></div><br />
<div id="purification-box" class="descr-box"><a href="https://2014.igem.org/Team:Heidelberg/Project/Toolbox#Purification"><img src="/wiki/images/0/04/Heidelberg_Toolbox_Purification.png" id="purification-icon" class="toolbox-icon toolbox-icon-scale"/></a><div><h3>PURIFICATION</h3></div><span>Placeholder</span></div><br />
<div id="toolbox-text"><br />
the intein<br><br />
<span><a class="scheisslinkbleibweiss" href="/Team:Heidelberg/Project/Toolbox">toolbox</a><span><br />
</div><br />
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<a href="https://2014.igem.org/Team:Heidelberg/Toolbox/Induction" class="block" id="lightning"><br />
<img class="toolbox-icon-scale" style="height:110px;display: inline-block;" src="/wiki/images/8/83/Heidelberg_Project_Lightning.png"><span style="position: relative;top: 20px;display: inline-block;">&nbsp;inducible via<br><br />
<span style="font-weight:bold;position: relative;left: -18px;" class="red-text">light induction</span></span><br />
</a><br />
<a href="https://2014.igem.org/Team:Heidelberg/Project/PCR_2.0" class="block box" style="width:250px;" id="dnmt1-box"><br />
<img id="dnmt1-img" src="/wiki/images/a/a6/Heidelberg_Project_Dnmt1.png"><br />
<span style="position:relative; z-index:5;" class="block"><br />
<span style="font-size:1.5em;"><br />
<span class="red-text">Heat-stable</span><br> circular <br><span style="font-weight:bold;">DNA-<br/>Methyltransferase</span><br />
</span><br><br />
<span style="font-weight:bold;font-size: 2.5em; text-align:right;line-height: 35px;"><br />
<span class="red-text">PCR 2.0</span><br />
</span><br />
</span><br />
</a><br />
<a href="/Team:Heidelberg/Toolbox_Guide" class="block" id="toolbox"><br />
<span style="position:relative; display: inline-block;height:110px; width:110px; vertical-align: middle;"><br />
<img style="height:100%; position:absolute; top:0; left:0;" id="toolbox-img" src="/wiki/images/2/24/Heidelberg_Project_Toolbox_guide.png" /><br />
<img style="height:100%; position:absolute; top:0; left:0; display:none;" id="toolbox-img-hover" src="/wiki/images/4/4c/Heidelberg_Toolbox_guide_highlighted.png" /><br />
</span><br />
<span style="position: relative;top: 20px;display: inline-block; vertical-align:middle;"><br />
<span style="font-weight:bold;" class="red-text">modify your protein</span><br/><br />
using the toolbox guide<br />
</span><br />
</a><br />
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<div class="container" id="Achievements"><br />
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<li class="active" style="font-size:30px"><a href="#Achievements-tab" role="tab" data-toggle="tab"><img src="https://static.igem.org/mediawiki/2014/2/22/Heidelberg_Achievements_red.png" height="50px" alt="Button"> Achievements</a></li><br />
<li style="font-size:30px"><a href="#Medal_Criteria-tab" role="tab" data-toggle="tab"> <img src="https://static.igem.org/mediawiki/2014/3/3f/Heidelberg_Gold_red.png" height="50px" alt="Button"> Medal Criteria</a></li><br />
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<p style="font-weight:light; font-size:25px">Establishing protein circularization as a NEW BIOENGINEERING TOOL in synthetic biology.</p> <br />
<p style="margin-left:50px; font-size:20px">Contributing to iGEM with a new foundational advance!</p><br />
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<img src="/wiki/images/7/7d/Check_new.png" style="height:100px; margin-right:15px;"><br />
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<p style="font-weight:light; font-size:25px">Providing a NEW COMPREHENSIVE TOOLBOX based on inteins for modifying proteins post-translationally.</p> <br />
<p style="margin-left:50px; font-size:20px">Sending 67 Biobricks to Registry of Biological parts!</p><br />
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<p style="font-weight:light; font-size:25px">Development of a NEW STANDARD to make the use of inteins easy and modular.</p> <br />
<p style="margin-left:50px; font-size:20px">Establishment of a new <a href="https://2014.igem.org/Team:Heidelberg/Parts/RFC">RFC</a>!</p><br />
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<img src="/wiki/images/7/7d/Check_new.png" style="height:100px; margin-right:15px;"><br />
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<p style="font-weight:light; font-size:25px">Showing that the toolbox WORKS: proteins are circularized and split fluorescent proteins are reconstituted.</p> <br />
<p style="margin-left:50px; font-size:20px">Making Gels, Western Blots, Fluorescence-based Assays and Mass spectrometry to prove it! </p><br />
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<img src="/wiki/images/7/7d/Check_new.png" style="height:100px; margin-right:15px;"><br />
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<p style="font-weight:light; font-size:25px">Creating circular DNMT1 and showing that it is ACTIVE.</p> <br />
<p style="margin-left:50px; font-size:20px">For the first time achieving the circularization of a large protein!</p><br />
<p style="margin-left:50px; font-size:20px">Circularizing LYSOZYME and XYLANASE, two very important proteins for<br />
research and industry!</p><br />
</div><br />
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<img src="/wiki/images/7/7d/Check_new.png" style="height:100px; margin-right:15px;"><br />
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<p style="font-weight:light; font-size:25px">Developing a NEW SOFTWARE to calculate customized linkers to circularize proteins.</p> <br />
<p style="margin-left:50px; font-size:20px">Making CRAUT open-source for the scientific community.</p><br />
</div><br />
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<img src="/wiki/images/7/7d/Check_new.png" style="height:100px; margin-right:15px;"><br />
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<img src="https://static.igem.org/mediawiki/2014/e/e5/Heidelberg_Frontpage_igemathome.png" class="img-responsive" style="margin-left:15px;"> <br />
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<p style="font-weight:light; font-size:25px"> Establishing a distributed computing platform called <a href="https://2014.igem.org/Team:Heidelberg/Software/igemathome">iGEM@home</a>.</p> <br />
<p style="margin-left:50px; font-size:20px">Using this platform as an entirely new way to reach out to the world with synthetic biology concepts!</p><br />
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<img src="/wiki/images/7/7d/Check_new.png" style="height:100px; margin-right:15px;"><br />
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<p style="font-weight:light; font-size:25px">Creating a NEW SOFTWARE to display the notebook on the wiki.</p> <br />
<p style="margin-left:50px; font-size:20px">Distributing MidNightDOC to the iGEM community to help future teams organize their protocols!</p><br />
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<h1>Bronze</h1><br />
<br><br />
<ul><br />
<li>Please find a comprehensive compilation of <a href="https://2014.igem.org/Team:Heidelberg/Team/Sponsoring">sponsors</a>, partners and scientific contributors on our <a href="https://2014.igem.org/Team:Heidelberg/Team/Attributions">acknowledgements page</a>. </li><br />
<br/><br />
<li>We also encourage you to take notice of the projects “Photo-intein” and “Mito-intein” by <a href="https://2014.igem.org/Team:Queens_Canada/Project">iGEM team Queens from Canada</a> that may supply you with complementary information and tools for the use of inteins in synthetic biology!</li><br />
<br/><br />
<li>A list of links to more than 60 parts in the registry submitted by our team (being or not being part of the new intein toolbox) can be found <a href="https://2014.igem.org/Team:Heidelberg/Parts#allParts">here</a>.</li><br />
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<h1>Silver</h1><br />
<br><br />
<ul><br />
<li>We experimentally validated that our biobricks <a href="http://parts.igem.org/Part:BBa_K1362000">BBa_K1362000</a>, <a href="http://parts.igem.org/Part:BBa_K1362100">BBa_K1362100 </a>and <a href="http://parts.igem.org/Part:BBa_K1362101">BBa_K1362101</a> work as expected. For more information on the <a href="https://2014.igem.org/Team:Heidelberg/Parts#Favorite Parts">parts</a> please visit the corresponding main pages in the parts registry or explore their involvement in our subprojects.</li><br />
<br/><br />
<li>Religious perceptions of synthetic biology have been part of several surveys during the past ten years of iGEM and Human Practices projects. Since religious groups cover the majority of worlds population, deliver moral values and wield power at the same time, we decided to dedicate a whole event on the topic of religion, philosophy and ethics regarding synthetic biology. Please find an <a href="https://2014.igem.org/Team:Heidelberg/Human_Practice/Ethics">evaluation of our event</a> on the corresponding Human Practices pages. <!--In order to reassure ourselves about the acceptability of our project and synthetic biology in general, we also used this opportunity to build up on the work of the iGEM Team Heidelberg 2013 and conducted a survey that addresses basic questions regarding the public reflection of our work.--><br />
</li><br />
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<h1>Gold</h1><br />
<ul><br />
<li>We improved the function of the <b>already existing</b> biobrick part <a href="http://parts.igem.org/Part:BBa_K117505">BBa_K1175005 </a>by optimizing and resubmitting the corresponding sequence of B. subtilis xylanase to the registry (Part:<a href="http://parts.igem.org/Part:BBa_K1362020"> BBa_K1362020</a>). In addition, we submitted a new part for expression of <a href="http://parts.igem.org/Part:BBa_K1362022">circularized xylanase </a>(BBa_K1362022) that might be used in future applications with need for refined enzyme stability.</li><br />
<br/><br />
<li>Despite the fact that we focused on building a set of powerful soft- and wetware tools to help future iGEM-teams developing and realizing projects in synthetic biology, we are happy to announce that we were also able to help out several team during the course of our project, aspecially with sending <a href="https://2014.igem.org/Team:Heidelberg/Parts#Backbones"> our expression vectors</a>. Read more abou in in our <a href="https://2014.igem.org/Team:Heidelberg/Team/Collaborations">Collaborations</a>.</li><br />
<br/><br />
<li>In the style of of the new iGEM community labs track that involves science amateurs “beyond the accolades of scientific publishing and economic reward”, we sought for a new way to involve laymen in actual science and build a strong community of well informed supporters and communicators of synthetic biology at the same time. Now we proudly present the crowd sourcing and communication platform <a href="https://2014.igem.org/Team:Heidelberg/Human_Practice/igemathome">iGEM@home</a>.</li><br />
</ul><br />
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<div><br/><br />
In the variety of proteins that exist, inteins are clearly among the most mysterious.<br />
They behave in such a peculiarly way that scientists are still puzzle about their initial role in the host organisms and are even more astonished when discovering their endless seeming abilities.<br />
<br />
Inteins are integrated as extraneous polypeptide sequences into ordinary proteins. They do not contribute to the original protein function but perform an autocatalytic splicing reaction after protein translation. Analog to intron splicing on RNA level, this posttranslational modification was named protein splicing. Consequentially the protein segments were called inteins, derived from “internal protein sequence”, and the flanking protein chains exteins, “external protein sequences”. Inteins excise themselves out of the host protein while reconnecting the remaining N and C exteins via a new peptide bond. Despite this immense invasion the original protein regains its normal structure and function after splicing [[#References|[1]]]. <br />
<br />
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{{:Team:Heidelberg/templates/image-half|<br />
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caption=Figure 1) Trans-splicing mechanism reaction by split inteins. |<br />
descr=The N-Intein is fused to the C’ terminal end of the N-extein. Complementary, C-Intein is located at the N’ end of the C-extein. After assembly of the two intein fragments, a splicing reaction takes place, where the intein removes itself from the precursor protein and simultaneously ligates the exteins via a peptide bond.|<br />
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<span style="font-weight:bold;">Trans-splicing mechanism reaction by split inteins.</span><br />
<p>Animation of the split intein splicing reaction</p><br />
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<br />
==History==<br />
<br />
Inteins were discovered in 1990 when dissimilarities between the mature protein sequence of the yeast vacuolar ATPase (Vma1) and its corresponding mRNA were investigated. Surprisingly the mature protein had a lower molecular weight than expected from the encoding sequence, indicating the loss of one part of the protein after translation. <br />
Indeed, a region of 454 amino acids was found to be translated and subsequently removed from the Vma1 protein [[#References|[2]]] [[#References|[3]]]. Since then, over 600 different inteins have been reported in all three domains of life as well as in viruses [[#References|[4]]]. <br />
<br />
Dependent on the organism they belong to, the intein’s name consists out of the genus and species abbreviation followed by the host gene [[#References|[5]]]. For example, the "golden standard" split-intein, NpuDnaE has derived from the DNA polymerase III (DnaE) in <i>Nostoc punctiforme</i> PCC73102 (<i>Npu</i>).<br />
<br />
==Evolution: Inteins as parasitic genes==<br />
Despite many years of research the initial role of inteins in their host organism remains still unclear.<br />
Many natural inteins contain a homing endonuclease (HEN) domain, an enzyme that cleaves DNA and introduces new sequences at homing sites via homologous recombination or reverse transcription. This characteristic led to the perception of the intein as selfish DNA sequence - a gene generating copies of itself without an advantage for the host organism [[#References|[6]]]. This allows horizontal gene transfer of inteins. [[#References|[7]]]. Data showing that 27% of the intein's host proteins are related to DNA metabolism and involved in DNA replication or repair is further supporting the "selfish gene" hypothesis. This ensures expression of the inteins and the HEN domain during DNA replication, where they can take advantage of the DNA replication and repair system for introducing changes in the DNA [[#References|[8]]].<br />
<br />
However recent work on conditional protein splicing has shown sensitivity of some inteins to redox state, temperature and small molecules (reviewed in [[#References|[9]]], [[#References|[10]]]). <br />
This might be a sign for inteins having a role in post-translational protein regulation upon environmental signals. Inserted close to the catalytic core of an protein the intein deactivates the protein function until the splicing reaction has taken place. Evolutionary said, selfish inteins might have adapted due to positive selection pressure to provide a beneficial mechanism for the host cell, thereby becoming selfless [[#References|[8]]].<br />
<br />
==Structure and Classification==<br />
<br />
The structure of inteins contains several conserved motifs. The splicing domains are located at the N and C terminal ends. Before splicing takes place the intein rearranges itself from its initial linear structure into to a horseshoe like structure where the termini are brought in close proximity making up the catalytic core [[#References|[14]]]. <br />
Inteins are divided into three groups: bifunctional (large) inteins, mini inteins and split inteins.<br />
<br />
Large inteins carry both a splicing domain and an endonuclease (HEN) domain whereas mini inteins lack the HEN domain [[#References|[11]]]. <br />
The most promising inteins for biotechnology are split inteins, which are basically mini-inteins, just divided in two fragments and expressed separately connected different proteins. After translation, they assemble with high affinity to become catalytic active and perform a splicing reaction. Split enzymes occure naturally [[#References|[12]]] but can also be engineered artificially [[#References|[13]]]. <br />
<br />
In our project we focused on split inteins, as they present a powerful tool to insert posttranslational modifications, offering a plethora of applications in biotechnology. We have characterized the most promising inteins in our [[Team:Heidelberg/Parts#Intein_Library|Intein Library]].<br />
<br />
==A detailed description of the trans-splicing reaction==<br />
<br />
In contrast to the mini and large inteins that mediate cis-splicing reactions, split inteins are responsible for trans-splicing and fusion of protein parts. The trans-splicing reaction can be divided into the following steps:<br />
<br />
# N-Intein and C-Intein first assemble together to form a dimer like structure with a newly formed catalytic core next to the exteins.<br />
# The tertiary structure of the intein, once correctly formed, facilitates an ''N&rarr;O/S'' acyl rearrangement at its N-terminal serine or cysteine residue. The result is an ester or thioester bond, respectively between the side-chain and the peptide backbone of the N-extein.<br />
# The two exteins are then linked by trans(thio)esterification involving the N-terminal serine or cysteine residue of the C-extein. The C-terminus of the N-extein is now covalently bound to the N-terminal side-chain of the C-extein, while its backbone still retains its normal peptide bond to the intein.<br />
# The C-terminal asparagine of the intein undergoes self-cyclisation to form a succinimidyl moiety. The peptide bond between the intein and the extein is thereby broken, resolving the branched intermediate.<br />
# In a final reaction, an ''O/S&rarr;N'' acyl shift results in the two exteins now being linked by an amide bond, indistinguishable from a ribosome-assembled fusion protein.<br />
(as reviewed in [[#References|[15]]]) <br />
<br />
{{:Team:Heidelberg/templates/image-full|<br />
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|descr=<br />
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<br />
For intein efficiency not only the structure of the intein domain itself is important but also the nature of the flanking extein residues, as they are heavily involved in the splicing reaction. It has been shown, that the first residue has to be a nucleophilic amino acid, preferable Cysteine, Serine or Threonine. Effectiveness of the intein can be mold by changing the these amino acids [[#References|[17]]].<br />
<br />
==Use of inteins in molecular biology and biotechnology==<br />
<br />
Due to their distinct characteristics intein are a powerful tool for molecular biology and biotechnology. Performing an autocatalytic reaction, the inteins are neither dependent on their host protein nor on any other additional substrate. This makes it them broadly applicable for in vivo and in vitro applications. [[#References|[16]]].<br />
<br />
Inteins have been used for protein purification, achieving a higher yield of protein due to more specific targeting. The production of semi-synthetic proteins and the attachment of synthetic groups is of huge interest for recombinant proteins. [[#References|[16]]]. Split Intein-mediated Circular Ligation Of Peptides a ProteinS (SICLOPPS) is a method to produce circular peptides of eight amino acids length, which could be potent therapeutic drugs [[#References|[18]]]. Inteins, fused to split fluorescent proteins as reporter and to the protein of investigation have been used to detect protein-protein interactions in vivo (reviewed in [[#References|[10]]]).<br />
Nevertheless most of this applications are performed in vitro and do not exploit the full potential of inteins as regulatory element for post-translational modification. The iGEM Team Heidelberg employs this excellent mechanism of protein splicing to specifically change whole amino-acid sequences and thereby regulate proteins via (dis-) assembly, protein cleavage or circularization of enzymes in vivo. All this and much more was embedded into our versatile intein toolbox. With this universal toolkit we provide a foundational advance for protein control - introducing the full potential of post-translational modification and therefore a new dimension of genetic engineering to Synthetic Biology. There is much more to [[Team:Heidelberg/Project|explore]]!<br />
<br />
=References=<br />
<br />
[1] Perler FB, Davis EO, Dean GE, Gimble FS, Jack WE, et al. (1994) Protein splicing elements: inteins and exteins–a definition of terms and recommended nomenclature. Nucleic Acids Res 22: 1125–1127<br />
<br />
[2] Kane P.M., et al. Protein Splicing Converts the Yeast TEPI Gene Product to the 69-kD Subunit of the Vacuolar. 13253, (1990)<br />
<br />
[3] Hirata, R. et al. Molecular structure of a gene, VMA1, encoding the catalytic subunit of H(+)-translocating adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae. J. Biol. Chem. 265 , 6726–6733 (1990)<br />
<br />
[4] Perler, F. B. InBase : the Intein Database. 30, 383–384 (2002)<br />
<br />
[5] Perler, F. B. et al. Protein splicing elements : inteins and exteins — a definition of terms and recommended nomenclature. 22, 1125–1127 (1994)<br />
<br />
[6] Barzel, A., Naor, A., Privman, E., Kupiec, M. & Gophna, U. Homing endonucleases residing within inteins: evolutionary puzzles awaiting genetic solutions. Biochem. Soc. Trans. 39, 169–73 (2011)<br />
<br />
[7] Pietrokovski, S. Intein spread and extinction in evolution. Trends Genet. 17, 465-472 (2001)<br />
<br />
[8] Novikova, O., Topilina, N. & Belfort, M. Enigmatic distribution, evolution and function of inteins. J. Biol. Chem. (2014)<br />
<br />
[9] Shah, N. H., and Muir, T. W. Inteins: nature's gift to protein chemists. Chem. Sci. 5, 446- 461 (2014) <br />
<br />
[10] Topilina, N. I. & Mills, K. V. Recent advances in in vivo applications of intein-mediated protein splicing. Mob. DNA 5, 5 (2014)<br />
<br />
[11] Eryilmaz, E., Shah, N., Muir, T. & Cowburn, D. Structural and Dynamical Features of Inteins and Implications on Protein Splicing. J. Biol. Chem. (2014)<br />
<br />
[12] Carvajal-Vallejos, P., Pallissé, R., Mootz, H. D. & Schmidt, S. R. Unprecedented rates and efficiencies revealed for new natural split inteins from metagenomic sources. J. Biol. Chem. 287, 28686–96 (2012) <br />
<br />
[13] Lin, Y. et al. Protein trans-splicing of multiple atypical split inteins engineered from natural inteins. PLoS One 8, e59516 (2013). <br />
<br />
[14] Eryilmaz, E., Shah, N., Muir, T. & Cowburn, D. Structural and Dynamical Features of Inteins and Implications on Protein Splicing. J. Biol. Chem. (2014)<br />
<br />
[15] Mills, K. V, Johnson, M. a & Perler, F. B. Protein Splicing: How Inteins Escape from Precursor Proteins. J. Biol. Chem. (2014)<br />
<br />
[16] Mootz, H. D. Split inteins as versatile tools for protein semisynthesis. Chembiochem 10, 2579–89 (2009).<br />
<br />
[17] Amitai, G., Callahan, B. P., Stanger, M. J., Belfort, G. & Belfort, M. Modulation of intein activity by its neighboring extein substrates. Proc. Natl. Acad. Sci. U. S. A. 106, 11005–10 (2009).<br />
<br />
[18] Tavassoli, A. & Benkovic, S. J. Split-intein mediated circular ligation used in the synthesis of cyclic peptide libraries in E. coli. Nat. Protoc. 2, 1126–1133 (2007).</div>Maexlichhttp://2014.igem.org/Team:Heidelberg/ProjectTeam:Heidelberg/Project2014-10-18T03:53:26Z<p>Maexlich: </p>
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<h4><img id="dropdownImg" src="/wiki/images/7/70/Heidelberg_Abstract-dropdown.png"/>&nbsp;Project overview</h4><br />
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<div id="abstract-content" class="col-lg-12" ><br />
<p>Proteins are the functional basis of all biological processes and being able to control and improve their functions through design and engineering is one of the fundamental goals of Synthetic Biology. Protein splicing, a process in which a catalytically active internal polypeptide termed INTEIN excises itself from a precursor protein, has been used by our team to alter the functionalities of proteins in various ways post-translationally. <br />
<br></br><br />
We have developed a comprehensive <a href="/Team:Heidelberg/Project/Toolbox">toolbox</a> compromising five sets of <a href="/Team:Heidelberg/Parts">standardized parts</a> that provide standard mechanisms for specific modifications of proteins: <br />
(1) A <a href="/Team:Heidelberg/Project/Toolbox#Purification">purification</a> standard to faciliate purification of recombinant proteins. <br />
(2) A construct that allows for post-translational <a href="/Team:Heidelberg/Project/Toolbox#Fusion_and_Tagging">fusion</a> of protein domains to produce synthetic proteins<br />
(3) A standard that allows for switching proteins <a href="/Team:Heidelberg/Project/Toolbox#On_Off">on and off</a>.<br />
(4) One to <a href="/Team:Heidelberg/Project/Toolbox#Oligomerization">oligomerize</a> protein monomers.<br />
And (5) perhaps the most intriguing standard construct of this toolbox that was built to produce heat stable proteins, which can be achieved by <a href="/Team:Heidelberg/Toolbox/Circularization">circularization</a>.<br />
In order to offer a comfortable way to apply any of the above mechanisms to a protein and a biological system we designed a <a href="/Team:Heidelberg/Toolbox_Guide">toolbox guide</a> that provides step by step instructions for cloning based on the intein standard parts described in our <a href="/Team:Heidelberg/Parts/RFC">RFC</a>.<br />
<br></br><br />
Besides achieving a solid and broad foundation for future intein applications in synthetic biology, we aimed on deeply exploring one of the functions provided in our toolbox: circularization of proteins. In head to tail circularized peptides the terminal amino acids are joined together just like in the rest of the chain, forming a circular structure. Such peptides have been discovered in all kingdoms of life during the past years and they are unified by an extreme stability towards high temperatures, proteases and changes in pH.<br />
Synthetically connecting a protein's termini without disrupting its 3D structure and function is, however, a delicate task which has so far been accomplished only for relatively small proteins whose ends lie close to each other. With <a href="/Team:Heidelberg/Modeling/Linker_Modeling">CRAUT</a> we have brought into existence a powerful open-source software to predict an optimal rigid linker to support stabilizing circularization of a protein preserving its 3D structure and function. <br />
In order to provide us - and future iGEM teams - with a standard way to conduct the needed consuming modelling calculations we deployed a distributed computing platform we call <a href="/Team:Heidelberg/Human_Practice/igemathome">iGEM@home</a> that we also developed into a powerful science communication platform.<br />
As an evaluation of the linker software we screened linkers generated by the software by circularizing <a href="/Team:Heidelberg/Project/Linker_Screening">Lambda Lysozyme</a> and experimentally evaluating the heat stability of the products.<br />
<br></br><br />
Based on the calibrated software, we constructed linkers to circularize the 871 a.a. long methyltransferase Dnmt1 and provide data suggesting that circular DNMT1 is more functional than its linear counterpart at high temperatures. Our results have strong implications for developing an innovative PCR-based technique that could revolutionize epigenetic studies and cancer research by maintaining the methylation pattern of the DNA template during amplification.<br />
Besides a protein with potential medical (lysozyme) and one with biotechnological (DNMT1) applications we chose the hemicellulose xylanase as a third target for circularization, that has applications in large scale production in paper and food industry.<br />
<br></br><br />
As an additional feature we decided to add another dimension to intein mediated modifications of our toolbox by developing optically induced small inteins that are photocaged by an <a href=”/Team:Heidelberg/Project/LOV”>As LOV2</a> domain. While sterically hampering intein dimerization in the dark, the LOV domain opens up when exposed to blue light, releasing the intein to proceed the splicing reaction.<br />
<br></br><br />
Setting new standards also for wiki documentation we created and developed a new way of documenting collaborative lab work by bringing the MidnightDoc to life.<br />
<br></br><br />
Finally we complemented our already elaborate Human Practice activities with two events on education and religion, philosophy and ethics in synthetic biology.<br />
<br />
<br />
<br />
</p><br />
</div><br />
</div><br />
<div class="graphicalAbstract"><br />
<div id="redOverlay"></div><br />
<div style="z-index:3;position:relative; height:100%;"><br />
<div id="linker-links"><br />
<a href="https://2014.igem.org/Team:Heidelberg/Software/igemathome" class="block box" id="calibrated"><br />
<img style="height:60px;display: inline-block;" src="/wiki/images/e/ea/Heidelberg_Project_Computer.png"><br />
<span style="position: relative;top: 10px;display: inline-block;"><br />
calibrated<br><br />
<span class="red-text">in silico</span></span><br />
</a><br />
<a href="https://2014.igem.org/Team:Heidelberg/Project/Linker_Screening" class="block box" id="screened"><br />
<span style="position: relative;top:10px;display: inline-block;"><br />
screened <span class="red-text">in vitro</span><br><br />
with lysozyme</span><br />
<img style="height:60px; display:inline-block;" src="/wiki/images/d/df/Heidelberg_Lysozyme.png" /><br />
</a><br />
<a href="/Team:Heidelberg/Software/Linker_Software" class="block box" style="bottom:0; left:-20px; position:absolute;width:170px;"><br />
<img src="/wiki/images/4/42/Craut_small.png" alt="..." style="width:100%;"/><br />
<span class="red-text" >circularize</span> it<br><br />
with calculated linkers<br />
</a><br />
</div><br />
<div class="ringbox"><br />
<img src="/wiki/images/0/0d/Heidelberg_Firering_red.png" id="ring-background" /><br />
<div id="circ-box" class="descr-box"><br />
<a href="/Team:Heidelberg/Toolbox/Circularization"><br />
<img src="/wiki/images/5/58/Heidelberg_Toolbox_Circularization.png" id="circ-icon" class="toolbox-icon toolbox-icon-scale"/><br />
</a><br />
<h3>CIRCULARIZATION</h3><br />
<div><span>Create a linker with our crowd computing software and make your protein heat stable</span></div><br />
</div><br />
<div id="oligo-box" class="descr-box"><a href="https://2014.igem.org/Team:Heidelberg/Project/Toolbox#Oligomerization"><img src="/wiki/images/4/40/Oligomerization.png" id="oligo-icon" class="toolbox-icon toolbox-icon-scale"/></a><h3>OLIGOMERIZATION</h3><div><span>Fuse multiple Proteins or Domains using Inteins</span></div><br />
</div><br />
<div id="fusion-box" class="descr-box"><a href="https://2014.igem.org/Team:Heidelberg/Project/Toolbox#Fusion_and_Tagging"><img src="/wiki/images/8/87/Heidelberg_Toolbox_Fusion.png" id="fusion-icon" class="toolbox-icon toolbox-icon-scale"/></a><h3>FUSION</h3><div><span>Fuse two Proteins or Domains together using Inteins</span></div><br />
</div><br />
<div id="onoff-box" class="descr-box"><a href="https://2014.igem.org/Team:Heidelberg/Project/Toolbox#On_Off"><img src="/wiki/images/c/c2/Heidelberg_Toolbox_On-Off.png" id="onoff-icon" class="toolbox-icon toolbox-icon-scale"/></a><h3>ON/OFF</h3><div><span>Activate or deactivate Proteins using Inteins</span></div></div><br />
<div id="purification-box" class="descr-box"><a href="https://2014.igem.org/Team:Heidelberg/Project/Toolbox#Purification"><img src="/wiki/images/0/04/Heidelberg_Toolbox_Purification.png" id="purification-icon" class="toolbox-icon toolbox-icon-scale"/></a><div><h3>PURIFICATION</h3></div><span>Placeholder</span></div><br />
<div id="toolbox-text"><br />
the intein<br><br />
<span><a class="scheisslinkbleibweiss" href="/Team:Heidelberg/Project/Toolbox">toolbox</a><span><br />
</div><br />
</div><br />
<a href="https://2014.igem.org/Team:Heidelberg/Toolbox/Induction" class="block" id="lightning"><br />
<img class="toolbox-icon-scale" style="height:110px;display: inline-block;" src="/wiki/images/8/83/Heidelberg_Project_Lightning.png"><span style="position: relative;top: 20px;display: inline-block;">&nbsp;inducible via<br><br />
<span style="font-weight:bold;position: relative;left: -18px;" class="red-text">light induction</span></span><br />
</a><br />
<a href="https://2014.igem.org/Team:Heidelberg/Project/PCR_2.0" class="block box" style="width:250px;" id="dnmt1-box"><br />
<img id="dnmt1-img" src="/wiki/images/a/a6/Heidelberg_Project_Dnmt1.png"><br />
<span style="position:relative; z-index:5;" class="block"><br />
<span style="font-size:1.5em;"><br />
<span class="red-text">Heat-stable</span><br> circular <br><span style="font-weight:bold;">DNA-<br/>Methyltransferase</span><br />
</span><br><br />
<span style="font-weight:bold;font-size: 2.5em; text-align:right;line-height: 35px;"><br />
<span class="red-text">PCR 2.0</span><br />
</span><br />
</span><br />
</a><br />
<a href="/Team:Heidelberg/Toolbox_Guide" class="block" id="toolbox"><br />
<span style="position:relative; display: inline-block;height:110px; width:110px; vertical-align: middle;"><br />
<img style="height:100%; position:absolute; top:0; left:0;" id="toolbox-img" src="/wiki/images/2/24/Heidelberg_Project_Toolbox_guide.png" /><br />
<img style="height:100%; position:absolute; top:0; left:0; display:none;" id="toolbox-img-hover" src="/wiki/images/4/4c/Heidelberg_Toolbox_guide_highlighted.png" /><br />
</span><br />
<span style="position: relative;top: 20px;display: inline-block; vertical-align:middle;"><br />
<span style="font-weight:bold;" class="red-text">modify your protein</span><br/><br />
using the toolbox guide<br />
</span><br />
</a><br />
<div class="clearfix"></div><br />
</div><br />
</div><br />
</div><br />
<div class="container" id="Achievements"><br />
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<li class="active" style="font-size:30px"><a href="#Achievements-tab" role="tab" data-toggle="tab"><img src="https://static.igem.org/mediawiki/2014/2/22/Heidelberg_Achievements_red.png" height="50px" alt="Button"> Achievements</a></li><br />
<li style="font-size:30px"><a href="#Medal_Criteria-tab" role="tab" data-toggle="tab"> <img src="https://static.igem.org/mediawiki/2014/3/3f/Heidelberg_Gold_red.png" height="50px" alt="Button"> Medal Criteria</a></li><br />
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<br />
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<p style="font-weight:light; font-size:25px">Establishing protein circularization as a NEW BIOENGINEERING TOOL in synthetic biology.</p> <br />
<p style="margin-left:50px; font-size:20px">Contributing to iGEM with a new foundational advance!</p><br />
</div><br />
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<div class="col-xs-3"><br />
<img src="/wiki/images/7/7d/Check_new.png" style="height:100px; margin-right:15px;"><br />
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<p style="font-weight:light; font-size:25px">Providing a NEW COMPREHENSIVE TOOLBOX based on inteins for modifying proteins post-translationally.</p> <br />
<p style="margin-left:50px; font-size:20px">Sending 67 Biobricks to Registry of Biological parts!</p><br />
</div><br />
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<div class="col-xs-3"><br />
<img src="/wiki/images/7/7d/Check_new.png" style="height:100px; margin-right:15px;"><br />
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<p style="font-weight:light; font-size:25px">Development of a NEW STANDARD to make the use of inteins easy and modular.</p> <br />
<p style="margin-left:50px; font-size:20px">Establishment of a new <a href="https://2014.igem.org/Team:Heidelberg/Parts/RFC">RFC</a>!</p><br />
</div><br />
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<div class="col-xs-3"><br />
<img src="/wiki/images/7/7d/Check_new.png" style="height:100px; margin-right:15px;"><br />
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<div class="linie"></div><br />
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<img src="https://static.igem.org/mediawiki/2014/1/1b/Achievements_toolbox.png" class="img-responsive" style="margin-left:15px;"> <br />
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<p style="font-weight:light; font-size:25px">Showing that the toolbox WORKS: proteins are circularized and split fluorescent proteins are reconstituted.</p> <br />
<p style="margin-left:50px; font-size:20px">Making Gels, Western Blots, Fluorescence-based Assays and Mass spectrometry to prove it! </p><br />
</div><br />
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<div class="col-xs-3"><br />
<img src="/wiki/images/7/7d/Check_new.png" style="height:100px; margin-right:15px;"><br />
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<p style="font-weight:light; font-size:25px">Creating circular DNMT1 and showing that it is ACTIVE.</p> <br />
<p style="margin-left:50px; font-size:20px">For the first time achieving the circularization of a large protein!</p><br />
<p style="margin-left:50px; font-size:20px">Circularizing LYSOZYME and XYLANASE, two very important proteins for<br />
research and industry!</p><br />
</div><br />
<br />
<div class="col-xs-3"><br />
<img src="/wiki/images/7/7d/Check_new.png" style="height:100px; margin-right:15px;"><br />
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<p style="font-weight:light; font-size:25px">Developing a NEW SOFTWARE to calculate customized linkers to circularize proteins.</p> <br />
<p style="margin-left:50px; font-size:20px">Making CRAUT open-source for the scientific community.</p><br />
</div><br />
<br />
<div class="col-xs-3"><br />
<img src="/wiki/images/7/7d/Check_new.png" style="height:100px; margin-right:15px;"><br />
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<img src="https://static.igem.org/mediawiki/2014/e/e5/Heidelberg_Frontpage_igemathome.png" class="img-responsive" style="margin-left:15px;"> <br />
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<br />
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<p style="font-weight:light; font-size:25px"> Establishing a distributed computing platform called <a href="https://2014.igem.org/Team:Heidelberg/Software/igemathome">iGEM@home</a>.</p> <br />
<p style="margin-left:50px; font-size:20px">Using this platform as an entirely new way to reach out to the world with synthetic biology concepts!</p><br />
</div><br />
<br />
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<div class="col-xs-3"><br />
<img src="/wiki/images/7/7d/Check_new.png" style="height:100px; margin-right:15px;"><br />
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<p style="font-weight:light; font-size:25px">Creating a NEW SOFTWARE to display the notebook on the wiki.</p> <br />
<p style="margin-left:50px; font-size:20px">Distributing MidNightDOC to the iGEM community to help future teams organize their protocols!</p><br />
</div><br />
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<img src="/wiki/images/7/7d/Check_new.png" style="height:100px; margin-right:15px;"><br />
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<img class="medal" src="https://static.igem.org/mediawiki/2014/6/63/Heidelberg_Bronze.png"><br />
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<br />
<div class="col-md-9 col-xs-12"><br />
<h1>Bronze</h1><br />
<br><br />
<ul><br />
<li>Please find a comprehensive compilation of <a href="https://2014.igem.org/Team:Heidelberg/Team/Sponsoring">sponsors</a>, partners and scientific contributors on our <a href="https://2014.igem.org/Team:Heidelberg/Team/Attributions">acknowledgements page</a>. </li><br />
<br/><br />
<li>We also encourage you to take notice of the projects “Photo-intein” and “Mito-intein” by <a href="https://2014.igem.org/Team:Queens_Canada/Project">iGEM team Queens from Canada</a> that may supply you with complementary information and tools for the use of inteins in synthetic biology!</li><br />
<br/><br />
<li>A list of links to more than 60 parts in the registry submitted by our team (being or not being part of the new intein toolbox) can be found <a href="https://2014.igem.org/Team:Heidelberg/Parts#allParts">here</a>.</li><br />
</ul><br />
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<div class="col-md-9 col-xs-12"><br />
<h1>Silver</h1><br />
<br><br />
<ul><br />
<li>We experimentally validated that our biobricks <a href="http://parts.igem.org/Part:BBa_K1362000">BBa_K1362000</a>, <a href="http://parts.igem.org/Part:BBa_K1362100">BBa_K1362100 </a>and <a href="http://parts.igem.org/Part:BBa_K1362101">BBa_K1362101</a> work as expected. For more information on the <a href="https://2014.igem.org/Team:Heidelberg/Parts#Favorite Parts">parts</a> please visit the corresponding main pages in the parts registry or explore their involvement in our subprojects.</li><br />
<br/><br />
<li>Religious perceptions of synthetic biology have been part of several surveys during the past ten years of iGEM and Human Practices projects. Since religious groups cover the majority of worlds population, deliver moral values and wield power at the same time, we decided to dedicate a whole event on the topic of religion, philosophy and ethics regarding synthetic biology. Please find an <a href="https://2014.igem.org/Team:Heidelberg/Human_Practice/Ethics">evaluation of our event</a> on the corresponding Human Practices pages. <!--In order to reassure ourselves about the acceptability of our project and synthetic biology in general, we also used this opportunity to build up on the work of the iGEM Team Heidelberg 2013 and conducted a survey that addresses basic questions regarding the public reflection of our work.--><br />
</li><br />
</ul><br />
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<h1>Gold</h1><br />
<ul><br />
<li>We improved the function of the <b>already existing</b> biobrick part <a href="http://parts.igem.org/Part:BBa_K117505">BBa_K1175005 </a>by optimizing and resubmitting the corresponding sequence of B. subtilis xylanase to the registry (Part:<a href="http://parts.igem.org/Part:BBa_K1362020"> BBa_K1362020</a>). In addition, we submitted a new part for expression of <a href="http://parts.igem.org/Part:BBa_K1362022">circularized xylanase </a>(BBa_K1362022) that might be used in future applications with need for refined enzyme stability.</li><br />
<br/><br />
<li>Despite the fact that we focused on building a set of powerful soft- and wetware tools to help future iGEM-teams developing and realizing projects in synthetic biology, we are happy to announce that we were also able to help out several team during the course of our project, aspecially with sending <a href="https://2014.igem.org/Team:Heidelberg/Parts#Backbones"> our expression vectors</a>. Read more abou in in our <a href="https://2014.igem.org/Team:Heidelberg/Team/Collaborations">Collaborations</a>.</li><br />
<br/><br />
<li>In the style of of the new iGEM community labs track that involves science amateurs “beyond the accolades of scientific publishing and economic reward”, we sought for a new way to involve laymen in actual science and build a strong community of well informed supporters and communicators of synthetic biology at the same time. Now we proudly present the crowd sourcing and communication platform <a href="https://2014.igem.org/Team:Heidelberg/Human_Practice/igemathome">iGEM@home</a>.</li><br />
</ul><br />
</div><br />
</div><br />
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<div class="container main" style="color: white;"><br />
<div id="abstract-dropdown" class="row abstract-special dark-grey"><br />
<div class="col-lg-offset-1 col-lg-11"><br />
<h4><img id="dropdownImg" src="/wiki/images/7/70/Heidelberg_Abstract-dropdown.png"/>&nbsp;Project overview</h4><br />
</div><br />
<!--<div class="col-lg-9"><br />
<span>Click to read the project overview</span><br />
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<div id="abstract-content" class="col-lg-12" ><br />
<p>Proteins are the functional basis of all biological processes and being able to control and improve their functions through design and engineering is one of the fundamental goals of synthetic biology. Protein splicing, a process in which a catalytically active internal polypeptide termed INTEIN excises itself from a precursor protein, has been used by our team to alter the functionalities of proteins in various ways post-translationally. <br />
<br></br><br />
We have developed a comprehensive <a href=”/Team:Heidelberg/Project/Toolbox">toolbox</a> compromising five sets of <a href=”/Team:Heidelberg/Parts">standardized parts</a> that provide standard mechanisms for specific modifications of proteins: <br />
(1) A <a href=”/Team:Heidelberg/Project/Toolbox#Purification">purification</a> standard to faciliate purification of recombinant proteins. <br />
(2) A construct that allows for post-translational <a href=”/Team:Heidelberg/Project/Toolbox#Fusion_and_Tagging">fusion</a> of protein domains to produce synthetic proteins<br />
(3) A standard that allows for switching proteins <a href=”/Team:Heidelberg/Project/Toolbox#On_Off">on and off</a>.<br />
(4) One to <a href=”/Team:Heidelberg/Project/Toolbox#Oligomerization">oligomerize</a> protein monomers.<br />
And (5) perhaps the most intriguing standard construct of this toolbox that was built to produce heat stable proteins, which can be achieved by <a href=”/Team:Heidelberg/Toolbox/Circularization">circularization</a>.<br />
In order to offer a comfortable way to apply any of the above mechanisms to a protein and a biological system we designed a <a href=”/Team:Heidelberg/Toolbox_Guide”>toolbox guide</a> that provides step by step instructions for cloning based on the intein standard parts described in our <a href=”/Team:Heidelberg/Parts/RFC”>RFC</a>.<br />
<br></br><br />
Besides achieving a solid and broad foundation for future intein applications in synthetic biology, we aimed on deeply exploring one of the functions provided in our toolbox: circularization of proteins. In head to tail circularized peptides the terminal amino acids are joined together just like in the rest of the chain, forming a circular structure. Such peptides have been discovered in all kingdoms of life during the past years and they are unified by an extreme stability towards high temperatures, proteases and changes in pH.<br />
Synthetically connecting a protein's termini without disrupting its 3D structure and function is, however, a delicate task which has so far been accomplished only for relatively small proteins whose ends lie close to each other. With CRAUT we have brought into existence a powerful open-source software to predict an optimal rigid linker to support stabilizing circularization of a protein preserving its 3D structure and function. <br />
In order to provide us - and future iGEM teams - with a standard way to conduct the needed consuming modelling calculations we deployed a distributed computing platform we call iGEM@home that we also developed into a powerful science communication platform.<br />
As an evaluation of the linker software we screened linkers generated by the software by circularizing lamda lysozyme and experimentally evaluating the heat stability of the products.<br />
<br></br><br />
Based on the calibrated software, we constructed linkers to circularize the 871 a.a. long methyltransferase Dnmt1 and provide data suggesting that circular DNMT1 is more functional than its linear counterpart at high temperatures. Our results have strong implications for developing an innovative PCR-based technique that could revolutionize epigenetic studies and cancer research by maintaining the methylation pattern of the DNA template during amplification.<br />
Besides a protein with potential medical (lysozyme) and one with biotechnological (DNMT1) applications we chose the hemicellulose xylanase as a third target for circularization, that has applications in large scale production in paper and food industry.<br />
<br></br><br />
As an additional feature we decided to add another dimension to intein mediated modifications of our toolbox by developing optically induced small inteins that are photocaged by an <a href=”/Team:Heidelberg/Project/LOV”>As LOV2</a> domain. While sterically hampering intein dimerization in the dark, the LOV domain opens up when exposed to blue light, releasing the intein to proceed the splicing reaction.<br />
<br></br><br />
Setting new standards also for wiki documentation we created and developed a new way of documenting collaborative lab work by bringing the MidnightDoc to life.<br />
<br></br><br />
Finally we complemented our already elaborate Human Practice activities with two events on education and religion, philosophy and ethics in synthetic biology.<br />
<br />
<br />
<br />
</p><br />
</div><br />
</div><br />
<div class="graphicalAbstract"><br />
<div id="redOverlay"></div><br />
<div style="z-index:3;position:relative; height:100%;"><br />
<div id="linker-links"><br />
<a href="https://2014.igem.org/Team:Heidelberg/Software/igemathome" class="block box" id="calibrated"><br />
<img style="height:60px;display: inline-block;" src="/wiki/images/e/ea/Heidelberg_Project_Computer.png"><br />
<span style="position: relative;top: 10px;display: inline-block;"><br />
calibrated<br><br />
<span class="red-text">in silico</span></span><br />
</a><br />
<a href="https://2014.igem.org/Team:Heidelberg/Project/Linker_Screening" class="block box" id="screened"><br />
<span style="position: relative;top:10px;display: inline-block;"><br />
screened <span class="red-text">in vitro</span><br><br />
with lysozyme</span><br />
<img style="height:60px; display:inline-block;" src="/wiki/images/d/df/Heidelberg_Lysozyme.png" /><br />
</a><br />
<a href="/Team:Heidelberg/Software/Linker_Software" class="block box" style="bottom:0; left:-20px; position:absolute;width:170px;"><br />
<img src="/wiki/images/4/42/Craut_small.png" alt="..." style="width:100%;"/><br />
<span class="red-text" >circularize</span> it<br><br />
with calculated linkers<br />
</a><br />
</div><br />
<div class="ringbox"><br />
<img src="/wiki/images/0/0d/Heidelberg_Firering_red.png" id="ring-background" /><br />
<div id="circ-box" class="descr-box"><br />
<a href="/Team:Heidelberg/Toolbox/Circularization"><br />
<img src="/wiki/images/5/58/Heidelberg_Toolbox_Circularization.png" id="circ-icon" class="toolbox-icon toolbox-icon-scale"/><br />
</a><br />
<h3>CIRCULARIZATION</h3><br />
<div><span>Create a linker with our crowd computing software and make your protein heat stable</span></div><br />
</div><br />
<div id="oligo-box" class="descr-box"><a href="https://2014.igem.org/Team:Heidelberg/Project/Toolbox#Oligomerization"><img src="/wiki/images/4/40/Oligomerization.png" id="oligo-icon" class="toolbox-icon toolbox-icon-scale"/></a><h3>OLIGOMERIZATION</h3><div><span>Fuse multiple Proteins or Domains using Inteins</span></div><br />
</div><br />
<div id="fusion-box" class="descr-box"><a href="https://2014.igem.org/Team:Heidelberg/Project/Toolbox#Fusion_and_Tagging"><img src="/wiki/images/8/87/Heidelberg_Toolbox_Fusion.png" id="fusion-icon" class="toolbox-icon toolbox-icon-scale"/></a><h3>FUSION</h3><div><span>Fuse two Proteins or Domains together using Inteins</span></div><br />
</div><br />
<div id="onoff-box" class="descr-box"><a href="https://2014.igem.org/Team:Heidelberg/Project/Toolbox#On_Off"><img src="/wiki/images/c/c2/Heidelberg_Toolbox_On-Off.png" id="onoff-icon" class="toolbox-icon toolbox-icon-scale"/></a><h3>ON/OFF</h3><div><span>Activate or deactivate Proteins using Inteins</span></div></div><br />
<div id="purification-box" class="descr-box"><a href="https://2014.igem.org/Team:Heidelberg/Project/Toolbox#Purification"><img src="/wiki/images/0/04/Heidelberg_Toolbox_Purification.png" id="purification-icon" class="toolbox-icon toolbox-icon-scale"/></a><div><h3>PURIFICATION</h3></div><span>Placeholder</span></div><br />
<div id="toolbox-text"><br />
the intein<br><br />
<span><a class="scheisslinkbleibweiss" href="/Team:Heidelberg/Project/Toolbox">toolbox</a><span><br />
</div><br />
</div><br />
<a href="https://2014.igem.org/Team:Heidelberg/Toolbox/Induction" class="block" id="lightning"><br />
<img class="toolbox-icon-scale" style="height:110px;display: inline-block;" src="/wiki/images/8/83/Heidelberg_Project_Lightning.png"><span style="position: relative;top: 20px;display: inline-block;">&nbsp;inducible via<br><br />
<span style="font-weight:bold;position: relative;left: -18px;" class="red-text">light induction</span></span><br />
</a><br />
<a href="https://2014.igem.org/Team:Heidelberg/Project/PCR_2.0" class="block box" style="width:250px;" id="dnmt1-box"><br />
<img id="dnmt1-img" src="/wiki/images/a/a6/Heidelberg_Project_Dnmt1.png"><br />
<span style="position:relative; z-index:5;" class="block"><br />
<span style="font-size:1.5em;"><br />
<span class="red-text">Heat-stable</span><br> circular <br><span style="font-weight:bold;">DNA-<br/>Methyltransferase</span><br />
</span><br><br />
<span style="font-weight:bold;font-size: 2.5em; text-align:right;line-height: 35px;"><br />
<span class="red-text">PCR 2.0</span><br />
</span><br />
</span><br />
</a><br />
<a href="/Team:Heidelberg/Toolbox_Guide" class="block" id="toolbox"><br />
<span style="position:relative; display: inline-block;height:110px; width:110px; vertical-align: middle;"><br />
<img style="height:100%; position:absolute; top:0; left:0;" id="toolbox-img" src="/wiki/images/2/24/Heidelberg_Project_Toolbox_guide.png" /><br />
<img style="height:100%; position:absolute; top:0; left:0; display:none;" id="toolbox-img-hover" src="/wiki/images/4/4c/Heidelberg_Toolbox_guide_highlighted.png" /><br />
</span><br />
<span style="position: relative;top: 20px;display: inline-block; vertical-align:middle;"><br />
<span style="font-weight:bold;" class="red-text">modify your protein</span><br/><br />
using the toolbox guide<br />
</span><br />
</a><br />
<div class="clearfix"></div><br />
</div><br />
</div><br />
</div><br />
<div class="container" id="Achievements"><br />
<ul class="nav nav-tabs" role="tablist"><br />
<li class="active" style="font-size:30px"><a href="#Achievements-tab" role="tab" data-toggle="tab"><img src="https://static.igem.org/mediawiki/2014/2/22/Heidelberg_Achievements_red.png" height="50px" alt="Button"> Achievements</a></li><br />
<li style="font-size:30px"><a href="#Medal_Criteria-tab" role="tab" data-toggle="tab"> <img src="https://static.igem.org/mediawiki/2014/3/3f/Heidelberg_Gold_red.png" height="50px" alt="Button"> Medal Criteria</a></li><br />
</ul><br />
<div class="tab-content"><br />
<div class="tab-pane fade in active" style="background-color:white; color:black;" id="Achievements-tab"><br />
<br />
<div class="row" style="margin-top: 20px; background-color:white"><br />
</div><br />
<div class="row" style="margin:0;"><br />
<div class="col-md-3 col-xs-12 vcenter-cell"><br />
<img src="https://static.igem.org/mediawiki/2014/a/ae/Achievements_3.png" class="img-responsive" style="margin-left:15px;"><br />
</div><br />
<br />
<div class="col-md-9 col-xs-12 vcenter-cell"><br />
<div class="col-xs-9"><br />
<p style="font-weight:light; font-size:25px">Establishing protein circularization as a NEW BIOENGINEERING TOOL in synthetic biology.</p> <br />
<p style="margin-left:50px; font-size:20px">Contributing to iGEM with a new foundational advance!</p><br />
</div><br />
<br />
<div class="col-xs-3"><br />
<img src="/wiki/images/7/7d/Check_new.png" style="height:100px; margin-right:15px;"><br />
</div><br />
<br />
</div><br />
</div><br />
<div class="linie"></div><br />
<br />
<div class="row" style="margin:0;"><br />
<div class="col-md-3 col-xs-12 vcenter-cell"><br />
<img src="https://static.igem.org/mediawiki/2014/1/11/Achievement_1.png" class="img-responsive" style="margin-left:15px;"><br />
</div><br />
<br />
<div class="col-md-9 col-xs-12 vcenter-cell"><br />
<div class="col-xs-9"><br />
<p style="font-weight:light; font-size:25px">Providing a NEW COMPREHENSIVE TOOLBOX based on inteins for modifying proteins post-translationally.</p> <br />
<p style="margin-left:50px; font-size:20px">Sending 67 Biobricks to Registry of Biological parts!</p><br />
</div><br />
<br />
<div class="col-xs-3"><br />
<img src="/wiki/images/7/7d/Check_new.png" style="height:100px; margin-right:15px;"><br />
</div><br />
<br />
</div><br />
</div><br />
<div class="linie"></div><br />
<br />
<div class="row vcenter-table" style="margin:0;"><br />
<div class="col-md-3 col-xs-12 vcenter-cell"><br />
<img src="https://static.igem.org/mediawiki/2014/f/f9/Achievement_2.png" class="img-responsive" style="margin-left:15px;"> <br />
</div><br />
<br />
<div class="col-md-9 col-xs-12 vcenter-cell"><br />
<div class="col-xs-9"><br />
<p style="font-weight:light; font-size:25px">Development of a NEW STANDARD to make the use of inteins easy and modular.</p> <br />
<p style="margin-left:50px; font-size:20px">Establishment of a new <a href="https://2014.igem.org/Team:Heidelberg/Parts/RFC">RFC</a>!</p><br />
</div><br />
<br />
<div class="col-xs-3"><br />
<img src="/wiki/images/7/7d/Check_new.png" style="height:100px; margin-right:15px;"><br />
</div><br />
<br />
</div><br />
</div><br />
<div class="linie"></div><br />
<br />
<br />
<div class="row vcenter-table" style="margin:0;"><br />
<div class="col-md-3 col-xs-12 vcenter-cell"><br />
<img src="https://static.igem.org/mediawiki/2014/1/1b/Achievements_toolbox.png" class="img-responsive" style="margin-left:15px;"> <br />
</div><br />
<br />
<div class="col-md-9 col-xs-12 vcenter-cell"><br />
<div class="col-xs-9"><br />
<p style="font-weight:light; font-size:25px">Showing that the toolbox WORKS: proteins are circularized and split fluorescent proteins are reconstituted.</p> <br />
<p style="margin-left:50px; font-size:20px">Making Gels, Western Blots, Fluorescence-based Assays and Mass spectrometry to prove it! </p><br />
</div><br />
<br />
<div class="col-xs-3"><br />
<img src="/wiki/images/7/7d/Check_new.png" style="height:100px; margin-right:15px;"><br />
</div><br />
<br />
</div><br />
</div><br />
<div class="linie"></div><br />
<br />
<div class="row vcenter-table" style="margin:0;"><br />
<div class="col-md-3 col-xs-12 vcenter-cell"><br />
<img src="https://static.igem.org/mediawiki/2014/2/26/Achievements_4.png" class="img-responsive" style="margin-left:15px;"> <br />
</div><br />
<br />
<div class="col-md-9 col-xs-12 vcenter-cell"><br />
<div class="col-xs-9"><br />
<p style="font-weight:light; font-size:25px">Creating circular DNMT1 and showing that it is ACTIVE.</p> <br />
<p style="margin-left:50px; font-size:20px">For the first time achieving the circularization of a large protein!</p><br />
<p style="margin-left:50px; font-size:20px">Circularizing LYSOZYME and XYLANASE, two very important proteins for<br />
research and industry!</p><br />
</div><br />
<br />
<div class="col-xs-3"><br />
<img src="/wiki/images/7/7d/Check_new.png" style="height:100px; margin-right:15px;"><br />
</div><br />
<br />
</div><br />
</div><br />
<div class="linie"></div><br />
<br />
<div class="row vcenter-table" style="margin:0;"><br />
<div class="col-md-3 col-xs-12 vcenter-cell"><br />
<img src="https://static.igem.org/mediawiki/2014/5/5a/Achievements_craut.png" class="img-responsive" style="margin-left:15px;"> <br />
</div><br />
<br />
<div class="col-md-9 col-xs-12 vcenter-cell"><br />
<div class="col-xs-9"><br />
<p style="font-weight:light; font-size:25px">Developing a NEW SOFTWARE to calculate customized linkers to circularize proteins.</p> <br />
<p style="margin-left:50px; font-size:20px">Making CRAUT open-source for the scientific community.</p><br />
</div><br />
<br />
<div class="col-xs-3"><br />
<img src="/wiki/images/7/7d/Check_new.png" style="height:100px; margin-right:15px;"><br />
</div><br />
<br />
</div><br />
</div><br />
<div class="linie"></div><br />
<br />
<div class="row vcenter-table" style="margin:0;"><br />
<div class="col-md-3 col-xs-12 vcenter-cell"><br />
<img src="https://static.igem.org/mediawiki/2014/e/e5/Heidelberg_Frontpage_igemathome.png" class="img-responsive" style="margin-left:15px;"> <br />
</div><br />
<br />
<div class="col-md-9 col-xs-12 vcenter-cell"><br />
<div class="col-xs-9"><br />
<p style="font-weight:light; font-size:25px"> Establishing a distributed computing platform called <a href="https://2014.igem.org/Team:Heidelberg/Software/igemathome">iGEM@home</a>.</p> <br />
<p style="margin-left:50px; font-size:20px">Using this platform as an entirely new way to reach out to the world with synthetic biology concepts!</p><br />
</div><br />
<br />
<br />
<div class="col-xs-3"><br />
<img src="/wiki/images/7/7d/Check_new.png" style="height:100px; margin-right:15px;"><br />
</div><br />
</div><br />
</div><br />
<div class="linie"></div><br />
<div class="row" style="margin:0;"><br />
<div class="col-md-3 col-xs-12 vcenter-cell"><br />
<img src="https://static.igem.org/mediawiki/2014/1/18/Achievements_md.png" class="img-responsive" style="margin-left:15px;"> <br />
</div><br />
<br />
<div class="col-md-9 col-xs-12 vcenter-cell"><br />
<div class="col-xs-9"><br />
<p style="font-weight:light; font-size:25px">Creating a NEW SOFTWARE to display the notebook on the wiki.</p> <br />
<p style="margin-left:50px; font-size:20px">Distributing MidNightDOC to the iGEM community to help future teams organize their protocols!</p><br />
</div><br />
<br />
<br />
<div class="col-xs-3"><br />
<img src="/wiki/images/7/7d/Check_new.png" style="height:100px; margin-right:15px;"><br />
</div><br />
<br />
</div><br />
</div><br />
</div><br />
<br />
<div class="tab-pane fade" style="color:white;" id="Medal_Criteria-tab"><br />
<br />
<div class="col-md-12 col-sm-12 col-xs-12" style="margin-top: 20px"><br />
</div><br />
<div class="row "> <br />
<div class="col-md-3 col-xs-12"><br />
<img class="medal" src="https://static.igem.org/mediawiki/2014/6/63/Heidelberg_Bronze.png"><br />
</div><br />
<br />
<div class="col-md-9 col-xs-12"><br />
<h1>Bronze</h1><br />
<br><br />
<ul><br />
<li>Please find a comprehensive compilation of <a href="https://2014.igem.org/Team:Heidelberg/Team/Sponsoring">sponsors</a>, partners and scientific contributors on our <a href="https://2014.igem.org/Team:Heidelberg/Team/Attributions">acknowledgements page</a>. </li><br />
<br/><br />
<li>We also encourage you to take notice of the projects “Photo-intein” and “Mito-intein” by <a href="https://2014.igem.org/Team:Queens_Canada/Project">iGEM team Queens from Canada</a> that may supply you with complementary information and tools for the use of inteins in synthetic biology!</li><br />
<br/><br />
<li>A list of links to more than 60 parts in the registry submitted by our team (being or not being part of the new intein toolbox) can be found <a href="https://2014.igem.org/Team:Heidelberg/Parts#allParts">here</a>.</li><br />
</ul><br />
</div><br />
</div><br />
<br />
<div class="row "><br />
<div class="col-md-3 col-xs-12"><br />
<img class="medal" src="https://static.igem.org/mediawiki/2014/b/bf/Heidelberg_Silver.png"><br />
</div><br />
<br />
<div class="col-md-9 col-xs-12"><br />
<h1>Silver</h1><br />
<br><br />
<ul><br />
<li>We experimentally validated that our biobricks <a href="http://parts.igem.org/Part:BBa_K1362000">BBa_K1362000</a>, <a href="http://parts.igem.org/Part:BBa_K1362100">BBa_K1362100 </a>and <a href="http://parts.igem.org/Part:BBa_K1362101">BBa_K1362101</a> work as expected. For more information on the <a href="https://2014.igem.org/Team:Heidelberg/Parts#Favorite Parts">parts</a> please visit the corresponding main pages in the parts registry or explore their involvement in our subprojects.</li><br />
<br/><br />
<li>Religious perceptions of synthetic biology have been part of several surveys during the past ten years of iGEM and Human Practices projects. Since religious groups cover the majority of worlds population, deliver moral values and wield power at the same time, we decided to dedicate a whole event on the topic of religion, philosophy and ethics regarding synthetic biology. Please find an <a href="https://2014.igem.org/Team:Heidelberg/Human_Practice/Ethics">evaluation of our event</a> on the corresponding Human Practices pages. <!--In order to reassure ourselves about the acceptability of our project and synthetic biology in general, we also used this opportunity to build up on the work of the iGEM Team Heidelberg 2013 and conducted a survey that addresses basic questions regarding the public reflection of our work.--><br />
</li><br />
</ul><br />
</div><br />
</div><br />
<br />
<div class="row"><br />
<div class="col-md-3 col-xs-12"><br />
<img class="medal" src="https://static.igem.org/mediawiki/2014/0/0a/Heidelberg_Gold.png"><br />
</div><br />
<br />
<div class="col-md-9 col-xs-12"><br />
<h1>Gold</h1><br />
<ul><br />
<li>We improved the function of the <b>already existing</b> biobrick part <a href="http://parts.igem.org/Part:BBa_K117505">BBa_K1175005 </a>by optimizing and resubmitting the corresponding sequence of B. subtilis xylanase to the registry (Part:<a href="http://parts.igem.org/Part:BBa_K1362020"> BBa_K1362020</a>). In addition, we submitted a new part for expression of <a href="http://parts.igem.org/Part:BBa_K1362022">circularized xylanase </a>(BBa_K1362022) that might be used in future applications with need for refined enzyme stability.</li><br />
<br/><br />
<li>Despite the fact that we focused on building a set of powerful soft- and wetware tools to help future iGEM-teams developing and realizing projects in synthetic biology, we are happy to announce that we were also able to help out several team during the course of our project, aspecially with sending <a href="https://2014.igem.org/Team:Heidelberg/Parts#Backbones"> our expression vectors</a>. Read more abou in in our <a href="https://2014.igem.org/Team:Heidelberg/Team/Collaborations">Collaborations</a>.</li><br />
<br/><br />
<li>In the style of of the new iGEM community labs track that involves science amateurs “beyond the accolades of scientific publishing and economic reward”, we sought for a new way to involve laymen in actual science and build a strong community of well informed supporters and communicators of synthetic biology at the same time. Now we proudly present the crowd sourcing and communication platform <a href="https://2014.igem.org/Team:Heidelberg/Human_Practice/igemathome">iGEM@home</a>.</li><br />
</ul><br />
</div><br />
</div><br />
<br />
</div><br />
</div><br />
<br />
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</html></div>Maexlichhttp://2014.igem.org/File:Heidelberg_half_Splicingmechanism.pngFile:Heidelberg half Splicingmechanism.png2014-10-18T03:48:53Z<p>Maexlich: Thumbnail (620px X 883 px) of Heidelberg_orig_Splicingmechanism.png</p>
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<div>Thumbnail (620px X 883 px) of Heidelberg_orig_Splicingmechanism.png</div>Maexlichhttp://2014.igem.org/File:Heidelberg_full_Splicingmechanism.pngFile:Heidelberg full Splicingmechanism.png2014-10-18T03:48:51Z<p>Maexlich: Thumbnail (1070px X 1524 px) of Heidelberg_orig_Splicingmechanism.png</p>
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<div>Thumbnail (1070px X 1524 px) of Heidelberg_orig_Splicingmechanism.png</div>Maexlichhttp://2014.igem.org/File:Heidelberg_quarter_Splicingmechanism.pngFile:Heidelberg quarter Splicingmechanism.png2014-10-18T03:48:48Z<p>Maexlich: Thumbnail (430px X 612 px) of Heidelberg_orig_Splicingmechanism.png</p>
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<div>Thumbnail (430px X 612 px) of Heidelberg_orig_Splicingmechanism.png</div>Maexlichhttp://2014.igem.org/File:Heidelberg_orig_Splicingmechanism.pngFile:Heidelberg orig Splicingmechanism.png2014-10-18T03:48:47Z<p>Maexlich: Original File Heidelberg_orig_Splicingmechanism.png</p>
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<div>Original File Heidelberg_orig_Splicingmechanism.png</div>Maexlichhttp://2014.igem.org/Team:Heidelberg/Toolbox_Guide/LocalizationTeam:Heidelberg/Toolbox Guide/Localization2014-10-18T03:46:51Z<p>Maexlich: </p>
<hr />
<div>{{:Team:Heidelberg/Templates/MainTemplate}}<br />
{{:Team:Heidelberg/Templates/IncludeCSS|:Team:Heidelberg/css/bootstrapcss}}<br />
{{:Team:Heidelberg/Templates/IncludeCSS|:Team:Heidelberg/css/bootstraptheme}}<br />
{{:Team:Heidelberg/Templates/IncludeCSS|:Team:Heidelberg/css/overrides}}<br />
{{:Team:Heidelberg/Templates/IncludeJS|:Team:Heidelberg/js/jquery}}<br />
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{{:Team:Heidelberg/Templates/IncludeJS|:Team:Heidelberg/js/wikipage}}<br />
{{:Team:Heidelberg/Templates/IncludeJS|:Team:Heidelberg/js/scrollTo}}<br />
{{:Team:Heidelberg/Templates/IncludeJS|:Team:Heidelberg/js/knockout}}<br />
<br />
<html><br />
<style type="text/css"><br />
#myContainer {<br />
padding:0;<br />
background-color: black;<br />
background-image: url(/wiki/images/6/6a/Heidelberg_epic_background.jpg);<br />
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-o-transform: scale(1.15);<br />
-ms-transform: scale(1.15);<br />
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<br />
.toolbox-icons-bar a img {<br />
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background-color: rgb(81,81,81);<br />
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margin-bottom: 40px;<br />
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<br />
<br />
</style><br />
<div id="myContainer" class="container"><br />
</html><br />
{{:Team:Heidelberg/Templates/BootstrapNav|<br />
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}}<br />
<html><br />
<div class="container main" style="color: white;"><br />
<div class="row"><br />
<div class="col-lg-8"><br />
<div class="row"><br />
<div class="col-lg-12"><br />
<span class="middle">use the <span class="red-text">intein</span></span><br />
</div> <br />
</div><br />
<div class="row"><br />
<div class="col-lg-12" style="position: relative; top: -20px;"><br />
<span class="large">TOOLBOX&nbsp;</span><span class="middle" >to</span><br />
</div> <br />
</div><br />
</div><br />
<div class="col-lg-4 toolbox-icons-bar"><br />
<div class="col-lg-6 col-md-6 col-sm-6 col-xs-12"><br />
<a href="https://2014.igem.org/Team:Heidelberg/Toolbox/Circularization"><img src="/wiki/images/3/3a/Heidelberg_Localize_button.png" class="img-responsive">LOCALIZE</a><br />
</div><br />
</div><br />
</div><br />
<div class="col-lg-12" style="color:white;"><br />
<h3>First of all, check the DNA sequence of your proteins for EcoRI, XbaI, SpeI, PstI and BsaI sites. If there are E/X/S/P sites, you might have problems to change your backbone or add a promotor. If there is a BsaI site, the cloning will be more difficult.</h3><br />
<div class="panel panel-default"><br />
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<div class="radio"><br />
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<input type="radio" data-bind="checked: data.hasBSAI, checkedValue: false, click: data.q0A.bind(null, true)" /><br />
There is no BsaI site.<br />
</label><br />
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<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.hasBSAI, checkedValue: true, click: data.q0A.bind(null, true)" /><br />
There is a BsaI site.<br />
</label><br />
</div><br />
</div><br />
</div><br />
<div data-bind="if: data.q0A"><br />
<h3>Choose the tag you want to add or remove</h3><br />
<div class="panel panel-default"><br />
<div class="panel-body"><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.isiGEMHD, checkedValue: true, click: data.q1A.bind(null, true)" /><br />
iGEMHD Tags (collection of usefull tags based on Biobricks)<br />
</label><br />
</div><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.isiGEMHD, checkedValue: false, click: data.q1A.bind(null, true)" /><br />
Custom Tag<br />
</label><br />
</div><br />
</div><br />
</div><br />
</div><br />
<br />
<!-- ko if: data.isiGEMHD --><br />
<div data-bind="if: data.q1A"><br />
<h3>Please select the organism in that the tag should be used</h3><br />
<div class="panel panel-default"><br />
<div class="panel-body"><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.inEColi, checkedValue: true, click: data.q2A.bind(null, true)" /><br />
<i>Escherichia coli</i><br />
</label><br />
</div><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.inEColi, checkedValue: false, click: data.q2A.bind(null, true)" /><br />
eukaryotic organism<br />
</label><br />
</div><br />
</div><br />
</div><br />
</div><br />
<br />
<div data-bind="if: data.q2A()"><br />
<h3>Which Tag would you like to use?</h3><br />
<div class="panel panel-default"><br />
<div class="panel-body"><br />
<!-- ko foreach: data.inEColi() ? EColiTags : EucarioticTags --><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: $parent.data.selectedTag, checkedValue: $data, click: $parent.data.q4A.bind(null, true)" /><br />
<span data-bind="text: tagname"></span> (<span data-bind="text: descr"></span>)<br />
</label><br />
</div><br />
<!-- /ko --><br />
</div><br />
</div><br />
</div><br />
<!-- /ko --><br />
<br />
<br />
<br />
<div data-bind="if: false"><br />
<h3>Do you want to add or remove the Tag</h3><br />
<div class="panel panel-default"><br />
<div class="panel-body"><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.addTag, checkedValue: true, click: data.q4A.bind(null, true)" /><br />
Add tag to protein<br />
</label><br />
</div><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.addTag, checkedValue: false, click: data.q4A.bind(null, true)" /><br />
Remove tag via intein protease<br />
</label><br />
</div><br />
</div><br />
</div><br />
</div><br />
<br />
<div data-bind="if: data.q1A() &amp;&amp; !data.isiGEMHD()"><br />
<h3>At which Poition do you want to <span data-bind="if: data.addTag">add</span><span data-bind="if: !data.addTag()">remove</span> the tag?</h3><br />
<div class="panel panel-default"><br />
<div class="panel-body"><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.terminus, checkedValue: 'N', click: data.q5A.bind(null, true)" /><br />
N-Terminus<br />
</label><br />
</div><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.terminus, checkedValue: 'C', click: data.q5A.bind(null, true)" /><br />
C-Terminus<br />
</label><br />
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</div><br />
</div><br />
</div><br />
<br />
<div data-bind="if: (data.isiGEMHD() &amp;&amp; data.q3A() &amp;&amp; data.selectedTag().askFirstStop) || (data.q5A() &amp;&amp; !data.isiGEMHD())"><br />
<h3>Does your protein of interest contain methionin as a first amino acid?</h3><br />
<div class="panel panel-default"><br />
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<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.methionin, checkedValue: true, click: data.q6A.bind(null, true)" /><br />
Yes<br />
</label><br />
</div><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.methionin, checkedValue: false, click: data.q6A.bind(null, true)" /><br />
No<br />
</label><br />
</div><br />
</div><br />
</div><br />
<!-- ko if: data.q6A --><br />
<h3>Does your protein of interest contain a stop codon?</h3><br />
<div class="panel panel-default"><br />
<div class="panel-body"><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.stopCodon, checkedValue: true, click: data.q7A.bind(null, true)" /><br />
Yes<br />
</label><br />
</div><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.stopCodon, checkedValue: false, click: data.q7A.bind(null, true)" /><br />
No<br />
</label><br />
</div><br />
</div><br />
</div><br />
<!-- /ko --><br />
</div><br />
<br />
<div data-bind="if: (!data.isiGEMHD() &amp;&amp; data.q7A()) || (data.isiGEMHD() &amp;&amp; data.q7A() &amp;&amp; data.selectedTag().askFirstStop) || (data.q4A() &amp;&amp; data.isiGEMHD() &amp;&amp; !data.selectedTag().askFirstStop )"><br />
<h3>How do you want to induce the trans-splicing reaction?</h3><br />
<div class="panel panel-default"><br />
<div class="panel-body"><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.lightInduced, checkedValue: false, click: data.q8A.bind(null, true)" /><br />
regulation of gene expression<br />
</label><br />
</div><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.lightInduced, checkedValue: true, click: data.q8A.bind(null, true)" /><br />
light induction<br />
</label><br />
</div><br />
</div><br />
</div><br />
</div><br />
<div data-bind="if: data.q8A"><br />
<h1>Protocol</h1><br />
<ul><br />
<li>Get BBa_K1362105 (RBS + NpuDnaE C-intein RFC [<span data-bind="text: RFCNumber"></span>] assembly construct) from the registry of standard biological parts.</li><br />
<li>Get BBa_K1362104 (RBS +NpuDnaE N-intein RFC [<span data-bind="text: RFCNumber"></span>] assembly construct) from the registry registry of standard biological parts.</li><br />
<li>Get <span data-bind="if: data.isiGEMHD"><span data-bind="text: data.selectedTag().Biobrick"></span></span><span data-bind="if: !data.isiGEMHD">your tag</span> from the registry of standard biological parts.</li><br />
<li>Get the DNA of your protein of interest.</li><br />
<!-- ko if: data.addTag --><br />
<!-- ko if: data.isiGEMHD --><br />
<li><span data-bind="if: data.isiGEMHD() &amp;&amp; data.selectedTag().tagname == 'MinD MTS'"><br />
Use Golden Gate Assembly to replace the mRFP selection marker in BBa_K1362105 (RBS + NpuDnaE C-intein RFC [<span data-bind="text: RFCNumber"></span>] assembly construct) with the C-terminal tag.<br />
</span><br />
<span data-bind="if: data.isiGEMHD() &amp;&amp; data.selectedTag().tagname != 'MinD MTS'"><br />
Use Golden Gate Assembly to replace the mRFP selection marker in BBa_K1362104 (RBS + NpuDnaE N-intein RFC [<span data-bind="text: RFCNumber"></span>] assembly construct) with the N-terminal tag.<br />
</span><br />
</li><br />
<!-- /ko --><br />
<!-- /ko --><br />
<!-- ko if: !data.isiGEMHD() --><br />
<li><br />
<p>Design and order primers</p><br />
<p>In 5'-3 direction, the forward primer should have the following sequence:</p><br />
<p><br />
<span data-bind="if: data.terminus() == 'C' &amp;&amp; data.addTag()">NNNNNNNNGGTCTCCCAACTGCTGGGAA + binding part</span><br />
<span data-bind="if: data.terminus() == 'N' &amp;&amp; data.addTag() &amp;&amp; !data.methionin()">NNNNNNNNGGTCTCAGATG + binding part</span><br />
<span data-bind="if: data.terminus() == 'N' &amp;&amp; data.addTag() &amp;&amp; data.methionin()">NNNNNNNNGGTCTCAGATG + binding part</span><!-- 2.1.1.2 --><br />
</p><br />
<p>In 5'-3' direction, the reverse primer should have the following sequence:</p><br />
<p><br />
<span data-bind="if: data.terminus() == 'C' &amp;&amp; data.addTag()">binding part + NNNNNNNNGGTCTCTATTA</span><br />
<span data-bind="if: data.terminus() == 'N' &amp;&amp; data.addTag()">binding part + NNNNNNNNGGTCTCGAGCACTTGCCCCT</span><br><br />
<span data-bind="if: data.terminus() == 'N' &amp;&amp; data.addTag() &amp;&amp; data.stopCodon()">Be careful not to include stop codons of your protein of interest into the binding part.</span><br />
</p><br />
</li><br />
<li><br />
<p>Use PCR to create your insert and purify it.</p><br />
</li><br />
<li><br />
<p>Use Golden Gate Assembly to replace the mRFP selection marker in <br />
<span data-bind="if: data.terminus() == 'C' &amp;&amp; data.addTag()">BBa_K1362105 (RBS + SspDnaE C-intein RFC [<span data-bind="text: RFCNumber"></span>] assembly construct) with your C-terminal tag</span><br />
<span data-bind="if: data.terminus() == 'N' &amp;&amp; data.addTag()">BBa_K1362104 (RBS + SspDnaE N-intein RFC [<span data-bind="text: RFCNumber"></span>] assembly construct). With your N-terminal tag.</span><br />
</p><br />
</li><br />
<!-- /ko --><br />
<li><br />
<p>Backtranslate the amino acid sequence of your protein of interest into a nucleotide</p><br />
</li><br />
<li><br />
<p>Design and order primers:</p><br />
<p>In 5'-3' direction, the forward primer should have the following sequence:</p><br />
<span data-bind="if: data.addTag() &amp;&amp; ((data.isiGEMHD() &amp;&amp; data.selectedTag().tagname == 'MinD MTS') || (!data.isiGEMHD() &amp;&amp; data.terminus() == 'C'))"><br />
<p><br />
<span data-bind="if: !data.methionin()">NNNNNNGGTCTCAGATG + binding part</span><br />
<span data-bind="if: data.methionin()">NNNNNNGGTCTCAG + binding part</span><br />
</p><br />
<p>In 5'-3' direction, the reverse primer should have the following sequence:</p><br />
<p>NNNNNNNNGGTCTCGAGCACTTGCCCCT + binding part</p><br />
<span data-bind="if: data.stopCodon"><br />
Be careful not to include stop codons of your protein of interest into the binding part.<br />
</span> <br />
</span><br />
<span data-bind="if: (data.isiGEMHD() &amp;&amp; data.selectedTag().tagname != 'MinD MTS') || (!data.isiGEMHD() &amp;&amp; data.terminus() == 'N')"><br />
<p>In 5'-3' direction, the forward primer should have the following sequence:</p><br />
<p>NNNNNNGGTCTCCCAACTGCTGGGAA + binding part</p><br />
<p>In 5'-3' direction, the reverse primer should have the following sequence:</p><br />
<p>NNNNNNGGTCTCTATTA + binding part</p><br />
</span><br />
</li><br />
<li><br />
<p>Use PCR to create your insert and purify it.</p><br />
</li><br />
<li><br />
<p>Use Golden Gate assembly to to exchange the mRFP selection marker in <br />
<span data-bind="if: data.addTag() &amp;&amp; ((data.isiGEMHD() &amp;&amp; data.selectedTag().tagname == 'MinD MTS') || (!data.isiGEMHD() &amp;&amp; data.terminus() == 'C'))"><br />
BBa_K1362104 ( SspDnaE N-intein RFC [<span data-bind="text: RFCNumber"></span>] assembly construct) with your insert.</span><br />
<span data-bind="if: (data.isiGEMHD() &amp;&amp; data.selectedTag().tagname != 'MinD MTS') || (!data.isiGEMHD() &amp;&amp; data.terminus() == 'N')"><br />
BBa_K1362105 ( SspDnaE C-intein RFC [<span data-bind="text: RFCNumber"></span>] assembly construct) with your insert.<br />
</span> <br />
<span data-bind="if: data.hasBSAI"><p>Since there is a BsaI site in your protein, you have to religate after the Golden Gate reaction.</p></span><br />
</p><br />
</li><br />
<li><br />
<span data-bind="if: !data.lightInduced()"><br />
<p>Clone both assembly constructs into compatible expression vectors.</p><br />
</span><br />
<span data-bind="if: data.lightInduced()"><br />
<p>Please use: <br />
<a href="/Team:Heidelberg/Project/LOV">LOV</a> to induce splicing<br />
</p><br />
</span><br />
</li><br />
</ul><br />
<br />
</div><br />
<br />
</div><br />
</div><br />
<br />
<script type="text/javascript"><br />
$(document).ready(function(){<br />
vm = new TaggingViewModel();<br />
ko.applyBindings(vm);<br />
});<br />
</script><br />
</html><br />
{{:Team:Heidelberg/Templates/IncludeJS|:Team:Heidelberg/js/toolboxguide}}</div>Maexlichhttp://2014.igem.org/Team:Heidelberg/Toolbox_Guide/LocalizationTeam:Heidelberg/Toolbox Guide/Localization2014-10-18T03:46:30Z<p>Maexlich: Undo revision 399553 by Maexlich (talk)</p>
<hr />
<div>{{:Team:Heidelberg/Templates/MainTemplate}}<br />
{{:Team:Heidelberg/Templates/IncludeCSS|:Team:Heidelberg/css/bootstrapcss}}<br />
{{:Team:Heidelberg/Templates/IncludeCSS|:Team:Heidelberg/css/bootstraptheme}}<br />
{{:Team:Heidelberg/Templates/IncludeCSS|:Team:Heidelberg/css/overrides}}<br />
{{:Team:Heidelberg/Templates/IncludeJS|:Team:Heidelberg/js/jquery}}<br />
{{:Team:Heidelberg/Templates/IncludeJS|:Team:Heidelberg/js/bootstrapjs}}<br />
{{:Team:Heidelberg/Templates/IncludeJS|:Team:Heidelberg/js/wikipage}}<br />
{{:Team:Heidelberg/Templates/IncludeJS|:Team:Heidelberg/js/scrollTo}}<br />
{{:Team:Heidelberg/Templates/IncludeJS|:Team:Heidelberg/js/knockout}}<br />
<br />
<html><br />
<style type="text/css"><br />
#myContainer {<br />
padding:0;<br />
background-color: black;<br />
background-image: url(/wiki/images/6/6a/Heidelberg_epic_background.jpg);<br />
background-repeat: no-repeat;<br />
background-size: 100% auto;<br />
}<br />
<br />
#light:hover {<br />
color: #FF7E25;<br />
}<br />
<br />
.main {<br />
margin-top: 25px;<br />
}<br />
<br />
.middle {<br />
font-size: 3em;<br />
}<br />
<br />
.large {<br />
font-size: 5em;<br />
font-weight: bold;<br />
}<br />
<br />
/* Enlarge Icons on hover */<br />
.toolbox-icons-bar a:hover img {<br />
transform: scale(1.15);<br />
-webkit-transform: scale(1.15);<br />
-moz-transform: scale(1.15);<br />
-o-transform: scale(1.15);<br />
-ms-transform: scale(1.15);<br />
}<br />
<br />
.toolbox-icons-bar a img {<br />
transition:transform 0.15s ease;<br />
-webkit-transition:-webkit-transform 0.15s ease;<br />
-moz-transition:-moz-transform 0.15s ease;<br />
-o-transition:-o-transform 0.15s ease;<br />
}<br />
<br />
.panel.panel-default {<br />
border: solid 2px #de4230;<br />
font-size: 1.2em;<br />
background-color: rgb(81,81,81);<br />
background-color: rgba(81,81,81,0.7);<br />
margin-bottom: 40px;<br />
}<br />
<br />
<br />
</style><br />
<div id="myContainer" class="container"><br />
</html><br />
{{:Team:Heidelberg/Templates/BootstrapNav|<br />
red=|<br />
white=true|<br />
red-logo=true|<br />
white-logo=|<br />
header-bg=|<br />
header-img=|<br />
title=<br />
}}<br />
<html><br />
<div class="container main" style="color: white;"><br />
<div class="row"><br />
<div class="col-lg-8"><br />
<div class="row"><br />
<div class="col-lg-12"><br />
<span class="middle">use the <span class="red-text">intein</span></span><br />
</div> <br />
</div><br />
<div class="row"><br />
<div class="col-lg-12" style="position: relative; top: -20px;"><br />
<span class="large">TOOLBOX&nbsp;</span><span class="middle" >to</span><br />
</div> <br />
</div><br />
</div><br />
<div class="col-lg-4 toolbox-icons-bar"><br />
<div class="col-lg-6 col-md-6 col-sm-6 col-xs-12"><br />
<a href="https://2014.igem.org/Team:Heidelberg/Toolbox/Circularization"><img src="/wiki/images/4/40/Oligomerization.png" class="img-responsive">LOCALIZE</a><br />
</div><br />
</div><br />
</div><br />
<div class="col-lg-12" style="color:white;"><br />
<h3>First of all, check the DNA sequence of your proteins for EcoRI, XbaI, SpeI, PstI and BsaI sites. If there are E/X/S/P sites, you might have problems to change your backbone or add a promotor. If there is a BsaI site, the cloning will be more difficult.</h3><br />
<div class="panel panel-default"><br />
<div class="panel-body"><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.hasBSAI, checkedValue: false, click: data.q0A.bind(null, true)" /><br />
There is no BsaI site.<br />
</label><br />
</div><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.hasBSAI, checkedValue: true, click: data.q0A.bind(null, true)" /><br />
There is a BsaI site.<br />
</label><br />
</div><br />
</div><br />
</div><br />
<div data-bind="if: data.q0A"><br />
<h3>Choose the tag you want to add or remove</h3><br />
<div class="panel panel-default"><br />
<div class="panel-body"><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.isiGEMHD, checkedValue: true, click: data.q1A.bind(null, true)" /><br />
iGEMHD Tags (collection of usefull tags based on Biobricks)<br />
</label><br />
</div><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.isiGEMHD, checkedValue: false, click: data.q1A.bind(null, true)" /><br />
Custom Tag<br />
</label><br />
</div><br />
</div><br />
</div><br />
</div><br />
<br />
<!-- ko if: data.isiGEMHD --><br />
<div data-bind="if: data.q1A"><br />
<h3>Please select the organism in that the tag should be used</h3><br />
<div class="panel panel-default"><br />
<div class="panel-body"><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.inEColi, checkedValue: true, click: data.q2A.bind(null, true)" /><br />
<i>Escherichia coli</i><br />
</label><br />
</div><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.inEColi, checkedValue: false, click: data.q2A.bind(null, true)" /><br />
eukaryotic organism<br />
</label><br />
</div><br />
</div><br />
</div><br />
</div><br />
<br />
<div data-bind="if: data.q2A()"><br />
<h3>Which Tag would you like to use?</h3><br />
<div class="panel panel-default"><br />
<div class="panel-body"><br />
<!-- ko foreach: data.inEColi() ? EColiTags : EucarioticTags --><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: $parent.data.selectedTag, checkedValue: $data, click: $parent.data.q4A.bind(null, true)" /><br />
<span data-bind="text: tagname"></span> (<span data-bind="text: descr"></span>)<br />
</label><br />
</div><br />
<!-- /ko --><br />
</div><br />
</div><br />
</div><br />
<!-- /ko --><br />
<br />
<br />
<br />
<div data-bind="if: false"><br />
<h3>Do you want to add or remove the Tag</h3><br />
<div class="panel panel-default"><br />
<div class="panel-body"><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.addTag, checkedValue: true, click: data.q4A.bind(null, true)" /><br />
Add tag to protein<br />
</label><br />
</div><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.addTag, checkedValue: false, click: data.q4A.bind(null, true)" /><br />
Remove tag via intein protease<br />
</label><br />
</div><br />
</div><br />
</div><br />
</div><br />
<br />
<div data-bind="if: data.q1A() &amp;&amp; !data.isiGEMHD()"><br />
<h3>At which Poition do you want to <span data-bind="if: data.addTag">add</span><span data-bind="if: !data.addTag()">remove</span> the tag?</h3><br />
<div class="panel panel-default"><br />
<div class="panel-body"><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.terminus, checkedValue: 'N', click: data.q5A.bind(null, true)" /><br />
N-Terminus<br />
</label><br />
</div><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.terminus, checkedValue: 'C', click: data.q5A.bind(null, true)" /><br />
C-Terminus<br />
</label><br />
</div><br />
</div><br />
</div><br />
</div><br />
<br />
<div data-bind="if: (data.isiGEMHD() &amp;&amp; data.q3A() &amp;&amp; data.selectedTag().askFirstStop) || (data.q5A() &amp;&amp; !data.isiGEMHD())"><br />
<h3>Does your protein of interest contain methionin as a first amino acid?</h3><br />
<div class="panel panel-default"><br />
<div class="panel-body"><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.methionin, checkedValue: true, click: data.q6A.bind(null, true)" /><br />
Yes<br />
</label><br />
</div><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.methionin, checkedValue: false, click: data.q6A.bind(null, true)" /><br />
No<br />
</label><br />
</div><br />
</div><br />
</div><br />
<!-- ko if: data.q6A --><br />
<h3>Does your protein of interest contain a stop codon?</h3><br />
<div class="panel panel-default"><br />
<div class="panel-body"><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.stopCodon, checkedValue: true, click: data.q7A.bind(null, true)" /><br />
Yes<br />
</label><br />
</div><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.stopCodon, checkedValue: false, click: data.q7A.bind(null, true)" /><br />
No<br />
</label><br />
</div><br />
</div><br />
</div><br />
<!-- /ko --><br />
</div><br />
<br />
<div data-bind="if: (!data.isiGEMHD() &amp;&amp; data.q7A()) || (data.isiGEMHD() &amp;&amp; data.q7A() &amp;&amp; data.selectedTag().askFirstStop) || (data.q4A() &amp;&amp; data.isiGEMHD() &amp;&amp; !data.selectedTag().askFirstStop )"><br />
<h3>How do you want to induce the trans-splicing reaction?</h3><br />
<div class="panel panel-default"><br />
<div class="panel-body"><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.lightInduced, checkedValue: false, click: data.q8A.bind(null, true)" /><br />
regulation of gene expression<br />
</label><br />
</div><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.lightInduced, checkedValue: true, click: data.q8A.bind(null, true)" /><br />
light induction<br />
</label><br />
</div><br />
</div><br />
</div><br />
</div><br />
<div data-bind="if: data.q8A"><br />
<h1>Protocol</h1><br />
<ul><br />
<li>Get BBa_K1362105 (RBS + NpuDnaE C-intein RFC [<span data-bind="text: RFCNumber"></span>] assembly construct) from the registry of standard biological parts.</li><br />
<li>Get BBa_K1362104 (RBS +NpuDnaE N-intein RFC [<span data-bind="text: RFCNumber"></span>] assembly construct) from the registry registry of standard biological parts.</li><br />
<li>Get <span data-bind="if: data.isiGEMHD"><span data-bind="text: data.selectedTag().Biobrick"></span></span><span data-bind="if: !data.isiGEMHD">your tag</span> from the registry of standard biological parts.</li><br />
<li>Get the DNA of your protein of interest.</li><br />
<!-- ko if: data.addTag --><br />
<!-- ko if: data.isiGEMHD --><br />
<li><span data-bind="if: data.isiGEMHD() &amp;&amp; data.selectedTag().tagname == 'MinD MTS'"><br />
Use Golden Gate Assembly to replace the mRFP selection marker in BBa_K1362105 (RBS + NpuDnaE C-intein RFC [<span data-bind="text: RFCNumber"></span>] assembly construct) with the C-terminal tag.<br />
</span><br />
<span data-bind="if: data.isiGEMHD() &amp;&amp; data.selectedTag().tagname != 'MinD MTS'"><br />
Use Golden Gate Assembly to replace the mRFP selection marker in BBa_K1362104 (RBS + NpuDnaE N-intein RFC [<span data-bind="text: RFCNumber"></span>] assembly construct) with the N-terminal tag.<br />
</span><br />
</li><br />
<!-- /ko --><br />
<!-- /ko --><br />
<!-- ko if: !data.isiGEMHD() --><br />
<li><br />
<p>Design and order primers</p><br />
<p>In 5'-3 direction, the forward primer should have the following sequence:</p><br />
<p><br />
<span data-bind="if: data.terminus() == 'C' &amp;&amp; data.addTag()">NNNNNNNNGGTCTCCCAACTGCTGGGAA + binding part</span><br />
<span data-bind="if: data.terminus() == 'N' &amp;&amp; data.addTag() &amp;&amp; !data.methionin()">NNNNNNNNGGTCTCAGATG + binding part</span><br />
<span data-bind="if: data.terminus() == 'N' &amp;&amp; data.addTag() &amp;&amp; data.methionin()">NNNNNNNNGGTCTCAGATG + binding part</span><!-- 2.1.1.2 --><br />
</p><br />
<p>In 5'-3' direction, the reverse primer should have the following sequence:</p><br />
<p><br />
<span data-bind="if: data.terminus() == 'C' &amp;&amp; data.addTag()">binding part + NNNNNNNNGGTCTCTATTA</span><br />
<span data-bind="if: data.terminus() == 'N' &amp;&amp; data.addTag()">binding part + NNNNNNNNGGTCTCGAGCACTTGCCCCT</span><br><br />
<span data-bind="if: data.terminus() == 'N' &amp;&amp; data.addTag() &amp;&amp; data.stopCodon()">Be careful not to include stop codons of your protein of interest into the binding part.</span><br />
</p><br />
</li><br />
<li><br />
<p>Use PCR to create your insert and purify it.</p><br />
</li><br />
<li><br />
<p>Use Golden Gate Assembly to replace the mRFP selection marker in <br />
<span data-bind="if: data.terminus() == 'C' &amp;&amp; data.addTag()">BBa_K1362105 (RBS + SspDnaE C-intein RFC [<span data-bind="text: RFCNumber"></span>] assembly construct) with your C-terminal tag</span><br />
<span data-bind="if: data.terminus() == 'N' &amp;&amp; data.addTag()">BBa_K1362104 (RBS + SspDnaE N-intein RFC [<span data-bind="text: RFCNumber"></span>] assembly construct). With your N-terminal tag.</span><br />
</p><br />
</li><br />
<!-- /ko --><br />
<li><br />
<p>Backtranslate the amino acid sequence of your protein of interest into a nucleotide</p><br />
</li><br />
<li><br />
<p>Design and order primers:</p><br />
<p>In 5'-3' direction, the forward primer should have the following sequence:</p><br />
<span data-bind="if: data.addTag() &amp;&amp; ((data.isiGEMHD() &amp;&amp; data.selectedTag().tagname == 'MinD MTS') || (!data.isiGEMHD() &amp;&amp; data.terminus() == 'C'))"><br />
<p><br />
<span data-bind="if: !data.methionin()">NNNNNNGGTCTCAGATG + binding part</span><br />
<span data-bind="if: data.methionin()">NNNNNNGGTCTCAG + binding part</span><br />
</p><br />
<p>In 5'-3' direction, the reverse primer should have the following sequence:</p><br />
<p>NNNNNNNNGGTCTCGAGCACTTGCCCCT + binding part</p><br />
<span data-bind="if: data.stopCodon"><br />
Be careful not to include stop codons of your protein of interest into the binding part.<br />
</span> <br />
</span><br />
<span data-bind="if: (data.isiGEMHD() &amp;&amp; data.selectedTag().tagname != 'MinD MTS') || (!data.isiGEMHD() &amp;&amp; data.terminus() == 'N')"><br />
<p>In 5'-3' direction, the forward primer should have the following sequence:</p><br />
<p>NNNNNNGGTCTCCCAACTGCTGGGAA + binding part</p><br />
<p>In 5'-3' direction, the reverse primer should have the following sequence:</p><br />
<p>NNNNNNGGTCTCTATTA + binding part</p><br />
</span><br />
</li><br />
<li><br />
<p>Use PCR to create your insert and purify it.</p><br />
</li><br />
<li><br />
<p>Use Golden Gate assembly to to exchange the mRFP selection marker in <br />
<span data-bind="if: data.addTag() &amp;&amp; ((data.isiGEMHD() &amp;&amp; data.selectedTag().tagname == 'MinD MTS') || (!data.isiGEMHD() &amp;&amp; data.terminus() == 'C'))"><br />
BBa_K1362104 ( SspDnaE N-intein RFC [<span data-bind="text: RFCNumber"></span>] assembly construct) with your insert.</span><br />
<span data-bind="if: (data.isiGEMHD() &amp;&amp; data.selectedTag().tagname != 'MinD MTS') || (!data.isiGEMHD() &amp;&amp; data.terminus() == 'N')"><br />
BBa_K1362105 ( SspDnaE C-intein RFC [<span data-bind="text: RFCNumber"></span>] assembly construct) with your insert.<br />
</span> <br />
<span data-bind="if: data.hasBSAI"><p>Since there is a BsaI site in your protein, you have to religate after the Golden Gate reaction.</p></span><br />
</p><br />
</li><br />
<li><br />
<span data-bind="if: !data.lightInduced()"><br />
<p>Clone both assembly constructs into compatible expression vectors.</p><br />
</span><br />
<span data-bind="if: data.lightInduced()"><br />
<p>Please use: <br />
<a href="/Team:Heidelberg/Project/LOV">LOV</a> to induce splicing<br />
</p><br />
</span><br />
</li><br />
</ul><br />
<br />
</div><br />
<br />
</div><br />
</div><br />
<br />
<script type="text/javascript"><br />
$(document).ready(function(){<br />
vm = new TaggingViewModel();<br />
ko.applyBindings(vm);<br />
});<br />
</script><br />
</html><br />
{{:Team:Heidelberg/Templates/IncludeJS|:Team:Heidelberg/js/toolboxguide}}</div>Maexlichhttp://2014.igem.org/Team:Heidelberg/Toolbox_Guide/LocalizationTeam:Heidelberg/Toolbox Guide/Localization2014-10-18T03:43:48Z<p>Maexlich: </p>
<hr />
<div>{{:Team:Heidelberg/Templates/MainTemplate}}<br />
{{:Team:Heidelberg/Templates/IncludeCSS|:Team:Heidelberg/css/bootstrapcss}}<br />
{{:Team:Heidelberg/Templates/IncludeCSS|:Team:Heidelberg/css/bootstraptheme}}<br />
{{:Team:Heidelberg/Templates/IncludeCSS|:Team:Heidelberg/css/overrides}}<br />
{{:Team:Heidelberg/Templates/IncludeJS|:Team:Heidelberg/js/jquery}}<br />
{{:Team:Heidelberg/Templates/IncludeJS|:Team:Heidelberg/js/bootstrapjs}}<br />
{{:Team:Heidelberg/Templates/IncludeJS|:Team:Heidelberg/js/wikipage}}<br />
{{:Team:Heidelberg/Templates/IncludeJS|:Team:Heidelberg/js/scrollTo}}<br />
{{:Team:Heidelberg/Templates/IncludeJS|:Team:Heidelberg/js/knockout}}<br />
<br />
<html><br />
<style type="text/css"><br />
#myContainer {<br />
padding:0;<br />
background-color: black;<br />
background-image: url(/wiki/images/6/6a/Heidelberg_epic_background.jpg);<br />
background-repeat: no-repeat;<br />
background-size: 100% auto;<br />
}<br />
<br />
#light:hover {<br />
color: #FF7E25;<br />
}<br />
<br />
.main {<br />
margin-top: 25px;<br />
}<br />
<br />
.middle {<br />
font-size: 3em;<br />
}<br />
<br />
.large {<br />
font-size: 5em;<br />
font-weight: bold;<br />
}<br />
<br />
/* Enlarge Icons on hover */<br />
.toolbox-icons-bar a:hover img {<br />
transform: scale(1.15);<br />
-webkit-transform: scale(1.15);<br />
-moz-transform: scale(1.15);<br />
-o-transform: scale(1.15);<br />
-ms-transform: scale(1.15);<br />
}<br />
<br />
.toolbox-icons-bar a img {<br />
transition:transform 0.15s ease;<br />
-webkit-transition:-webkit-transform 0.15s ease;<br />
-moz-transition:-moz-transform 0.15s ease;<br />
-o-transition:-o-transform 0.15s ease;<br />
}<br />
<br />
.panel.panel-default {<br />
border: solid 2px #de4230;<br />
font-size: 1.2em;<br />
background-color: rgb(81,81,81);<br />
background-color: rgba(81,81,81,0.7);<br />
margin-bottom: 40px;<br />
}<br />
<br />
<br />
</style><br />
<div id="myContainer" class="container"><br />
</html><br />
{{:Team:Heidelberg/Templates/BootstrapNav|<br />
red=|<br />
white=true|<br />
red-logo=true|<br />
white-logo=|<br />
header-bg=|<br />
header-img=|<br />
title=<br />
}}<br />
<html><br />
<div class="container main" style="color: white;"><br />
<div class="row"><br />
<div class="col-lg-8"><br />
<div class="row"><br />
<div class="col-lg-12"><br />
<span class="middle">use the <span class="red-text">intein</span></span><br />
</div> <br />
</div><br />
<div class="row"><br />
<div class="col-lg-12" style="position: relative; top: -20px;"><br />
<span class="large">TOOLBOX&nbsp;</span><span class="middle" >to</span><br />
</div> <br />
</div><br />
</div><br />
<div class="col-lg-4 toolbox-icons-bar"><br />
<div class="col-lg-6 col-md-6 col-sm-6 col-xs-12"><br />
<a href="/wiki/images/3/3a/Heidelberg_Localize_button.png" class="img-responsive">LOCALIZE</a><br />
</div><br />
</div><br />
</div><br />
<div class="col-lg-12" style="color:white;"><br />
<h3>First of all, check the DNA sequence of your proteins for EcoRI, XbaI, SpeI, PstI and BsaI sites. If there are E/X/S/P sites, you might have problems to change your backbone or add a promotor. If there is a BsaI site, the cloning will be more difficult.</h3><br />
<div class="panel panel-default"><br />
<div class="panel-body"><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.hasBSAI, checkedValue: false, click: data.q0A.bind(null, true)" /><br />
There is no BsaI site.<br />
</label><br />
</div><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.hasBSAI, checkedValue: true, click: data.q0A.bind(null, true)" /><br />
There is a BsaI site.<br />
</label><br />
</div><br />
</div><br />
</div><br />
<div data-bind="if: data.q0A"><br />
<h3>Choose the tag you want to add or remove</h3><br />
<div class="panel panel-default"><br />
<div class="panel-body"><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.isiGEMHD, checkedValue: true, click: data.q1A.bind(null, true)" /><br />
iGEMHD Tags (collection of usefull tags based on Biobricks)<br />
</label><br />
</div><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.isiGEMHD, checkedValue: false, click: data.q1A.bind(null, true)" /><br />
Custom Tag<br />
</label><br />
</div><br />
</div><br />
</div><br />
</div><br />
<br />
<!-- ko if: data.isiGEMHD --><br />
<div data-bind="if: data.q1A"><br />
<h3>Please select the organism in that the tag should be used</h3><br />
<div class="panel panel-default"><br />
<div class="panel-body"><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.inEColi, checkedValue: true, click: data.q2A.bind(null, true)" /><br />
<i>Escherichia coli</i><br />
</label><br />
</div><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.inEColi, checkedValue: false, click: data.q2A.bind(null, true)" /><br />
eukaryotic organism<br />
</label><br />
</div><br />
</div><br />
</div><br />
</div><br />
<br />
<div data-bind="if: data.q2A()"><br />
<h3>Which Tag would you like to use?</h3><br />
<div class="panel panel-default"><br />
<div class="panel-body"><br />
<!-- ko foreach: data.inEColi() ? EColiTags : EucarioticTags --><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: $parent.data.selectedTag, checkedValue: $data, click: $parent.data.q4A.bind(null, true)" /><br />
<span data-bind="text: tagname"></span> (<span data-bind="text: descr"></span>)<br />
</label><br />
</div><br />
<!-- /ko --><br />
</div><br />
</div><br />
</div><br />
<!-- /ko --><br />
<br />
<br />
<br />
<div data-bind="if: false"><br />
<h3>Do you want to add or remove the Tag</h3><br />
<div class="panel panel-default"><br />
<div class="panel-body"><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.addTag, checkedValue: true, click: data.q4A.bind(null, true)" /><br />
Add tag to protein<br />
</label><br />
</div><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.addTag, checkedValue: false, click: data.q4A.bind(null, true)" /><br />
Remove tag via intein protease<br />
</label><br />
</div><br />
</div><br />
</div><br />
</div><br />
<br />
<div data-bind="if: data.q1A() &amp;&amp; !data.isiGEMHD()"><br />
<h3>At which Poition do you want to <span data-bind="if: data.addTag">add</span><span data-bind="if: !data.addTag()">remove</span> the tag?</h3><br />
<div class="panel panel-default"><br />
<div class="panel-body"><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.terminus, checkedValue: 'N', click: data.q5A.bind(null, true)" /><br />
N-Terminus<br />
</label><br />
</div><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.terminus, checkedValue: 'C', click: data.q5A.bind(null, true)" /><br />
C-Terminus<br />
</label><br />
</div><br />
</div><br />
</div><br />
</div><br />
<br />
<div data-bind="if: (data.isiGEMHD() &amp;&amp; data.q3A() &amp;&amp; data.selectedTag().askFirstStop) || (data.q5A() &amp;&amp; !data.isiGEMHD())"><br />
<h3>Does your protein of interest contain methionin as a first amino acid?</h3><br />
<div class="panel panel-default"><br />
<div class="panel-body"><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.methionin, checkedValue: true, click: data.q6A.bind(null, true)" /><br />
Yes<br />
</label><br />
</div><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.methionin, checkedValue: false, click: data.q6A.bind(null, true)" /><br />
No<br />
</label><br />
</div><br />
</div><br />
</div><br />
<!-- ko if: data.q6A --><br />
<h3>Does your protein of interest contain a stop codon?</h3><br />
<div class="panel panel-default"><br />
<div class="panel-body"><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.stopCodon, checkedValue: true, click: data.q7A.bind(null, true)" /><br />
Yes<br />
</label><br />
</div><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.stopCodon, checkedValue: false, click: data.q7A.bind(null, true)" /><br />
No<br />
</label><br />
</div><br />
</div><br />
</div><br />
<!-- /ko --><br />
</div><br />
<br />
<div data-bind="if: (!data.isiGEMHD() &amp;&amp; data.q7A()) || (data.isiGEMHD() &amp;&amp; data.q7A() &amp;&amp; data.selectedTag().askFirstStop) || (data.q4A() &amp;&amp; data.isiGEMHD() &amp;&amp; !data.selectedTag().askFirstStop )"><br />
<h3>How do you want to induce the trans-splicing reaction?</h3><br />
<div class="panel panel-default"><br />
<div class="panel-body"><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.lightInduced, checkedValue: false, click: data.q8A.bind(null, true)" /><br />
regulation of gene expression<br />
</label><br />
</div><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.lightInduced, checkedValue: true, click: data.q8A.bind(null, true)" /><br />
light induction<br />
</label><br />
</div><br />
</div><br />
</div><br />
</div><br />
<div data-bind="if: data.q8A"><br />
<h1>Protocol</h1><br />
<ul><br />
<li>Get BBa_K1362105 (RBS + NpuDnaE C-intein RFC [<span data-bind="text: RFCNumber"></span>] assembly construct) from the registry of standard biological parts.</li><br />
<li>Get BBa_K1362104 (RBS +NpuDnaE N-intein RFC [<span data-bind="text: RFCNumber"></span>] assembly construct) from the registry registry of standard biological parts.</li><br />
<li>Get <span data-bind="if: data.isiGEMHD"><span data-bind="text: data.selectedTag().Biobrick"></span></span><span data-bind="if: !data.isiGEMHD">your tag</span> from the registry of standard biological parts.</li><br />
<li>Get the DNA of your protein of interest.</li><br />
<!-- ko if: data.addTag --><br />
<!-- ko if: data.isiGEMHD --><br />
<li><span data-bind="if: data.isiGEMHD() &amp;&amp; data.selectedTag().tagname == 'MinD MTS'"><br />
Use Golden Gate Assembly to replace the mRFP selection marker in BBa_K1362105 (RBS + NpuDnaE C-intein RFC [<span data-bind="text: RFCNumber"></span>] assembly construct) with the C-terminal tag.<br />
</span><br />
<span data-bind="if: data.isiGEMHD() &amp;&amp; data.selectedTag().tagname != 'MinD MTS'"><br />
Use Golden Gate Assembly to replace the mRFP selection marker in BBa_K1362104 (RBS + NpuDnaE N-intein RFC [<span data-bind="text: RFCNumber"></span>] assembly construct) with the N-terminal tag.<br />
</span><br />
</li><br />
<!-- /ko --><br />
<!-- /ko --><br />
<!-- ko if: !data.isiGEMHD() --><br />
<li><br />
<p>Design and order primers</p><br />
<p>In 5'-3 direction, the forward primer should have the following sequence:</p><br />
<p><br />
<span data-bind="if: data.terminus() == 'C' &amp;&amp; data.addTag()">NNNNNNNNGGTCTCCCAACTGCTGGGAA + binding part</span><br />
<span data-bind="if: data.terminus() == 'N' &amp;&amp; data.addTag() &amp;&amp; !data.methionin()">NNNNNNNNGGTCTCAGATG + binding part</span><br />
<span data-bind="if: data.terminus() == 'N' &amp;&amp; data.addTag() &amp;&amp; data.methionin()">NNNNNNNNGGTCTCAGATG + binding part</span><!-- 2.1.1.2 --><br />
</p><br />
<p>In 5'-3' direction, the reverse primer should have the following sequence:</p><br />
<p><br />
<span data-bind="if: data.terminus() == 'C' &amp;&amp; data.addTag()">binding part + NNNNNNNNGGTCTCTATTA</span><br />
<span data-bind="if: data.terminus() == 'N' &amp;&amp; data.addTag()">binding part + NNNNNNNNGGTCTCGAGCACTTGCCCCT</span><br><br />
<span data-bind="if: data.terminus() == 'N' &amp;&amp; data.addTag() &amp;&amp; data.stopCodon()">Be careful not to include stop codons of your protein of interest into the binding part.</span><br />
</p><br />
</li><br />
<li><br />
<p>Use PCR to create your insert and purify it.</p><br />
</li><br />
<li><br />
<p>Use Golden Gate Assembly to replace the mRFP selection marker in <br />
<span data-bind="if: data.terminus() == 'C' &amp;&amp; data.addTag()">BBa_K1362105 (RBS + SspDnaE C-intein RFC [<span data-bind="text: RFCNumber"></span>] assembly construct) with your C-terminal tag</span><br />
<span data-bind="if: data.terminus() == 'N' &amp;&amp; data.addTag()">BBa_K1362104 (RBS + SspDnaE N-intein RFC [<span data-bind="text: RFCNumber"></span>] assembly construct). With your N-terminal tag.</span><br />
</p><br />
</li><br />
<!-- /ko --><br />
<li><br />
<p>Backtranslate the amino acid sequence of your protein of interest into a nucleotide</p><br />
</li><br />
<li><br />
<p>Design and order primers:</p><br />
<p>In 5'-3' direction, the forward primer should have the following sequence:</p><br />
<span data-bind="if: data.addTag() &amp;&amp; ((data.isiGEMHD() &amp;&amp; data.selectedTag().tagname == 'MinD MTS') || (!data.isiGEMHD() &amp;&amp; data.terminus() == 'C'))"><br />
<p><br />
<span data-bind="if: !data.methionin()">NNNNNNGGTCTCAGATG + binding part</span><br />
<span data-bind="if: data.methionin()">NNNNNNGGTCTCAG + binding part</span><br />
</p><br />
<p>In 5'-3' direction, the reverse primer should have the following sequence:</p><br />
<p>NNNNNNNNGGTCTCGAGCACTTGCCCCT + binding part</p><br />
<span data-bind="if: data.stopCodon"><br />
Be careful not to include stop codons of your protein of interest into the binding part.<br />
</span> <br />
</span><br />
<span data-bind="if: (data.isiGEMHD() &amp;&amp; data.selectedTag().tagname != 'MinD MTS') || (!data.isiGEMHD() &amp;&amp; data.terminus() == 'N')"><br />
<p>In 5'-3' direction, the forward primer should have the following sequence:</p><br />
<p>NNNNNNGGTCTCCCAACTGCTGGGAA + binding part</p><br />
<p>In 5'-3' direction, the reverse primer should have the following sequence:</p><br />
<p>NNNNNNGGTCTCTATTA + binding part</p><br />
</span><br />
</li><br />
<li><br />
<p>Use PCR to create your insert and purify it.</p><br />
</li><br />
<li><br />
<p>Use Golden Gate assembly to to exchange the mRFP selection marker in <br />
<span data-bind="if: data.addTag() &amp;&amp; ((data.isiGEMHD() &amp;&amp; data.selectedTag().tagname == 'MinD MTS') || (!data.isiGEMHD() &amp;&amp; data.terminus() == 'C'))"><br />
BBa_K1362104 ( SspDnaE N-intein RFC [<span data-bind="text: RFCNumber"></span>] assembly construct) with your insert.</span><br />
<span data-bind="if: (data.isiGEMHD() &amp;&amp; data.selectedTag().tagname != 'MinD MTS') || (!data.isiGEMHD() &amp;&amp; data.terminus() == 'N')"><br />
BBa_K1362105 ( SspDnaE C-intein RFC [<span data-bind="text: RFCNumber"></span>] assembly construct) with your insert.<br />
</span> <br />
<span data-bind="if: data.hasBSAI"><p>Since there is a BsaI site in your protein, you have to religate after the Golden Gate reaction.</p></span><br />
</p><br />
</li><br />
<li><br />
<span data-bind="if: !data.lightInduced()"><br />
<p>Clone both assembly constructs into compatible expression vectors.</p><br />
</span><br />
<span data-bind="if: data.lightInduced()"><br />
<p>Please use: <br />
<a href="/Team:Heidelberg/Project/LOV">LOV</a> to induce splicing<br />
</p><br />
</span><br />
</li><br />
</ul><br />
<br />
</div><br />
<br />
</div><br />
</div><br />
<br />
<script type="text/javascript"><br />
$(document).ready(function(){<br />
vm = new TaggingViewModel();<br />
ko.applyBindings(vm);<br />
});<br />
</script><br />
</html><br />
{{:Team:Heidelberg/Templates/IncludeJS|:Team:Heidelberg/js/toolboxguide}}</div>Maexlichhttp://2014.igem.org/Team:Heidelberg/Toolbox_GuideTeam:Heidelberg/Toolbox Guide2014-10-18T03:43:25Z<p>Maexlich: </p>
<hr />
<div>{{:Team:Heidelberg/Templates/MainTemplate}}<br />
{{:Team:Heidelberg/Templates/IncludeCSS|:Team:Heidelberg/css/bootstrapcss}}<br />
{{:Team:Heidelberg/Templates/IncludeCSS|:Team:Heidelberg/css/bootstraptheme}}<br />
{{:Team:Heidelberg/Templates/IncludeCSS|:Team:Heidelberg/css/overrides}}<br />
{{:Team:Heidelberg/Templates/IncludeJS|:Team:Heidelberg/js/jquery}}<br />
{{:Team:Heidelberg/Templates/IncludeJS|:Team:Heidelberg/js/bootstrapjs}}<br />
{{:Team:Heidelberg/Templates/IncludeJS|:Team:Heidelberg/js/wikipage}}<br />
<html><br />
<style type="text/css"><br />
#myContainer {<br />
padding:0;<br />
background-color: black;<br />
background-image: url(/wiki/images/6/6a/Heidelberg_epic_background.jpg);<br />
background-repeat: no-repeat;<br />
background-size: 100% auto;<br />
}<br />
<br />
#light:hover {<br />
color: #FF7E25;<br />
}<br />
<br />
.main {<br />
margin-top: 25px;<br />
}<br />
<br />
.middle {<br />
font-size: 3em;<br />
}<br />
<br />
.large {<br />
font-size: 5em;<br />
font-weight: bold;<br />
}<br />
<br />
/* Enlarge Icons on hover */<br />
.toolbox-icons-bar a:hover img {<br />
transform: scale(1.15);<br />
-webkit-transform: scale(1.15);<br />
-moz-transform: scale(1.15);<br />
-o-transform: scale(1.15);<br />
-ms-transform: scale(1.15);<br />
}<br />
<br />
.toolbox-icons-bar a img {<br />
transition:transform 0.15s ease;<br />
-webkit-transition:-webkit-transform 0.15s ease;<br />
-moz-transition:-moz-transform 0.15s ease;<br />
-o-transition:-o-transform 0.15s ease;<br />
}<br />
<br />
<br />
</style><br />
<div id="myContainer" class="container"><br />
</html><br />
{{:Team:Heidelberg/Templates/BootstrapNav|<br />
red=|<br />
white=true|<br />
red-logo=true|<br />
white-logo=|<br />
header-bg=|<br />
header-img=|<br />
title=<br />
}}<br />
<html><br />
<div class="container main" style="color: white;"><br />
<div class="row"><br />
<div class="col-lg-12"><br />
<span class="middle">use the <span class="red-text">intein</span></span><br />
</div> <br />
</div><br />
<div class="row"><br />
<div class="col-lg-12" style="position: relative; top: -20px;"><br />
<span class="large">TOOLBOX&nbsp;</span><span class="middle" >to</span><br />
</div> <br />
</div><br />
<div class="row toolbox-icons-bar"><br />
<div class="col-lg-2 col-lg-offset-1 col-md-offset-1 col-md-2 col-sm-4 col-xs-6"><br />
<a href="https://2014.igem.org/Team:Heidelberg/Toolbox_Guide/Circularization"><span class="col-lg-offset-3 col-lg-6 col-md-offset-2 col-md-8 col-sm-12 nopadding nofloat"><img src="/wiki/images/5/58/Heidelberg_Toolbox_Circularization.png" class="img-responsive" /></span>CIRCULARIZE</a><br />
</div><br />
<div class="col-lg-2 col-md-2 col-sm-4 col-xs-6"><br />
<a href="https://2014.igem.org/Team:Heidelberg/Toolbox_Guide/Localization"><span class="col-lg-offset-3 col-lg-6 col-md-offset-2 col-md-8 col-sm-12 nopadding nofloat"><img src="/wiki/images/3/3a/Heidelberg_Localize_button.png" class="img-responsive" /></span>LOCALIZE</a><br />
</div><br />
<div class="col-lg-2 col-md-2 col-sm-4 col-xs-6"><br />
<a href="https://2014.igem.org/Team:Heidelberg/Toolbox_Guide/Fusion"><span class="col-lg-offset-3 col-lg-6 col-md-offset-2 col-md-8 col-sm-12 nopadding nofloat"><img src="/wiki/images/8/87/Heidelberg_Toolbox_Fusion.png" class="img-responsive" /></span>ATTACH</a><br />
</div><br />
<div class="col-lg-2 col-md-2 col-sm-4 col-xs-6"><br />
<a href="https://2014.igem.org/Team:Heidelberg/Toolbox_Guide/Purification"><span class="col-lg-offset-3 col-lg-6 col-md-offset-2 col-md-8 col-sm-12 nopadding nofloat"><img src="/wiki/images/0/04/Heidelberg_Toolbox_Purification.png" class="img-responsive" /></span>PURIFY</a><br />
</div><br />
<!--<div class="col-lg-2 col-md-2 col-sm-4 col-xs-6"><br />
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</div>--><br />
</div><br />
</div><br />
</div><br />
</html></div>Maexlichhttp://2014.igem.org/File:Heidelberg_Localize_button.pngFile:Heidelberg Localize button.png2014-10-18T03:43:02Z<p>Maexlich: </p>
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<div></div>Maexlichhttp://2014.igem.org/Team:Heidelberg/Toolbox_GuideTeam:Heidelberg/Toolbox Guide2014-10-18T03:41:54Z<p>Maexlich: </p>
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-webkit-transition:-webkit-transform 0.15s ease;<br />
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<html><br />
<div class="container main" style="color: white;"><br />
<div class="row"><br />
<div class="col-lg-12"><br />
<span class="middle">use the <span class="red-text">intein</span></span><br />
</div> <br />
</div><br />
<div class="row"><br />
<div class="col-lg-12" style="position: relative; top: -20px;"><br />
<span class="large">TOOLBOX&nbsp;</span><span class="middle" >to</span><br />
</div> <br />
</div><br />
<div class="row toolbox-icons-bar"><br />
<div class="col-lg-2 col-lg-offset-1 col-md-offset-1 col-md-2 col-sm-4 col-xs-6"><br />
<a href="https://2014.igem.org/Team:Heidelberg/Toolbox_Guide/Circularization"><span class="col-lg-offset-3 col-lg-6 col-md-offset-2 col-md-8 col-sm-12 nopadding nofloat"><img src="/wiki/images/5/58/Heidelberg_Toolbox_Circularization.png" class="img-responsive" /></span>CIRCULARIZE</a><br />
</div><br />
<div class="col-lg-2 col-md-2 col-sm-4 col-xs-6"><br />
<a href="https://2014.igem.org/Team:Heidelberg/Toolbox_Guide/Localization"><span class="col-lg-offset-3 col-lg-6 col-md-offset-2 col-md-8 col-sm-12 nopadding nofloat"><img src="/wiki/images/4/40/Oligomerization.png" class="img-responsive" /></span>LOCALIZE</a><br />
</div><br />
<div class="col-lg-2 col-md-2 col-sm-4 col-xs-6"><br />
<a href="https://2014.igem.org/Team:Heidelberg/Toolbox_Guide/Fusion"><span class="col-lg-offset-3 col-lg-6 col-md-offset-2 col-md-8 col-sm-12 nopadding nofloat"><img src="/wiki/images/8/87/Heidelberg_Toolbox_Fusion.png" class="img-responsive" /></span>ATTACH</a><br />
</div><br />
<div class="col-lg-2 col-md-2 col-sm-4 col-xs-6"><br />
<a href="https://2014.igem.org/Team:Heidelberg/Toolbox_Guide/Purification"><span class="col-lg-offset-3 col-lg-6 col-md-offset-2 col-md-8 col-sm-12 nopadding nofloat"><img src="/wiki/images/0/04/Heidelberg_Toolbox_Purification.png" class="img-responsive" /></span>PURIFY</a><br />
</div><br />
<!--<div class="col-lg-2 col-md-2 col-sm-4 col-xs-6"><br />
<a href="https://2014.igem.org/Team:Heidelberg/Toolbox_Guide/OnOff"><span class="col-lg-offset-3 col-lg-6 col-md-offset-2 col-md-8 col-sm-12 nopadding nofloat"><img src="/wiki/images/c/c2/Heidelberg_Toolbox_On-Off.png" class="img-responsive" /></span>ACTIVATE &<br />DEACTIVATE</a><br />
</div>--><br />
</div><br />
</div><br />
</div><br />
</html></div>Maexlichhttp://2014.igem.org/Team:Heidelberg/Toolbox_Guide/FusionTeam:Heidelberg/Toolbox Guide/Fusion2014-10-18T03:41:10Z<p>Maexlich: Created page with "{{:Team:Heidelberg/Templates/MainTemplate}} {{:Team:Heidelberg/Templates/IncludeCSS|:Team:Heidelberg/css/bootstrapcss}} {{:Team:Heidelberg/Templates/IncludeCSS|:Team:Heidelberg/c..."</p>
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transition:transform 0.15s ease;<br />
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<html><br />
<div class="container main" style="color: white;"><br />
<div class="row"><br />
<div class="col-lg-8"><br />
<div class="row"><br />
<div class="col-lg-12"><br />
<span class="middle">use the <span class="red-text">intein</span></span><br />
</div> <br />
</div><br />
<div class="row"><br />
<div class="col-lg-12" style="position: relative; top: -20px;"><br />
<span class="large">TOOLBOX&nbsp;</span><span class="middle" >to</span><br />
</div> <br />
</div><br />
</div><br />
<div class="col-lg-4 toolbox-icons-bar"><br />
<div class="col-lg-6 col-md-6 col-sm-6 col-xs-12"><br />
<a href="https://2014.igem.org/Team:Heidelberg/Toolbox/Circularization"><img src="/wiki/images/8/87/Heidelberg_Toolbox_Fusion.png" class="img-responsive">ATTACH</a><br />
</div><br />
</div><br />
</div><br />
<div class="col-lg-12" style="color:white;"><br />
<br />
<h3>First of all, check the DNA sequence of your proteins for EcoRI, XbaI, SpeI, PstI and BsaI sites. If there are E/X/S/P sites, you might have problems to change your backbone or add a promotor. If there is a BsaI site, the cloning will be more difficult.</h3><br />
<div class="panel panel-default"><br />
<div class="panel-body"><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.hasBSAI, checkedValue: false, click: data.q0A.bind(null, true)" /><br />
There is no BsaI site.<br />
</label><br />
</div><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.hasBSAI, checkedValue: true, click: data.q0A.bind(null, true)" /><br />
There is a BsaI site.<br />
</label><br />
</div><br />
</div><br />
</div><br />
<div data-bind="if: data.q0A"><br />
<h3>At which position do you want to add your posttranslational modification?</h3><br />
<div class="panel panel-default"><br />
<div class="panel-body"><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.isNterminal, checkedValue: true, click: data.q1A.bind(null, true)" /><br />
N-terminal<br />
</label><br />
</div><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.isNterminal, checkedValue: false, click: data.q1A.bind(null, true)" /><br />
C-terminal<br />
</label><br />
</div><br />
</div><br />
</div><br />
</div><br />
<div data-bind="if: data.q1A() &amp;&amp; !data.isNterminal()"><br />
<h3>Does your protein of interest have methionin as a first amino acid?</h3><br />
<div class="panel panel-default"><br />
<div class="panel-body"><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.methionin, checkedValue: false, click: data.q2A.bind(null, true)" /><br />
No<br />
</label><br />
</div><br />
<div class="radio"><br />
<label><br />
<input type="radio" data-bind="checked: data.methionin, checkedValue: true, click: data.q2A.bind(null, true)" /><br />
Yes<br />
</label><br />
</div><br />
</div><br />
</div><br />
</div><br />
<br />
<div data-bind="if: (data.q1A() &amp;&amp; data.isNterminal() ) || data.q2A()"><br />
<h1>Protocol</h1><br />
<ul><br />
<li><br />
<p><br />
<span data-bind="if: data.isNterminal"><br />
Get BBa_K1362141 (RBS + SspDnaB C-intein RFC [<span data-bind="text: RFCNumber"></span>] assembly construct) from the registry of standard biological parts.<br />
</span><br />
<span data-bind="if: !data.isNterminal()"><br />
Get BBa_K1362110 (RBS + SspDnaX-S11 N-intein RFC [<span data-bind="text: RFCNumber"></span>] assembly construct) from the registry of standard biological parts.<br />
</span><br />
</p><br />
</li><br />
<br />
<li><br />
<p>Divide your protein of interest into <br />
<span data-bind="if: data.isNterminal"><br />
a short N-terminal part including the synthetic attachment and a long C-terminal one.<br />
</span><br />
<span data-bind="if: !data.isNterminal()"><br />
a long N-terminal part including the synthetic attachment and a short C-terminal one.<br />
</span><br />
</p><br />
</li><br />
<br />
<li><p>Get the DNA of the long part of your protein of interest.</p></li><br />
<br />
<li><br />
<p>The long part of your protein of interest will be expressed recombinantly. Therefore design and order primers for this part:</p><br />
<ul><br />
<li><br />
<p>In 5&apos;-3&apos; direction, the forward primer should have the following sequence: </p><br />
<p><br />
<span data-bind="if: data.isNterminal">NNNNNNGGTCTCCCAACAGCATTCGCAGC+ binding part</span><br />
<span data-bind="if: !data.isNterminal()"><br />
<span data-bind="if: !data.methionin()">NNNNNNGGTCTCCGATG + binding part</span><br />
<span data-bind="if: data.methionin()">NNNNNNGGTCTCCG + binding part</span><br />
</span><br />
</p><br />
</li><br />
<li><br />
<p>In 5&apos;-3&apos; direction, the reverse primer should have the following sequence:</p> <br />
<p><br />
<span data-bind="if: data.isNterminal">NNNNNNGGTCTCTATTA + binding part</span><br />
<span data-bind="if: !data.isNterminal()">NNNNNNGGTCTCGAGCAACCAGATTCACGCAG + binding part</span><br />
</p><br />
</li><br />
</ul><br />
</li><br />
<br />
<!--ES FEHLT: Verweis oder Hinweise zu Primerdesign allgemein. --><br />
<li><p>Use PCR to create your insert and purify it.</p></li><br />
<!--ES FEHLT: Verweis oder Hinweise zu PCR bzw Twostep-PCR.--> <br />
<br />
<li><br />
<p>Use Golden Gate assembly to insert the recombinant part of your protein of interest into<br />
<span data-bind="if data.isNterminal">BBa_K1362141 (SspDnaB C-intein RFC [???] assembly construct)</span><br />
<span data-bind="if !data.isNterminal()">BBa_K1362110 (SspDnaX N-intein RFC [???] assembly construct)</span><br />
</p><br />
<p data-bind="if: data.hasBSAI"><br />
Since there is a BsaI site in your protein, you have to religate after the Golden Gate reaction.<br />
</p><br />
</li><br />
<!-- ES FEHLT: Verweis oder Hinweise zu GG --><br />
<li><p>Induce expression.</p></li><br />
<br />
<li><p>Purify the protein.</p></li><br />
<br />
<li><br />
<p>Synthesize the short part of your protein including the posttranslational modification together with the split intein sequence.</p><br />
<p>The sequence should look like this:</p><br />
<p><br />
<span data-bind="if: data.isNterminal">N-terminal protein part with modification - SEFSGCISGDSLISLA</span> <br />
<span data-bind="if: !data.isNterminal()">GLLVHNCHT - C-terminal protein part with modification</span><br />
</p><br />
</li><br />
<li><br />
<p>Mix both parts in vitro to induce protein trans-splicing.</p><br />
<p>Further information about this step can be found here:</p><br />
<p>Mootz, Henning: Split Inteins as Versatile Tools for Protein Semisynthesis. ChemBioChem <br />
2009, 10, 2579 - 2589 . DOI: 10.1002/cbic.200900370 .</p><br />
</li><br />
</ul><br />
</div><br />
</div><br />
</div><br />
</div><br />
<br />
<script type="text/javascript"><br />
$(document).ready(function(){<br />
vm = new SyntheticViewModel();<br />
ko.applyBindings(vm);<br />
});<br />
</script><br />
</html><br />
{{:Team:Heidelberg/Templates/IncludeJS|:Team:Heidelberg/js/toolboxguide}}</div>Maexlichhttp://2014.igem.org/File:Heidelberg_half_LOV_microscopy.pngFile:Heidelberg half LOV microscopy.png2014-10-18T03:40:14Z<p>Maexlich: uploaded a new version of &quot;File:Heidelberg half LOV microscopy.png&quot;: Thumbnail (620px X 273 px) of Heidelberg_orig_LOV_microscopy.png</p>
<hr />
<div>Thumbnail (620px X 279 px) of Heidelberg_orig_LOV_microscopy.png</div>Maexlichhttp://2014.igem.org/File:Heidelberg_full_LOV_microscopy.pngFile:Heidelberg full LOV microscopy.png2014-10-18T03:40:12Z<p>Maexlich: uploaded a new version of &quot;File:Heidelberg full LOV microscopy.png&quot;: Thumbnail (1070px X 471 px) of Heidelberg_orig_LOV_microscopy.png</p>
<hr />
<div>Thumbnail (1070px X 481 px) of Heidelberg_orig_LOV_microscopy.png</div>Maexlichhttp://2014.igem.org/File:Heidelberg_quarter_LOV_microscopy.pngFile:Heidelberg quarter LOV microscopy.png2014-10-18T03:40:09Z<p>Maexlich: uploaded a new version of &quot;File:Heidelberg quarter LOV microscopy.png&quot;: Thumbnail (430px X 189 px) of Heidelberg_orig_LOV_microscopy.png</p>
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<div>Thumbnail (430px X 193 px) of Heidelberg_orig_LOV_microscopy.png</div>Maexlichhttp://2014.igem.org/File:Heidelberg_orig_LOV_microscopy.pngFile:Heidelberg orig LOV microscopy.png2014-10-18T03:40:08Z<p>Maexlich: uploaded a new version of &quot;File:Heidelberg orig LOV microscopy.png&quot;: Original File Heidelberg_orig_LOV_microscopy.png</p>
<hr />
<div>Original File Heidelberg_orig_LOV_microscopy.png</div>Maexlichhttp://2014.igem.org/File:Heidelberg_half_LOV-sfGFP-FACS.pngFile:Heidelberg half LOV-sfGFP-FACS.png2014-10-18T03:40:04Z<p>Maexlich: Thumbnail (620px X 221 px) of Heidelberg_orig_LOV-sfGFP-FACS.png</p>
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<div>Thumbnail (620px X 221 px) of Heidelberg_orig_LOV-sfGFP-FACS.png</div>Maexlichhttp://2014.igem.org/File:Heidelberg_full_LOV-sfGFP-FACS.pngFile:Heidelberg full LOV-sfGFP-FACS.png2014-10-18T03:40:01Z<p>Maexlich: Thumbnail (1070px X 382 px) of Heidelberg_orig_LOV-sfGFP-FACS.png</p>
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<div>Thumbnail (1070px X 382 px) of Heidelberg_orig_LOV-sfGFP-FACS.png</div>Maexlichhttp://2014.igem.org/File:Heidelberg_quarter_LOV-sfGFP-FACS.pngFile:Heidelberg quarter LOV-sfGFP-FACS.png2014-10-18T03:39:59Z<p>Maexlich: Thumbnail (430px X 153 px) of Heidelberg_orig_LOV-sfGFP-FACS.png</p>
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<div>Thumbnail (430px X 153 px) of Heidelberg_orig_LOV-sfGFP-FACS.png</div>Maexlichhttp://2014.igem.org/File:Heidelberg_orig_LOV-sfGFP-FACS.pngFile:Heidelberg orig LOV-sfGFP-FACS.png2014-10-18T03:39:57Z<p>Maexlich: Original File Heidelberg_orig_LOV-sfGFP-FACS.png</p>
<hr />
<div>Original File Heidelberg_orig_LOV-sfGFP-FACS.png</div>Maexlichhttp://2014.igem.org/Team:Heidelberg/pages/CollaborationsTeam:Heidelberg/pages/Collaborations2014-10-18T03:34:24Z<p>Maexlich: /* iGEM Team Marburg */</p>
<hr />
<div><br/><br />
<br />
Besides spending a whole summer in the lab and developing an own project, iGEM is about meeting new people, people with the same interests, the same motivation, the same dedication. Collaborations between team ensure the exchange of ideas, therefore helping the field of Synthetic Biology to be interdisciplinary and creative - the best conditions to give rise to revolutionary projects. We are very happy to met some of the team members from Freiburg, Aachen, Tuebingen, Marburg and London. <br />
<br />
=iGEM Team Aachen=<br />
<br />
<br />
Michael from the iGEM team Aachen visited us in Heidelberg. Their team worked on the development of a real-time pathogen detection technique for what they have build their own fluorescence measurement camera. We provided our expression vectors to the iGEM team Aachen, which they used to express their part [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1319003 E1010-K1319003], the human galectin-3 protein fused to mRFP. Nicely seen in Figure 1 and 2 the the intensity of the red color is significantly higher for the induced sample with [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1362091 pSBX1A30] compared to the uninduced ones. To ensure the right molecular mass of the fusion protein, a SDS gel was run as well (Figure 3).<br />
<br />
<div><br />
{{:Team:Heidelberg/templates/image-quarter| align=right|descr=|<br />
caption=3) SDS-PAGE of K1319020 expression| file=AachenCo3.png}}<br />
{{:Team:Heidelberg/templates/image-quarter| align=right|descr=|<br />
caption=2) Expression of Galectin-3| file=AachenCo2.png}}<br />
{{:Team:Heidelberg/templates/image-quarter| align=right|descr=|<br />
caption=1) Expression of Galectin-3| file=AachenCo1.jpg}}<br />
</div><br />
<br />
More information about their project can be found on Aachens [https://2014.igem.org/Team:Aachen/Project/Gal3 Galectin-3 site].<br />
<br />
In exchange they offered to characterise these expression vectors with their own designed analyser by measuring the amount of produced fluorescent proteins. Unfortunately, transporting <i>E.colis</i> to another city wasn't as easy as expected, not all Bacteria were able to grow afterwards and we could only obtain sufficient data from pSBX4C5. <br />
<br />
<br/><br />
<br />
=iGEM Team Tuebingen=<br />
The project of iGEM Team Tuebingen is about blood types and the difficulties that arises during blood donation of the wrong blood type. They aim to develop an easy system to convert blood types A, B, AB to the rares but also most applicable blood type 0. They have applied the method of intein targeting, a method which can also be found in our intein toolbox, to the N-Acetyl-Galactosaminidase protein. More about their project can be read in the very nice [http://igem14-heidelberg.tumblr.com/post/98138990630/igem-team-tubingen article] they have wrote for our tumblr blog or on their own [[Team:Tuebingen|wiki]].<br />
<br />
In this collaboration, Tuebingen conducted a mass spectrometry analysis of our Lambda lysozyme to proof circularization. The data still has to be evaluated, but we will have them ready until the jamboree. There is a high demand for our expression vectors. Tuebingen had problems with expressing their constructs, therefore we send them some of our plasmids pSBX1K30 ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1362093 BBa_K1362093]) and pSBX4K50 ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1362097 BBa_K1362097]), as they showed to work really nicely even with big proteins like the [[Team:Heidelberg/Project/PCR2.0|Dnmt1]] with 105kDa.<br />
<br />
<br/><br />
<br />
=iGEM Team Freiburg=<br />
<br />
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<a class="img-enlarge" href="https://static.igem.org/mediawiki/2014/c/c6/Heidelberg_orig_Freiburg_Inductionbox2.jpg" target="_blank"><img src="" alt="Image"></a><br />
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<a class="img-enlarge" href="/wiki/images/6/68/Freiburg_Cellculture2.JPG" target="_blank"><img src="/wiki/images/6/68/Freiburg_Cellculture2.JPG" class="img-responsive" alt="Image"></a><br />
</div><br />
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</div><br />
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<br />
Three of our team members, Anna, Caro, and Charlotte visited the iGEM team Freiburg. We met the team right after a big team meeting, therefore we were able to hear their latest project developments. We spend the evening talking about experiences in the lab and about iGEM. It was relieving to hear that they faced similar difficulties in the lab. <br />
The morning after we presented our project to the iGEM team Freiburg. Freiburg was very interested in our program iGEM@home as we offered them to promote their own human practice projects. You can also find an [http://igem14-heidelberg.tumblr.com/post/99936805780/optogenetics-the-lock-and-key-for-daily-life article] about optogenetics on our tumblr blog.<br />
Both our teams are working with light inducible systems, manly the LOV domain. Although working in different organisms, the constructs were compatible, therefore we could interchange protocols for induction and preliminary results. Furthermore we gazed at their professional light induction box - we can definitely improved ours. <br />
<br />
After cooperation with the iGEM Team Freiburg for two years in a row we are hoping to keep Freiburg as a reliable partner in the next years as well.<br />
<br />
<br/><br />
<br />
=iGEM Team Marburg=<br />
<br />
The screensaver of [[Team:Heidelberg/Human_Practice/igemathome|iGEM@home]] is a great possibility to spread news and information about Synthetic Biology around the world. Lately, even other iGEM team have used this new option and filled our screensaver with slides about their own project. Marburg was able to promote their quiz app about Synthetic Biology.<br />
<br />
<html><br />
<style><br />
#propose {text-indent: 40px;<br />
}<br />
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}<br />
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<div id="socioeconomics"> <br />
<h1>A proposal of the German iGEM Teams concerning Intellectual Property</h1><br />
<p>During the meetup of the German iGEM teams from 23rd to 25th May also <a href="https://2014.igem.org/Meetups:May_LMU-Munich/Workshops">workshops</a> took place in which amongst others we discussed the topic of bioethics. Moral questions were addressed, regarding the value of life and human influence on it, as well as questions dealing with the possible socioeconomic effects of synthetic biology.<br />
</p><br />
<p><br />
Especially the topic of an open source vs. patent controlled field accounted for a large part of the discussion. During the discussion one student brought up the point that the legal status of parts in registry remains unclear and that there are parts (e.g. <a href="http://parts.igem.org/Part:BBa_K180009">BBa_K180009</a>) where only upon a closer look it becomes clear that the rights are company–owned. The issue that the legal status of parts in the registry remains uncertain is also mentioned in a recent article published by Nature (<a href="http://www.nature.com/news/synthetic-biology-cultural-divide-1.15149">Bryn Nelson ‘Synthetic Biology: Cultural Divide ’, Nature 509, 152–154, 08 May 2014</a>) :<br />
</p><br />
<p><br />
<i>"[N]o one can say with any certainty how many of these parts are themselves entirely free of patent claims."</i><br />
</p><br />
<p><br />
We, the German iGEM teams, therefore like to suggest the addition of a new feature to the parts registry:<br />
</p><br />
<p id="propose"> A dedicated data field of license information for each BioBrick part.</p><br />
<p> <br />
For the implementation, we propose to introduce two new fields to BioBrick part entries in the registry:<br />
</p><br />
<ol><br />
<li>A string property "LicenseInfo"</li><br />
<li>A traffic light property (grey, green, yellow, red) to indicate the level of legal protection (unknown, BPA-like, free for research purposes, heavily protected)</li><br />
</ol><br />
<br />
<br />
<img src="https://static.igem.org/mediawiki/2014/6/69/TU-BS_Part_licence_info.png" alt="This image shows a how the proposed field LicenceInfo could look like in the registry."> <br />
<br />
<p><br />
Implementing this feature would in our opinion further clarify and extend the parts info, provide a machine-readable format and thus improve future entries. With the emerging Entrepreneurship track and applications getting closer to industrial realization, the legal status becomes more and more important. Also it would raise awareness to the topic of the legal status of parts, leading to a debate which could further promote the idea of open source. At the same time we hope that examination of most parts will show that they are indeed free of restrictive legal protections.<br />
</p><br />
<p id="greetings"><br />
The German iGEM Teams,<br />
</p><br />
<ul class="supporter_team_list"><br />
<li><a href="https://2014.igem.org/Team:Aachen">Aachen </a></li><br />
<li><a href="https://2014.igem.org/Team:Berlin">Berlin </a></li><br />
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec">Bielefeld-CeBiTec </a></li><br />
<li><a href="https://2014.igem.org/Team:Braunschweig">Braunschweig </a></li><br />
<li><a href="https://2014.igem.org/Team:Freiburg">Freiburg </a></li><br />
<li><a href="https://2014.igem.org/Team:Goettingen">Goettingen </a></li><br />
<li><a href="https://2014.igem.org/Team:Hannover">Hannover </a></li><br />
<li><a href="https://2014.igem.org/Team:Heidelberg">Heidelberg </a></li><br />
<li><a href="https://2014.igem.org/Team:LMU-Munich">LMU-Munich </a></li><br />
<li><a href="https://2014.igem.org/Team:Marburg">Marburg </a></li><br />
<li><a href="https://2014.igem.org/Team:Saarland">Saarland </a></li><br />
<li><a href="https://2014.igem.org/Team:Tuebingen">Tuebingen </a></li><br />
<li><a href="https://2014.igem.org/Team:TU_Darmstadt">TU&nbsp;Darmstadt </a></li><br />
</ul><br />
</div><br />
</html></div>Maexlichhttp://2014.igem.org/Team:Heidelberg/pages/CollaborationsTeam:Heidelberg/pages/Collaborations2014-10-18T03:31:12Z<p>Maexlich: </p>
<hr />
<div><br/><br />
<br />
Besides spending a whole summer in the lab and developing an own project, iGEM is about meeting new people, people with the same interests, the same motivation, the same dedication. Collaborations between team ensure the exchange of ideas, therefore helping the field of Synthetic Biology to be interdisciplinary and creative - the best conditions to give rise to revolutionary projects. We are very happy to met some of the team members from Freiburg, Aachen, Tuebingen, Marburg and London. <br />
<br />
=iGEM Team Aachen=<br />
<br />
<br />
Michael from the iGEM team Aachen visited us in Heidelberg. Their team worked on the development of a real-time pathogen detection technique for what they have build their own fluorescence measurement camera. We provided our expression vectors to the iGEM team Aachen, which they used to express their part [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1319003 E1010-K1319003], the human galectin-3 protein fused to mRFP. Nicely seen in Figure 1 and 2 the the intensity of the red color is significantly higher for the induced sample with [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1362091 pSBX1A30] compared to the uninduced ones. To ensure the right molecular mass of the fusion protein, a SDS gel was run as well (Figure 3).<br />
<br />
<div><br />
{{:Team:Heidelberg/templates/image-quarter| align=right|descr=|<br />
caption=3) SDS-PAGE of K1319020 expression| file=AachenCo3.png}}<br />
{{:Team:Heidelberg/templates/image-quarter| align=right|descr=|<br />
caption=2) Expression of Galectin-3| file=AachenCo2.png}}<br />
{{:Team:Heidelberg/templates/image-quarter| align=right|descr=|<br />
caption=1) Expression of Galectin-3| file=AachenCo1.jpg}}<br />
</div><br />
<br />
More information about their project can be found on Aachens [https://2014.igem.org/Team:Aachen/Project/Gal3 Galectin-3 site].<br />
<br />
In exchange they offered to characterise these expression vectors with their own designed analyser by measuring the amount of produced fluorescent proteins. Unfortunately, transporting <i>E.colis</i> to another city wasn't as easy as expected, not all Bacteria were able to grow afterwards and we could only obtain sufficient data from pSBX4C5. <br />
<br />
<br/><br />
<br />
=iGEM Team Tuebingen=<br />
The project of iGEM Team Tuebingen is about blood types and the difficulties that arises during blood donation of the wrong blood type. They aim to develop an easy system to convert blood types A, B, AB to the rares but also most applicable blood type 0. They have applied the method of intein targeting, a method which can also be found in our intein toolbox, to the N-Acetyl-Galactosaminidase protein. More about their project can be read in the very nice [http://igem14-heidelberg.tumblr.com/post/98138990630/igem-team-tubingen article] they have wrote for our tumblr blog or on their own [[Team:Tuebingen|wiki]].<br />
<br />
In this collaboration, Tuebingen conducted a mass spectrometry analysis of our Lambda lysozyme to proof circularization. The data still has to be evaluated, but we will have them ready until the jamboree. There is a high demand for our expression vectors. Tuebingen had problems with expressing their constructs, therefore we send them some of our plasmids pSBX1K30 ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1362093 BBa_K1362093]) and pSBX4K50 ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1362097 BBa_K1362097]), as they showed to work really nicely even with big proteins like the [[Team:Heidelberg/Project/PCR2.0|Dnmt1]] with 105kDa.<br />
<br />
<br/><br />
<br />
=iGEM Team Freiburg=<br />
<br />
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</div><br />
<div class="item"><br />
<a class="img-enlarge" href="https://static.igem.org/mediawiki/2014/c/c6/Heidelberg_orig_Freiburg_Inductionbox2.jpg" target="_blank"><img src="" alt="Image"></a><br />
</div><br />
<div class="item"><br />
<a class="img-enlarge" href="/wiki/images/6/68/Freiburg_Cellculture2.JPG" target="_blank"><img src="/wiki/images/6/68/Freiburg_Cellculture2.JPG" class="img-responsive" alt="Image"></a><br />
</div><br />
<div class="item"><br />
<a class="img-enlarge" href="https://static.igem.org/mediawiki/2014/d/d1/Heidelberg_orig_Freiburg_Cellculture2.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2014/d/d1/Heidelberg_orig_Freiburg_Cellculture2.jpg" class="img-responsive" alt="Image"></a><br />
</div><br />
<div class="item"><br />
<a class="img-enlarge" href="https://static.igem.org/mediawiki/2014/7/7c/Heidelberg_orig_Freiburg_discussion2.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2014/7/7c/Heidelberg_orig_Freiburg_discussion2.jpg" class="img-responsive" alt="Image"></a><br />
</div><br />
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}}<br />
<br />
Three of our team members, Anna, Caro, and Charlotte visited the iGEM team Freiburg. We met the team right after a big team meeting, therefore we were able to hear their latest project developments. We spend the evening talking about experiences in the lab and about iGEM. It was relieving to hear that they faced similar difficulties in the lab. <br />
The morning after we presented our project to the iGEM team Freiburg. Freiburg was very interested in our program iGEM@home as we offered them to promote their own human practice projects. You can also find an [http://igem14-heidelberg.tumblr.com/post/99936805780/optogenetics-the-lock-and-key-for-daily-life article] about optogenetics on our tumblr blog.<br />
Both our teams are working with light inducible systems, manly the LOV domain. Although working in different organisms, the constructs were compatible, therefore we could interchange protocols for induction and preliminary results. Furthermore we gazed at their professional light induction box - we can definitely improved ours. <br />
<br />
After cooperation with the iGEM Team Freiburg for two years in a row we are hoping to keep Freiburg as a reliable partner in the next years as well.<br />
<br />
<br/><br />
<br />
=iGEM Team Marburg=<br />
<br />
The screensaver of [[Team:Heidelberg/Human_Practice/igemathome|iGEM@home]] is a great possibility to spread news and information about Synthetic Biology around the world. Lately, even other iGEM team have used this new option and filled our screensaver with slides about their own project. Marburg was able to promote their quiz app about Synthetic Biology.<br />
<br />
{{#tag:html|<br />
<style><br />
#propose {text-indent: 40px;<br />
}<br />
#greetings {text-indent: 20px;<br />
}<br />
ul.supporter_team_list { list-style-type: none;<br />
}<br />
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ul.supporter_team_list a:hover {text-decoration: underline;}<br />
ul.supporter_team_list > li {display:inline;margin: 30px;<br />
}<br />
</style><br />
<div id="socioeconomics"> <br />
<h1>A proposal of the German iGEM Teams concerning Intellectual Property</h1><br />
<p>During the meetup of the German iGEM teams from 23rd to 25th May also <a href="https://2014.igem.org/Meetups:May_LMU-Munich/Workshops">workshops</a> took place in which amongst others we discussed the topic of bioethics. Moral questions were addressed, regarding the value of life and human influence on it, as well as questions dealing with the possible socioeconomic effects of synthetic biology.<br />
</p><br />
<p><br />
Especially the topic of an open source vs. patent controlled field accounted for a large part of the discussion. During the discussion one student brought up the point that the legal status of parts in registry remains unclear and that there are parts (e.g. <a href="http://parts.igem.org/Part:BBa_K180009">BBa_K180009</a>) where only upon a closer look it becomes clear that the rights are company–owned. The issue that the legal status of parts in the registry remains uncertain is also mentioned in a recent article published by Nature (<a href="http://www.nature.com/news/synthetic-biology-cultural-divide-1.15149">Bryn Nelson ‘Synthetic Biology: Cultural Divide ’, Nature 509, 152–154, 08 May 2014</a>) :<br />
</p><br />
<p><br />
<i>"[N]o one can say with any certainty how many of these parts are themselves entirely free of patent claims."</i><br />
</p><br />
<p><br />
We, the German iGEM teams, therefore like to suggest the addition of a new feature to the parts registry:<br />
</p><br />
<p id="propose"> A dedicated data field of license information for each BioBrick part.</p><br />
<p> <br />
For the implementation, we propose to introduce two new fields to BioBrick part entries in the registry:<br />
</p><br />
<ol><br />
<li>A string property "LicenseInfo"</li><br />
<li>A traffic light property (grey, green, yellow, red) to indicate the level of legal protection (unknown, BPA-like, free for research purposes, heavily protected)</li><br />
</ol><br />
<br />
<br />
<img src="https://static.igem.org/mediawiki/2014/6/69/TU-BS_Part_licence_info.png" alt="This image shows a how the proposed field LicenceInfo could look like in the registry."> <br />
<br />
<p><br />
Implementing this feature would in our opinion further clarify and extend the parts info, provide a machine-readable format and thus improve future entries. With the emerging Entrepreneurship track and applications getting closer to industrial realization, the legal status becomes more and more important. Also it would raise awareness to the topic of the legal status of parts, leading to a debate which could further promote the idea of open source. At the same time we hope that examination of most parts will show that they are indeed free of restrictive legal protections.<br />
</p><br />
<p id="greetings"><br />
The German iGEM Teams,<br />
</p><br />
<ul class="supporter_team_list"><br />
<li><a href="https://2014.igem.org/Team:Aachen">Aachen </a></li><br />
<li><a href="https://2014.igem.org/Team:Berlin">Berlin </a></li><br />
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec">Bielefeld-CeBiTec </a></li><br />
<li><a href="https://2014.igem.org/Team:Braunschweig">Braunschweig </a></li><br />
<li><a href="https://2014.igem.org/Team:Freiburg">Freiburg </a></li><br />
<li><a href="https://2014.igem.org/Team:Goettingen">Goettingen </a></li><br />
<li><a href="https://2014.igem.org/Team:Hannover">Hannover </a></li><br />
<li><a href="https://2014.igem.org/Team:Heidelberg">Heidelberg </a></li><br />
<li><a href="https://2014.igem.org/Team:LMU-Munich">LMU-Munich </a></li><br />
<li><a href="https://2014.igem.org/Team:Marburg">Marburg </a></li><br />
<li><a href="https://2014.igem.org/Team:Saarland">Saarland </a></li><br />
<li><a href="https://2014.igem.org/Team:Tuebingen">Tuebingen </a></li><br />
<li><a href="https://2014.igem.org/Team:TU_Darmstadt">TU&nbsp;Darmstadt </a></li><br />
</ul><br />
</div><br />
</body><br />
}</div>Maexlichhttp://2014.igem.org/File:Heidelberg_Notebook_Data_General_summary_1410931800_1411088400.txtFile:Heidelberg Notebook Data General summary 1410931800 1411088400.txt2014-10-18T03:30:48Z<p>Maexlich: uploaded a new version of &quot;File:Heidelberg Notebook Data General summary 1410931800 1411088400.txt&quot;: Data for MidnightDoc of iGEM Team Heidelberg: Heidelberg_Notebook_Data_General_summary_1410931800_1411088400.txt</p>
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<div>Data for MidnightDoc of iGEM Team Heidelberg: Heidelberg_Notebook_Data_General_summary_1410931800_1411088400.txt</div>Maexlichhttp://2014.igem.org/File:Heidelberg_Notebook_Data_General_summary_1411754400_1411992000.txtFile:Heidelberg Notebook Data General summary 1411754400 1411992000.txt2014-10-18T03:30:46Z<p>Maexlich: uploaded a new version of &quot;File:Heidelberg Notebook Data General summary 1411754400 1411992000.txt&quot;: Data for MidnightDoc of iGEM Team Heidelberg: Heidelberg_Notebook_Data_General_summary_1411754400_1411992000.txt</p>
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<div>Data for MidnightDoc of iGEM Team Heidelberg: Heidelberg_Notebook_Data_General_summary_1411754400_1411992000.txt</div>Maexlichhttp://2014.igem.org/File:Heidelberg_Notebook_Data_General_summary_1407888900_1408130100.txtFile:Heidelberg Notebook Data General summary 1407888900 1408130100.txt2014-10-18T03:30:44Z<p>Maexlich: uploaded a new version of &quot;File:Heidelberg Notebook Data General summary 1407888900 1408130100.txt&quot;: Data for MidnightDoc of iGEM Team Heidelberg: Heidelberg_Notebook_Data_General_summary_1407888900_1408130100.txt</p>
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<div>Data for MidnightDoc of iGEM Team Heidelberg: Heidelberg_Notebook_Data_General_summary_1407888900_1408130100.txt</div>Maexlichhttp://2014.igem.org/File:Heidelberg_Notebook_Data_General_summary_1404897720_1405066500.txtFile:Heidelberg Notebook Data General summary 1404897720 1405066500.txt2014-10-18T03:30:42Z<p>Maexlich: uploaded a new version of &quot;File:Heidelberg Notebook Data General summary 1404897720 1405066500.txt&quot;: Data for MidnightDoc of iGEM Team Heidelberg: Heidelberg_Notebook_Data_General_summary_1404897720_1405066500.txt</p>
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<div>Data for MidnightDoc of iGEM Team Heidelberg: Heidelberg_Notebook_Data_General_summary_1404897720_1405066500.txt</div>Maexlichhttp://2014.igem.org/File:Heidelberg_Notebook_Data_General_summary_1408881600_1409058000.txtFile:Heidelberg Notebook Data General summary 1408881600 1409058000.txt2014-10-18T03:30:40Z<p>Maexlich: uploaded a new version of &quot;File:Heidelberg Notebook Data General summary 1408881600 1409058000.txt&quot;: Data for MidnightDoc of iGEM Team Heidelberg: Heidelberg_Notebook_Data_General_summary_1408881600_1409058000.txt</p>
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<div>Data for MidnightDoc of iGEM Team Heidelberg: Heidelberg_Notebook_Data_General_summary_1408881600_1409058000.txt</div>Maexlichhttp://2014.igem.org/File:Heidelberg_Notebook_Data_General_summary_1410206400_1410264000.txtFile:Heidelberg Notebook Data General summary 1410206400 1410264000.txt2014-10-18T03:30:38Z<p>Maexlich: uploaded a new version of &quot;File:Heidelberg Notebook Data General summary 1410206400 1410264000.txt&quot;: Data for MidnightDoc of iGEM Team Heidelberg: Heidelberg_Notebook_Data_General_summary_1410206400_1410264000.txt</p>
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<div>Data for MidnightDoc of iGEM Team Heidelberg: Heidelberg_Notebook_Data_General_summary_1410206400_1410264000.txt</div>Maexlichhttp://2014.igem.org/File:Heidelberg_Notebook_Data_General_summary_1406106000_1406235600.txtFile:Heidelberg Notebook Data General summary 1406106000 1406235600.txt2014-10-18T03:30:36Z<p>Maexlich: uploaded a new version of &quot;File:Heidelberg Notebook Data General summary 1406106000 1406235600.txt&quot;: Data for MidnightDoc of iGEM Team Heidelberg: Heidelberg_Notebook_Data_General_summary_1406106000_1406235600.txt</p>
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<div>Data for MidnightDoc of iGEM Team Heidelberg: Heidelberg_Notebook_Data_General_summary_1406106000_1406235600.txt</div>Maexlichhttp://2014.igem.org/File:Heidelberg_Notebook_Data_General_summary_1404587400_1404832260.txtFile:Heidelberg Notebook Data General summary 1404587400 1404832260.txt2014-10-18T03:30:35Z<p>Maexlich: uploaded a new version of &quot;File:Heidelberg Notebook Data General summary 1404587400 1404832260.txt&quot;: Data for MidnightDoc of iGEM Team Heidelberg: Heidelberg_Notebook_Data_General_summary_1404587400_1404832260.txt</p>
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<div>Data for MidnightDoc of iGEM Team Heidelberg: Heidelberg_Notebook_Data_General_summary_1404587400_1404832260.txt</div>Maexlichhttp://2014.igem.org/File:Heidelberg_Notebook_Data_General_summary_1401804000_1402056000.txtFile:Heidelberg Notebook Data General summary 1401804000 1402056000.txt2014-10-18T03:30:32Z<p>Maexlich: uploaded a new version of &quot;File:Heidelberg Notebook Data General summary 1401804000 1402056000.txt&quot;: Data for MidnightDoc of iGEM Team Heidelberg: Heidelberg_Notebook_Data_General_summary_1401804000_1402056000.txt</p>
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<div>Data for MidnightDoc of iGEM Team Heidelberg: Heidelberg_Notebook_Data_General_summary_1401804000_1402056000.txt</div>Maexlichhttp://2014.igem.org/File:Heidelberg_Notebook_Data_General_summary_1403301600_1403390400.txtFile:Heidelberg Notebook Data General summary 1403301600 1403390400.txt2014-10-18T03:30:31Z<p>Maexlich: uploaded a new version of &quot;File:Heidelberg Notebook Data General summary 1403301600 1403390400.txt&quot;: Data for MidnightDoc of iGEM Team Heidelberg: Heidelberg_Notebook_Data_General_summary_1403301600_1403390400.txt</p>
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<div>Data for MidnightDoc of iGEM Team Heidelberg: Heidelberg_Notebook_Data_General_summary_1403301600_1403390400.txt</div>Maexlichhttp://2014.igem.org/File:Heidelberg_Notebook_Data_General_summary_1403030400_1403172000.txtFile:Heidelberg Notebook Data General summary 1403030400 1403172000.txt2014-10-18T03:30:28Z<p>Maexlich: uploaded a new version of &quot;File:Heidelberg Notebook Data General summary 1403030400 1403172000.txt&quot;: Data for MidnightDoc of iGEM Team Heidelberg: Heidelberg_Notebook_Data_General_summary_1403030400_1403172000.txt</p>
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<div>Data for MidnightDoc of iGEM Team Heidelberg: Heidelberg_Notebook_Data_General_summary_1403030400_1403172000.txt</div>Maexlichhttp://2014.igem.org/File:Heidelberg_Notebook_Data_General_summary_1408234500_1408476000.txtFile:Heidelberg Notebook Data General summary 1408234500 1408476000.txt2014-10-18T03:30:26Z<p>Maexlich: uploaded a new version of &quot;File:Heidelberg Notebook Data General summary 1408234500 1408476000.txt&quot;: Data for MidnightDoc of iGEM Team Heidelberg: Heidelberg_Notebook_Data_General_summary_1408234500_1408476000.txt</p>
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<div>Data for MidnightDoc of iGEM Team Heidelberg: Heidelberg_Notebook_Data_General_summary_1408234500_1408476000.txt</div>Maexlichhttp://2014.igem.org/File:Heidelberg_Notebook_Data_General_summary_1411421400_1411421400.txtFile:Heidelberg Notebook Data General summary 1411421400 1411421400.txt2014-10-18T03:30:24Z<p>Maexlich: uploaded a new version of &quot;File:Heidelberg Notebook Data General summary 1411421400 1411421400.txt&quot;: Data for MidnightDoc of iGEM Team Heidelberg: Heidelberg_Notebook_Data_General_summary_1411421400_1411421400.txt</p>
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<div>Data for MidnightDoc of iGEM Team Heidelberg: Heidelberg_Notebook_Data_General_summary_1411421400_1411421400.txt</div>Maexlich