http://2014.igem.org/wiki/index.php?title=Special:Contributions/Nbailly&feed=atom&limit=50&target=Nbailly&year=&month=2014.igem.org - User contributions [en]2024-03-29T08:34:28ZFrom 2014.igem.orgMediaWiki 1.16.5http://2014.igem.org/Team:Aachen/AttributionsTeam:Aachen/Attributions2014-10-18T03:51:58Z<p>Nbailly: /* Members */</p>
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=Members=<br />
<span class="anchor" id="members"></span><br />
<br />
A core concept of iGEM is the collaboration within an interdisciplinary student team. Accordingly, students of different age, gender and field of study came together to found our team in Aachen, in order to realize our project with joint forces and enthusiasm for the overall goal. Working closely together, we therefore not only have the chance to learn from each other, but also to excel ourselves. Using our collective creativity and common responsibility, we will create something special in order to make our contribution to synthetic biology.<br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:Mosthege" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> Michael Osthege </b><br />
<br/><br/><br />
<i> Molecular and Applied Biotechnology </i><br />
<br/><br/><br />
Man for Everything<br />
<br/><br/><br />
<!-- click for more information --><br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/3/35/Aachen_team_member_Michael_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:Fgohr" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
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<b> Florian Gohr </b><br />
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<i> Molecular and Applied Biotechnology </i><br />
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King Gibson<br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/c/ca/Aachen_team_member_Florian_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%;height: 180px; width: 180px;"> </div></a><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:AZimmermann" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
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<b> Arne Zimmermann </b><br />
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<i> Molecular and Applied Biotechnology </i><br />
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The Patience itself<br />
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<!-- click for more information --><br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/8/88/Aachen_team_member_Arne_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
</li><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:Nbailly" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
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<b> Nina Bailly </b><br />
<br/><br/><br />
<i> Molecular and Applied Biotechnology </i><br />
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Flask Prep Princess<br />
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<!-- click for more information --><br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/4/48/Aachen_team_member_Nina_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
</li><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:VeraA" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
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<b> Vera Alexandrova </b><br />
<br/><br/><br />
<i> Biology </i><br />
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Goddess of Plasmid Prep<br />
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<!-- click for more information --><br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/8/81/Aachen_team_member_Vera_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:Pdemling" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
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<b> Philipp Demling </b><br />
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<i> Molecular and Applied Biotechnology </i><br />
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Prince of Bad Puns<br />
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<!-- click for more information --><br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/7/77/Aachen_team_member_Philipp_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:R.hanke" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
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<b> René Hanke </b><br />
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<i> Molecular and Applied Biotechnology </i><br />
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Creative Mind & Cook<br />
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<!-- click for more information --><br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/b/b7/Aachen_team_member_Rene_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:PatrickO" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
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<b> Patrick Opdenstein </b><br />
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<i> Molecular and Applied Biotechnology </i><br />
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Graphics God<br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/1/1a/Aachen_team_member_Patrick_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:Bpeeters" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
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<b> Björn Peeters </b><br />
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<i> Computational Engineering Science </i><br />
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Hardware Master<br />
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<!-- click for more information --><br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/8/8d/Aachen_team_member_Bjoern_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:Eshani.sood" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
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<b> Eshani Sood </b><br />
<br/><br/><br />
<i> Biomedical Engineering </i><br />
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Sunshine<br />
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<!-- click for more information --><br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/5/52/Aachen_team_member_Eshani_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:Mjoppich" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
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<b> Markus Joppich </b><br />
<br/><br/><br />
<i> Computer Science </i><br />
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Software Expert & Travel Assistant<br />
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<!-- click for more information --><br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/8/83/Aachen_team_member_Markus_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:Ansgar" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> Ansgar Niemöller </b><br />
<br/><br/><br />
<i> Computational Engineering Science </i><br />
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GUI Master<br />
<!-- click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/4/4e/Aachen_team_member_Ansgar_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:J.plum" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> Julia Plum </b><br />
<br/><br/><br />
<i> Biology and Business Administration </i><br />
<br/><br/><br />
Fundraising & Balancing<br />
<br/><br/><br />
<!-- click for more information --><br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/c/c9/Aachen_team_member_Julia_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
</li><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:StefanReinhold" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
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<b> Stefan Reinhold </b><br />
<br/><br/><br />
<i> Molecular and Applied Biotechnology </i><br />
<br/><br/><br />
Treasurer<br />
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<!-- click for more information --><br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/0/0c/Aachen_team_member_Stefan_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
</li><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:Aschechtel" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> Anna Schechtel </b><br />
<br/><br/><br />
<i> Biology </i><br />
<br/><br/><br />
Chip Queen<br />
<br/><br/><br />
<!-- click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/a/a4/Aachen_team_member_Anna_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
</li><br />
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= Advisors =<br />
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<html><img class="imgshadow" style="float: left;" src="https://static.igem.org/mediawiki/2014/5/57/Aachen_team_member_Suresh_01.jpg" width="200px" /></html><br />
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'''Dr.-Ing. Suresh Sudarsan'''<br />
<br />
I'm working as a post-doc with Prof. Blank at RWTH Aachen University. In my Ph.D. I worked under the guidance of Prof. Andreas Schmid (2008-2012) at TU Dortmund, and Prof. Matthias Reuss (2009-2011) at the University of Stuttgart, with a focus on elucidating the link between the central and aromatic metabolism of ''P. putida'' using a systems biology approach.<br />
<br />
I'm fascinated about understanding the collective behavior of microorganisms and their metabolic potential in different niches. In my research, I use tools in ''metabolic engineering & systems biology'' such as metabolomics, fast sampling, kinetic/dynamic modeling and metabolic flux analysis. <br />
<br />
In iGEM, I enjoy working with a group of young scientists from Aachen to achieve our common goal of detecting ''Pseudomonas aeruginosa'' on hard surfaces. Besides science, I try to get myself involved in adventurous sports...but honestly... I just like to relax my day with a delicious meal and a good sci-fi movie!<br />
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<html><img class="imgshadow" style="float: left;" src="https://static.igem.org/mediawiki/2014/c/c8/Aachen_team_member_Ljubica.jpg" width="200px" /></html><br />
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'''Dr. rer. nat. Ljubica Vojcic'''<br />
<br />
I am working as a Subgroup Leader in Prof. Schwaneberg research group at the Institute of Biotechnology, RWTH Aachen. The research area of the Schwaneberg group focuses on directed protein evolution by developing novel methods for generating diversity at the gene level, analyzing consequences of mutational biases on the protein level and developing novel high-throughput screening systems that will lead to improved biocatalysts for prominent applications in industry. In particular, my core expertise is development of high throughput screening systems for different enzyme classes in order to redefine the screening step no longer as bottleneck in directed evolution. <br />
<br />
In iGEM, I enjoyed very much to work with highly motivated and ambitious young scientists from RWTH Aachen University. I truly believe that our collaboration has just started and that we will enjoy jointly solving the scientific challenges in the near future. <br />
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=Instructors=<br />
<span class="anchor" id="instructors"></span><br />
== Prof. Dr.-Ing. Lars M. Blank ==<br />
==== RWTH Institute for Applied Microbiology (iAMB) ====<br />
<html><p><img class="imgshadow" style="float: left;" src="https://static.igem.org/mediawiki/2014/4/42/Aachen_LarsBlank.jpg" width="150px" /></html> <br />
Prof. Blank focuses his research on fundamental and applied aspects of microbial metabolism. Of specific interest is the interaction between the metabolic network and the introduced genetic and environmental perturbations. The research on in silico/in vivo metabolic network operations is aimed at a deeper understanding of cell function, with the ultimate goal of rational cell engineering.<br />
<br />
In his teaching, Prof. Blank focuses on the integration of biological concepts with the tools from bioinformatics and engineering. He believes that a sound knowledge base in life sciences is the key for creative and thus successful work in the areas of Metabolic Engineering and Synthetic Biology. Read more about Prof. Blank's work on the [http://www.iamb.rwth-aachen.de/html/members.php?s=det&id=3 iAMB's website].<br />
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== Prof. Dr. Wolfgang Wiechert ==<br />
==== Forschungszentrum Jülich, Institute of Bio- and Geosciences (IGB-1) ====<br />
<html><p><img class="imgshadow" style="float: left;" src="https://static.igem.org/mediawiki/2014/8/88/Aachen_WolfgangWiechert.jpg" width="150px" /></html> <br />
Prof. Wiechert's main area of work lies within the field of applied systems bio(techno)logy of microorganisms with a special focus on methodological developments for quantitative biology. Characteristics of his research work are a close integration of experimental and theoretical work within multi disciplinary projects. As the head of the Systems Biotechnology research division at Forschungszentrum Jülich, he is developing methods for quantitative metabolomics, fluxomics and proteomics including model based mathematical methods for experimental design, parameter estimation, and process optimization in biotechnological systems. Future work will also incorporate micro fluidic methods for single cell analysis. In general, all research results are used to drive forward the process of gaining knowledge in the course of an iterative improvement of industrial production systems. This proceeds in close cooperation of all working groups at the IBG-1. Together with industrial partners also diverse examples from industry are investigated and further developed. Read more about Prof. Wiechert's work on the [http://www.fz-juelich.de/SharedDocs/Personen/IBG/IBG-1/EN/Research_groups/general/wiechert.html/ IBG-1 website].<br />
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== Prof. Dr. Ulrich Schwaneberg ==<br />
==== RWTH Institute for Biotechnology, Leibniz Institute for Interactive Materials (DWI) ====<br />
<html><p><img class="imgshadow" style="float: left;" src="https://static.igem.org/mediawiki/2014/f/fe/Aachen_UlrichSchwaneberg.jpg" width="150px" /></html> <br />
The Schwaneberg Group seeks to be at the research frontier in the interdisciplinary field of directed protein evolution by developing novel methods for generating diversity at the gene level, analyzing consequence of mutational biases on the protein level and developing novel high-throughput screening systems that will ultimately lead to tailored-biocatalysts for significant applications in industry. They train students in the cutting edge technologies of laboratory evolution, biocatalyst engineering and high throughput screening methodologies. The Schwaneberg Group believes in integrating fundamental principles of protein design with environmental awareness in their research and seeks to promote international scientific collaborations. Read more about Prof. Schwaneberg and his work on the [http://www.biotec.rwth-aachen.de/ Schwaneberg Group's website].<br />
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<br />
This project would not have been possible without the generous help and advice from many great people and organizations. We thank all the partners listed below for their essential contributions and financial support that covered registration fees, travel costs, the organization of the meetup in September, laboratory materials and expenses for services and materials.<br />
<br />
=Partners=<br />
<span class="anchor" id="partners"></span><br />
<div align="center"><br />
{|cellpadding="25"<br />
|[[File:Logo_Buergerstiftung-Aachen_hoch.jpg|159px|center|link=http://www.buergerstiftung-aachen.de|Bürgerstiftung Aachen]]<br />
|[[File:Aachen_Niersverband_Firmenlogo.jpg|318px|link=http://www.niersverband.de/|Niersverband]]<br />
|}<br />
{|cellpadding="13"<br />
|[[File:Aachen_Genscript_Logo.png|240px|link=http://www.genscript.com/|Genscript]]<br />
|[[File:Aachen_Eurofinsgenomics.png|193px|link=http://www.eurofinsgenomics.eu/|Eurofins Genomics]]<br />
|[[File:Aachen_Labomedic_Logo.jpg|256px|link=http://www.labomedic.de/|Labomedic]]<br />
|}<br />
<br />
{|cellpadding="35"<br />
|[[File:Aachen_bmbf.jpg|326px|link=http://www.bmbf.de/de/24140.php|BMBF]]<br />
|[[File:Aachen_idt_.png|217px|link=http://www.idt-biologika.de/|IDT]]<br />
|}<br />
<br />
{|cellpadding="25"<br />
|[[File:M2p_labs_logo.jpg|103px|link=http://www.m2p-labs.com/|m2p labs]]<br />
|[[File:Aachen_Roth_Logo.png|109px|link=http://www.carlroth.com/pages/index/COM/index.jsp?market=COM&lang=en-com|Carl Roth]]<br />
|[[File:Aachen_ProRWTH_logo.png|121px|link=http://www.prorwth.de/|pro RWTH]]<br />
|}<br />
{|cellpadding="25"<br />
|[[File:Aachen_Logo_iAMB.png|229px|link=http://www.iamb.rwth-aachen.de|Institute of Applied Microbiology - iAMB]]<br />
|[[File:Aachen_Logo_bio7.png|229px|link=http://www.molbiotech.rwth-aachen.de|Institute for Molecular Biotechnology]]<br />
|[[File:Aachen_Logo_HISynBio.png|246px|link=http://www.helmholtz.de/en/about_us/initiating_and_networking/assuring_excellence/synthetic_biology|Helmholtz Association - Initiative on Synthetic Biology]]<br />
|}<br />
{|cellpadding="35"<br />
|[[File:Aachen_Logo_ABBt.png|209px|link=http://www.biologie.rwth-aachen.de|Aachen Biology and Biotechnology - ABBt]]<br />
|[[File:Aachen_juelich.png|239px|link=http://www.fz-juelich.de/|Forschungszentrum Jülich]]<br />
|[[File:Aachen_FabLabAachenLogo.jpg|209px|link=http://hci.rwth-aachen.de/fablab|Fab Lab Aachen]]<br />
|[[File:Aachen_Schwaneberg_Group.png|150px|link=http://www.biotec.rwth-aachen.de|Schwaneberg Group]]<br />
|}<br />
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<br />
=Attribution of Scientific and General Support=<br />
<span class="anchor" id="support"></span><br />
Great projects do not only depend on solid funding, but even more on the invaluable support by great people. We found great people not only in our 'home institute' the iAMB, but across many partner institutions.<br />
<br />
<br />
* '''RWTH Institute of Molecular Biotechnology''' (Biology VII)<br />
** Dr. Ulrich Commandeur ''for giving us access to essential resources of the bio7''<br />
** Christina Dickmeis M.Sc. ''who answered lots of questions''<br />
* '''RWTH Institute of Biotechnology''' (Biology VI)<br />
** David Schönauer and Alan Mertens M.Sc. ''who helped us to purify proteins''<br />
* '''Helmholtz Institute for Biomedical Engineering'''<br />
** Prof. Dr. Lothar Elling and Sophia Böcker, M.Sc. ''for giving us access to their gal-3 expression plasmid''<br />
* '''RWTH Institute of Applied Microbiology''' (iAMB/Biology IV)<br />
** Prof. Dr. Miriam Agler-Rosenbaum ''for scientific information on quorum sensing''<br />
** Dr. Martin Zimmermann ''who counseled us on balancing iGEM with our studies''<br />
** Dr. Andrea Germer ''for teaching us Gibson cloning''<br />
** Erick Bosire Maosa M.Sc. ''who advised us on cultivation of Pseudomonas''<br />
** Gisela Beissel, Annette Schreer and Kalle Hüser ''for their great patience with our entropy generation''<br />
* '''Forschungszentrum Jülich'''<br />
** Marianne Heß ''who was and is essential for travel organization''<br />
** Dr. Hanno Scharr ''for advice on image analysis''<br />
* '''Fab Lab Aachen'''<br />
** René Bohne, Jan Zimmermann and Jan Thar ''who generously gave us access to the lasercutter and 3D printer''<br />
* '''Others'''<br />
** Dr. Helen Rosenkranz, ABBt, ''for helping us with hundreds of administrative paperwork''<br />
** Dr. Heinz-Albert Becker, NEAnderLab, ''for his help with the organization of the school project''<br />
** Ulrike Eisel, Gymnasium am Neandertal, ''for her help with the organization of the school project''<br />
** Fachschaft Biowissenschaften ''for food supplies and access to their rooms''<br />
** Dr. Joachim Fröhlingsdorf, MakeLight, ''who inspired us to expand our OD/F project''<br />
** Univ.-Prof. Dr. Wolfgang Dott, Uniklinik RWTH Aachen, ''for helpful feedback regarding fields of application for our biosensor''<br />
<br />
Finally, we want to thank '''Simon&nbsp;Unthan''', '''Michael&nbsp;Limberg''', '''Henrik&nbsp;Cordes''', '''Sven&nbsp;Jager''' and '''Team&nbsp;Bielefeld''' for their advice guiding us through our first iGEM participation! <br />
<br />
Special thanks to '''Dr.-Ing. Suresh Sudarsan''' for a final check for typos on our wiki!<br />
<br />
<br />
{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/AttributionsTeam:Aachen/Attributions2014-10-18T03:51:41Z<p>Nbailly: /* Members */</p>
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# General Support<br />
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# Lab support<br />
# Difficult technique support<br />
# Project advisor support<br />
# Wiki support<br />
# Presentation coaching<br />
# Policy & Practices support<br />
# Thanks and acknowledgements for all other people involved in helping make a successful iGEM team.<br />
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=Members=<br />
<span class="anchor" id="members"></span><br />
<br />
A core concept of iGEM is the collaboration within an interdisciplinary student team. Accordingly, students of different age, gender and field of study came together to found our team in Aachen, in order to realize our project with joint forces and enthusiasm for the overall goal. Working closely together, we therefore not only have the chance to learn from each other, but also to excel ourselves. Using our collective creativity and common responsibility, we will create something special in order to make our contribution to synthetic biology.<br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:Mosthege" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> Michael Osthege </b><br />
<br/><br/><br />
<i> Molecular and Applied Biotechnology </i><br />
<br/><br/><br />
Man for Everything<br />
<br/><br/><br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/3/35/Aachen_team_member_Michael_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
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<a href="https://2014.igem.org/User:Fgohr" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
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<b> Florian Gohr </b><br />
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<i> Molecular and Applied Biotechnology </i><br />
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King Gibson<br />
<br/><br/><br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/c/ca/Aachen_team_member_Florian_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%;height: 180px; width: 180px;"> </div></a><br />
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<a href="https://2014.igem.org/User:AZimmermann" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> Arne Zimmermann </b><br />
<br/><br/><br />
<i> Molecular and Applied Biotechnology </i><br />
<br/><br/><br />
The Patience itself<br />
<br/><br/><br />
<!-- click for more information --><br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/8/88/Aachen_team_member_Arne_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:Nbailly" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> Nina Bailly </b><br />
<br/><br/><br />
<i> Molecular and Applied Biotechnology </i><br />
<br/><br/><br />
Flask Prep Princess<br />
<br/><br/><br />
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</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/4/48/Aachen_team_member_Nina_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
</li><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:VeraA" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> Vera Alexandrova </b><br />
<br/><br/><br />
<i> Biology </i><br />
<br/><br/><br />
Goddess of Plasmid Prep<br />
<br/><br/><br />
<!-- click for more information --><br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/8/81/Aachen_team_member_Vera_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
</li><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:Pdemling" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> Philipp Demling </b><br />
<br/><br/><br />
<i> Molecular and Applied Biotechnology </i><br />
<br/><br/><br />
Prince of Bad Puns<br />
<br/><br/><br />
<!-- click for more information --><br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/7/77/Aachen_team_member_Philipp_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
</li><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:R.hanke" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> René Hanke </b><br />
<br/><br/><br />
<i> Molecular and Applied Biotechnology </i><br />
<br/><br/><br />
Creative Mind & Cook<br />
<br/><br/><br />
<!-- click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/b/b7/Aachen_team_member_Rene_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
</li><br />
<br />
<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:PatrickO" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> Patrick Opdenstein </b><br />
<br/><br/><br />
<i> Molecular and Applied Biotechnology </i><br />
<br/><br/><br />
Graphics God<br />
<br/><br/><br />
<!-- click for more information --><br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/1/1a/Aachen_team_member_Patrick_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
</li><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:Bpeeters" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> Björn Peeters </b><br />
<br/><br/><br />
<i> Computational Engineering Science </i><br />
<br/><br/><br />
Hardware Master<br />
<br/><br/><br />
<!-- click for more information --><br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/8/8d/Aachen_team_member_Bjoern_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:Eshani.sood" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> Eshani Sood </b><br />
<br/><br/><br />
<i> Biomedical Engineering </i><br />
<br/><br/><br />
Sunshine<br />
<br/><br/><br />
<!-- click for more information --><br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/5/52/Aachen_team_member_Eshani_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
</li><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:Mjoppich" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> Markus Joppich </b><br />
<br/><br/><br />
<i> Computer Science </i><br />
<br/><br/><br />
Software Expert & Travel Assistant<br />
<br/><br/><br />
<!-- click for more information --><br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/8/83/Aachen_team_member_Markus_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
</li><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:Ansgar" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> Ansgar Niemöller </b><br />
<br/><br/><br />
<i> Computational Engineering Science </i><br />
<br/><br/><br />
GUI Master<br />
<!-- click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/4/4e/Aachen_team_member_Ansgar_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
</li><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:J.plum" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> Julia Plum </b><br />
<br/><br/><br />
<i> Biology and Business Administration </i><br />
<br/><br/><br />
Fundraising and Balancing<br />
<br/><br/><br />
<!-- click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/c/c9/Aachen_team_member_Julia_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
</li><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:StefanReinhold" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> Stefan Reinhold </b><br />
<br/><br/><br />
<i> Molecular and Applied Biotechnology </i><br />
<br/><br/><br />
Treasurer<br />
<br/><br/><br />
<!-- click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/0/0c/Aachen_team_member_Stefan_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
</li><br />
<br />
<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:Aschechtel" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> Anna Schechtel </b><br />
<br/><br/><br />
<i> Biology </i><br />
<br/><br/><br />
Chip Queen<br />
<br/><br/><br />
<!-- click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/a/a4/Aachen_team_member_Anna_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
</li><br />
</ul><br />
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= Advisors =<br />
<br />
<html><img class="imgshadow" style="float: left;" src="https://static.igem.org/mediawiki/2014/5/57/Aachen_team_member_Suresh_01.jpg" width="200px" /></html><br />
<br />
'''Dr.-Ing. Suresh Sudarsan'''<br />
<br />
I'm working as a post-doc with Prof. Blank at RWTH Aachen University. In my Ph.D. I worked under the guidance of Prof. Andreas Schmid (2008-2012) at TU Dortmund, and Prof. Matthias Reuss (2009-2011) at the University of Stuttgart, with a focus on elucidating the link between the central and aromatic metabolism of ''P. putida'' using a systems biology approach.<br />
<br />
I'm fascinated about understanding the collective behavior of microorganisms and their metabolic potential in different niches. In my research, I use tools in ''metabolic engineering & systems biology'' such as metabolomics, fast sampling, kinetic/dynamic modeling and metabolic flux analysis. <br />
<br />
In iGEM, I enjoy working with a group of young scientists from Aachen to achieve our common goal of detecting ''Pseudomonas aeruginosa'' on hard surfaces. Besides science, I try to get myself involved in adventurous sports...but honestly... I just like to relax my day with a delicious meal and a good sci-fi movie!<br />
<br />
<br />
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<html><img class="imgshadow" style="float: left;" src="https://static.igem.org/mediawiki/2014/c/c8/Aachen_team_member_Ljubica.jpg" width="200px" /></html><br />
<br />
'''Dr. rer. nat. Ljubica Vojcic'''<br />
<br />
I am working as a Subgroup Leader in Prof. Schwaneberg research group at the Institute of Biotechnology, RWTH Aachen. The research area of the Schwaneberg group focuses on directed protein evolution by developing novel methods for generating diversity at the gene level, analyzing consequences of mutational biases on the protein level and developing novel high-throughput screening systems that will lead to improved biocatalysts for prominent applications in industry. In particular, my core expertise is development of high throughput screening systems for different enzyme classes in order to redefine the screening step no longer as bottleneck in directed evolution. <br />
<br />
In iGEM, I enjoyed very much to work with highly motivated and ambitious young scientists from RWTH Aachen University. I truly believe that our collaboration has just started and that we will enjoy jointly solving the scientific challenges in the near future. <br />
<br />
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=Instructors=<br />
<span class="anchor" id="instructors"></span><br />
== Prof. Dr.-Ing. Lars M. Blank ==<br />
==== RWTH Institute for Applied Microbiology (iAMB) ====<br />
<html><p><img class="imgshadow" style="float: left;" src="https://static.igem.org/mediawiki/2014/4/42/Aachen_LarsBlank.jpg" width="150px" /></html> <br />
Prof. Blank focuses his research on fundamental and applied aspects of microbial metabolism. Of specific interest is the interaction between the metabolic network and the introduced genetic and environmental perturbations. The research on in silico/in vivo metabolic network operations is aimed at a deeper understanding of cell function, with the ultimate goal of rational cell engineering.<br />
<br />
In his teaching, Prof. Blank focuses on the integration of biological concepts with the tools from bioinformatics and engineering. He believes that a sound knowledge base in life sciences is the key for creative and thus successful work in the areas of Metabolic Engineering and Synthetic Biology. Read more about Prof. Blank's work on the [http://www.iamb.rwth-aachen.de/html/members.php?s=det&id=3 iAMB's website].<br />
<html><br><br><br></p></html><br />
<br />
== Prof. Dr. Wolfgang Wiechert ==<br />
==== Forschungszentrum Jülich, Institute of Bio- and Geosciences (IGB-1) ====<br />
<html><p><img class="imgshadow" style="float: left;" src="https://static.igem.org/mediawiki/2014/8/88/Aachen_WolfgangWiechert.jpg" width="150px" /></html> <br />
Prof. Wiechert's main area of work lies within the field of applied systems bio(techno)logy of microorganisms with a special focus on methodological developments for quantitative biology. Characteristics of his research work are a close integration of experimental and theoretical work within multi disciplinary projects. As the head of the Systems Biotechnology research division at Forschungszentrum Jülich, he is developing methods for quantitative metabolomics, fluxomics and proteomics including model based mathematical methods for experimental design, parameter estimation, and process optimization in biotechnological systems. Future work will also incorporate micro fluidic methods for single cell analysis. In general, all research results are used to drive forward the process of gaining knowledge in the course of an iterative improvement of industrial production systems. This proceeds in close cooperation of all working groups at the IBG-1. Together with industrial partners also diverse examples from industry are investigated and further developed. Read more about Prof. Wiechert's work on the [http://www.fz-juelich.de/SharedDocs/Personen/IBG/IBG-1/EN/Research_groups/general/wiechert.html/ IBG-1 website].<br />
<html><br><br><br></p></html><br />
<br />
== Prof. Dr. Ulrich Schwaneberg ==<br />
==== RWTH Institute for Biotechnology, Leibniz Institute for Interactive Materials (DWI) ====<br />
<html><p><img class="imgshadow" style="float: left;" src="https://static.igem.org/mediawiki/2014/f/fe/Aachen_UlrichSchwaneberg.jpg" width="150px" /></html> <br />
The Schwaneberg Group seeks to be at the research frontier in the interdisciplinary field of directed protein evolution by developing novel methods for generating diversity at the gene level, analyzing consequence of mutational biases on the protein level and developing novel high-throughput screening systems that will ultimately lead to tailored-biocatalysts for significant applications in industry. They train students in the cutting edge technologies of laboratory evolution, biocatalyst engineering and high throughput screening methodologies. The Schwaneberg Group believes in integrating fundamental principles of protein design with environmental awareness in their research and seeks to promote international scientific collaborations. Read more about Prof. Schwaneberg and his work on the [http://www.biotec.rwth-aachen.de/ Schwaneberg Group's website].<br />
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<br />
This project would not have been possible without the generous help and advice from many great people and organizations. We thank all the partners listed below for their essential contributions and financial support that covered registration fees, travel costs, the organization of the meetup in September, laboratory materials and expenses for services and materials.<br />
<br />
=Partners=<br />
<span class="anchor" id="partners"></span><br />
<div align="center"><br />
{|cellpadding="25"<br />
|[[File:Logo_Buergerstiftung-Aachen_hoch.jpg|159px|center|link=http://www.buergerstiftung-aachen.de|Bürgerstiftung Aachen]]<br />
|[[File:Aachen_Niersverband_Firmenlogo.jpg|318px|link=http://www.niersverband.de/|Niersverband]]<br />
|}<br />
{|cellpadding="13"<br />
|[[File:Aachen_Genscript_Logo.png|240px|link=http://www.genscript.com/|Genscript]]<br />
|[[File:Aachen_Eurofinsgenomics.png|193px|link=http://www.eurofinsgenomics.eu/|Eurofins Genomics]]<br />
|[[File:Aachen_Labomedic_Logo.jpg|256px|link=http://www.labomedic.de/|Labomedic]]<br />
|}<br />
<br />
{|cellpadding="35"<br />
|[[File:Aachen_bmbf.jpg|326px|link=http://www.bmbf.de/de/24140.php|BMBF]]<br />
|[[File:Aachen_idt_.png|217px|link=http://www.idt-biologika.de/|IDT]]<br />
|}<br />
<br />
{|cellpadding="25"<br />
|[[File:M2p_labs_logo.jpg|103px|link=http://www.m2p-labs.com/|m2p labs]]<br />
|[[File:Aachen_Roth_Logo.png|109px|link=http://www.carlroth.com/pages/index/COM/index.jsp?market=COM&lang=en-com|Carl Roth]]<br />
|[[File:Aachen_ProRWTH_logo.png|121px|link=http://www.prorwth.de/|pro RWTH]]<br />
|}<br />
{|cellpadding="25"<br />
|[[File:Aachen_Logo_iAMB.png|229px|link=http://www.iamb.rwth-aachen.de|Institute of Applied Microbiology - iAMB]]<br />
|[[File:Aachen_Logo_bio7.png|229px|link=http://www.molbiotech.rwth-aachen.de|Institute for Molecular Biotechnology]]<br />
|[[File:Aachen_Logo_HISynBio.png|246px|link=http://www.helmholtz.de/en/about_us/initiating_and_networking/assuring_excellence/synthetic_biology|Helmholtz Association - Initiative on Synthetic Biology]]<br />
|}<br />
{|cellpadding="35"<br />
|[[File:Aachen_Logo_ABBt.png|209px|link=http://www.biologie.rwth-aachen.de|Aachen Biology and Biotechnology - ABBt]]<br />
|[[File:Aachen_juelich.png|239px|link=http://www.fz-juelich.de/|Forschungszentrum Jülich]]<br />
|[[File:Aachen_FabLabAachenLogo.jpg|209px|link=http://hci.rwth-aachen.de/fablab|Fab Lab Aachen]]<br />
|[[File:Aachen_Schwaneberg_Group.png|150px|link=http://www.biotec.rwth-aachen.de|Schwaneberg Group]]<br />
|}<br />
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<br />
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<br />
=Attribution of Scientific and General Support=<br />
<span class="anchor" id="support"></span><br />
Great projects do not only depend on solid funding, but even more on the invaluable support by great people. We found great people not only in our 'home institute' the iAMB, but across many partner institutions.<br />
<br />
<br />
* '''RWTH Institute of Molecular Biotechnology''' (Biology VII)<br />
** Dr. Ulrich Commandeur ''for giving us access to essential resources of the bio7''<br />
** Christina Dickmeis M.Sc. ''who answered lots of questions''<br />
* '''RWTH Institute of Biotechnology''' (Biology VI)<br />
** David Schönauer and Alan Mertens M.Sc. ''who helped us to purify proteins''<br />
* '''Helmholtz Institute for Biomedical Engineering'''<br />
** Prof. Dr. Lothar Elling and Sophia Böcker, M.Sc. ''for giving us access to their gal-3 expression plasmid''<br />
* '''RWTH Institute of Applied Microbiology''' (iAMB/Biology IV)<br />
** Prof. Dr. Miriam Agler-Rosenbaum ''for scientific information on quorum sensing''<br />
** Dr. Martin Zimmermann ''who counseled us on balancing iGEM with our studies''<br />
** Dr. Andrea Germer ''for teaching us Gibson cloning''<br />
** Erick Bosire Maosa M.Sc. ''who advised us on cultivation of Pseudomonas''<br />
** Gisela Beissel, Annette Schreer and Kalle Hüser ''for their great patience with our entropy generation''<br />
* '''Forschungszentrum Jülich'''<br />
** Marianne Heß ''who was and is essential for travel organization''<br />
** Dr. Hanno Scharr ''for advice on image analysis''<br />
* '''Fab Lab Aachen'''<br />
** René Bohne, Jan Zimmermann and Jan Thar ''who generously gave us access to the lasercutter and 3D printer''<br />
* '''Others'''<br />
** Dr. Helen Rosenkranz, ABBt, ''for helping us with hundreds of administrative paperwork''<br />
** Dr. Heinz-Albert Becker, NEAnderLab, ''for his help with the organization of the school project''<br />
** Ulrike Eisel, Gymnasium am Neandertal, ''for her help with the organization of the school project''<br />
** Fachschaft Biowissenschaften ''for food supplies and access to their rooms''<br />
** Dr. Joachim Fröhlingsdorf, MakeLight, ''who inspired us to expand our OD/F project''<br />
** Univ.-Prof. Dr. Wolfgang Dott, Uniklinik RWTH Aachen, ''for helpful feedback regarding fields of application for our biosensor''<br />
<br />
Finally, we want to thank '''Simon&nbsp;Unthan''', '''Michael&nbsp;Limberg''', '''Henrik&nbsp;Cordes''', '''Sven&nbsp;Jager''' and '''Team&nbsp;Bielefeld''' for their advice guiding us through our first iGEM participation! <br />
<br />
Special thanks to '''Dr.-Ing. Suresh Sudarsan''' for a final check for typos on our wiki!<br />
<br />
<br />
{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/AttributionsTeam:Aachen/Attributions2014-10-18T03:51:02Z<p>Nbailly: /* Members */</p>
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# Wiki support<br />
# Presentation coaching<br />
# Policy & Practices support<br />
# Thanks and acknowledgements for all other people involved in helping make a successful iGEM team.<br />
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=Members=<br />
<span class="anchor" id="members"></span><br />
<br />
A core concept of iGEM is the collaboration within an interdisciplinary student team. Accordingly, students of different age, gender and field of study came together to found our team in Aachen, in order to realize our project with joint forces and enthusiasm for the overall goal. Working closely together, we therefore not only have the chance to learn from each other, but also to excel ourselves. Using our collective creativity and common responsibility, we will create something special in order to make our contribution to synthetic biology.<br />
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<a href="https://2014.igem.org/User:Mosthege" style="color:black"><br />
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<b> Michael Osthege </b><br />
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<i> Molecular and Applied Biotechnology </i><br />
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Man for Everything<br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/3/35/Aachen_team_member_Michael_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
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<a href="https://2014.igem.org/User:Fgohr" style="color:black"><br />
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<b> Florian Gohr </b><br />
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<i> Molecular and Applied Biotechnology </i><br />
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King Gibson<br />
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<a href="https://2014.igem.org/User:AZimmermann" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
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<b> Arne Zimmermann </b><br />
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<i> Molecular and Applied Biotechnology </i><br />
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The Patience itself<br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/8/88/Aachen_team_member_Arne_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:Nbailly" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
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<b> Nina Bailly </b><br />
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<i> Molecular and Applied Biotechnology </i><br />
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Flask Prep Princess<br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/4/48/Aachen_team_member_Nina_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:VeraA" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
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<b> Vera Alexandrova </b><br />
<br/><br/><br />
<i> Biology </i><br />
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Goddess of Plasmid Prep<br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/8/81/Aachen_team_member_Vera_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
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<a href="https://2014.igem.org/User:Pdemling" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
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<b> Philipp Demling </b><br />
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<i> Molecular and Applied Biotechnology </i><br />
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Prince of bad Puns<br />
<br/><br/><br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/7/77/Aachen_team_member_Philipp_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:R.hanke" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
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<b> René Hanke </b><br />
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<i> Molecular and Applied Biotechnology </i><br />
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Creative Mind & Cook<br />
<br/><br/><br />
<!-- click for more information --><br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/b/b7/Aachen_team_member_Rene_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:PatrickO" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
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<b> Patrick Opdenstein </b><br />
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<i> Molecular and Applied Biotechnology </i><br />
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Graphics God<br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/1/1a/Aachen_team_member_Patrick_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
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<a href="https://2014.igem.org/User:Bpeeters" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
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<b> Björn Peeters </b><br />
<br/><br/><br />
<i> Computational Engineering Science </i><br />
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Hardware Master<br />
<br/><br/><br />
<!-- click for more information --><br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/8/8d/Aachen_team_member_Bjoern_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:Eshani.sood" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
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<b> Eshani Sood </b><br />
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<i> Biomedical Engineering </i><br />
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Sunshine<br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:Mjoppich" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
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<b> Markus Joppich </b><br />
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<i> Computer Science </i><br />
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Software Expert and Travel Assistant<br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:Ansgar" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
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<b> Ansgar Niemöller </b><br />
<br/><br/><br />
<i> Computational Engineering Science </i><br />
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GUI Master<br />
<!-- click for more information --><br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/4/4e/Aachen_team_member_Ansgar_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
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<a href="https://2014.igem.org/User:J.plum" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
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<b> Julia Plum </b><br />
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<i> Biology and Business Administration </i><br />
<br/><br/><br />
Fundraising and Balancing<br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/c/c9/Aachen_team_member_Julia_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
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<a href="https://2014.igem.org/User:StefanReinhold" style="color:black"><br />
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<b> Stefan Reinhold </b><br />
<br/><br/><br />
<i> Molecular and Applied Biotechnology </i><br />
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Treasurer<br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/0/0c/Aachen_team_member_Stefan_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:Aschechtel" style="color:black"><br />
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<b> Anna Schechtel </b><br />
<br/><br/><br />
<i> Biology </i><br />
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Chip Queen<br />
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<!-- click for more information --><br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/a/a4/Aachen_team_member_Anna_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
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= Advisors =<br />
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<html><img class="imgshadow" style="float: left;" src="https://static.igem.org/mediawiki/2014/5/57/Aachen_team_member_Suresh_01.jpg" width="200px" /></html><br />
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'''Dr.-Ing. Suresh Sudarsan'''<br />
<br />
I'm working as a post-doc with Prof. Blank at RWTH Aachen University. In my Ph.D. I worked under the guidance of Prof. Andreas Schmid (2008-2012) at TU Dortmund, and Prof. Matthias Reuss (2009-2011) at the University of Stuttgart, with a focus on elucidating the link between the central and aromatic metabolism of ''P. putida'' using a systems biology approach.<br />
<br />
I'm fascinated about understanding the collective behavior of microorganisms and their metabolic potential in different niches. In my research, I use tools in ''metabolic engineering & systems biology'' such as metabolomics, fast sampling, kinetic/dynamic modeling and metabolic flux analysis. <br />
<br />
In iGEM, I enjoy working with a group of young scientists from Aachen to achieve our common goal of detecting ''Pseudomonas aeruginosa'' on hard surfaces. Besides science, I try to get myself involved in adventurous sports...but honestly... I just like to relax my day with a delicious meal and a good sci-fi movie!<br />
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<html><img class="imgshadow" style="float: left;" src="https://static.igem.org/mediawiki/2014/c/c8/Aachen_team_member_Ljubica.jpg" width="200px" /></html><br />
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'''Dr. rer. nat. Ljubica Vojcic'''<br />
<br />
I am working as a Subgroup Leader in Prof. Schwaneberg research group at the Institute of Biotechnology, RWTH Aachen. The research area of the Schwaneberg group focuses on directed protein evolution by developing novel methods for generating diversity at the gene level, analyzing consequences of mutational biases on the protein level and developing novel high-throughput screening systems that will lead to improved biocatalysts for prominent applications in industry. In particular, my core expertise is development of high throughput screening systems for different enzyme classes in order to redefine the screening step no longer as bottleneck in directed evolution. <br />
<br />
In iGEM, I enjoyed very much to work with highly motivated and ambitious young scientists from RWTH Aachen University. I truly believe that our collaboration has just started and that we will enjoy jointly solving the scientific challenges in the near future. <br />
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=Instructors=<br />
<span class="anchor" id="instructors"></span><br />
== Prof. Dr.-Ing. Lars M. Blank ==<br />
==== RWTH Institute for Applied Microbiology (iAMB) ====<br />
<html><p><img class="imgshadow" style="float: left;" src="https://static.igem.org/mediawiki/2014/4/42/Aachen_LarsBlank.jpg" width="150px" /></html> <br />
Prof. Blank focuses his research on fundamental and applied aspects of microbial metabolism. Of specific interest is the interaction between the metabolic network and the introduced genetic and environmental perturbations. The research on in silico/in vivo metabolic network operations is aimed at a deeper understanding of cell function, with the ultimate goal of rational cell engineering.<br />
<br />
In his teaching, Prof. Blank focuses on the integration of biological concepts with the tools from bioinformatics and engineering. He believes that a sound knowledge base in life sciences is the key for creative and thus successful work in the areas of Metabolic Engineering and Synthetic Biology. Read more about Prof. Blank's work on the [http://www.iamb.rwth-aachen.de/html/members.php?s=det&id=3 iAMB's website].<br />
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<br />
== Prof. Dr. Wolfgang Wiechert ==<br />
==== Forschungszentrum Jülich, Institute of Bio- and Geosciences (IGB-1) ====<br />
<html><p><img class="imgshadow" style="float: left;" src="https://static.igem.org/mediawiki/2014/8/88/Aachen_WolfgangWiechert.jpg" width="150px" /></html> <br />
Prof. Wiechert's main area of work lies within the field of applied systems bio(techno)logy of microorganisms with a special focus on methodological developments for quantitative biology. Characteristics of his research work are a close integration of experimental and theoretical work within multi disciplinary projects. As the head of the Systems Biotechnology research division at Forschungszentrum Jülich, he is developing methods for quantitative metabolomics, fluxomics and proteomics including model based mathematical methods for experimental design, parameter estimation, and process optimization in biotechnological systems. Future work will also incorporate micro fluidic methods for single cell analysis. In general, all research results are used to drive forward the process of gaining knowledge in the course of an iterative improvement of industrial production systems. This proceeds in close cooperation of all working groups at the IBG-1. Together with industrial partners also diverse examples from industry are investigated and further developed. Read more about Prof. Wiechert's work on the [http://www.fz-juelich.de/SharedDocs/Personen/IBG/IBG-1/EN/Research_groups/general/wiechert.html/ IBG-1 website].<br />
<html><br><br><br></p></html><br />
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== Prof. Dr. Ulrich Schwaneberg ==<br />
==== RWTH Institute for Biotechnology, Leibniz Institute for Interactive Materials (DWI) ====<br />
<html><p><img class="imgshadow" style="float: left;" src="https://static.igem.org/mediawiki/2014/f/fe/Aachen_UlrichSchwaneberg.jpg" width="150px" /></html> <br />
The Schwaneberg Group seeks to be at the research frontier in the interdisciplinary field of directed protein evolution by developing novel methods for generating diversity at the gene level, analyzing consequence of mutational biases on the protein level and developing novel high-throughput screening systems that will ultimately lead to tailored-biocatalysts for significant applications in industry. They train students in the cutting edge technologies of laboratory evolution, biocatalyst engineering and high throughput screening methodologies. The Schwaneberg Group believes in integrating fundamental principles of protein design with environmental awareness in their research and seeks to promote international scientific collaborations. Read more about Prof. Schwaneberg and his work on the [http://www.biotec.rwth-aachen.de/ Schwaneberg Group's website].<br />
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<br />
This project would not have been possible without the generous help and advice from many great people and organizations. We thank all the partners listed below for their essential contributions and financial support that covered registration fees, travel costs, the organization of the meetup in September, laboratory materials and expenses for services and materials.<br />
<br />
=Partners=<br />
<span class="anchor" id="partners"></span><br />
<div align="center"><br />
{|cellpadding="25"<br />
|[[File:Logo_Buergerstiftung-Aachen_hoch.jpg|159px|center|link=http://www.buergerstiftung-aachen.de|Bürgerstiftung Aachen]]<br />
|[[File:Aachen_Niersverband_Firmenlogo.jpg|318px|link=http://www.niersverband.de/|Niersverband]]<br />
|}<br />
{|cellpadding="13"<br />
|[[File:Aachen_Genscript_Logo.png|240px|link=http://www.genscript.com/|Genscript]]<br />
|[[File:Aachen_Eurofinsgenomics.png|193px|link=http://www.eurofinsgenomics.eu/|Eurofins Genomics]]<br />
|[[File:Aachen_Labomedic_Logo.jpg|256px|link=http://www.labomedic.de/|Labomedic]]<br />
|}<br />
<br />
{|cellpadding="35"<br />
|[[File:Aachen_bmbf.jpg|326px|link=http://www.bmbf.de/de/24140.php|BMBF]]<br />
|[[File:Aachen_idt_.png|217px|link=http://www.idt-biologika.de/|IDT]]<br />
|}<br />
<br />
{|cellpadding="25"<br />
|[[File:M2p_labs_logo.jpg|103px|link=http://www.m2p-labs.com/|m2p labs]]<br />
|[[File:Aachen_Roth_Logo.png|109px|link=http://www.carlroth.com/pages/index/COM/index.jsp?market=COM&lang=en-com|Carl Roth]]<br />
|[[File:Aachen_ProRWTH_logo.png|121px|link=http://www.prorwth.de/|pro RWTH]]<br />
|}<br />
{|cellpadding="25"<br />
|[[File:Aachen_Logo_iAMB.png|229px|link=http://www.iamb.rwth-aachen.de|Institute of Applied Microbiology - iAMB]]<br />
|[[File:Aachen_Logo_bio7.png|229px|link=http://www.molbiotech.rwth-aachen.de|Institute for Molecular Biotechnology]]<br />
|[[File:Aachen_Logo_HISynBio.png|246px|link=http://www.helmholtz.de/en/about_us/initiating_and_networking/assuring_excellence/synthetic_biology|Helmholtz Association - Initiative on Synthetic Biology]]<br />
|}<br />
{|cellpadding="35"<br />
|[[File:Aachen_Logo_ABBt.png|209px|link=http://www.biologie.rwth-aachen.de|Aachen Biology and Biotechnology - ABBt]]<br />
|[[File:Aachen_juelich.png|239px|link=http://www.fz-juelich.de/|Forschungszentrum Jülich]]<br />
|[[File:Aachen_FabLabAachenLogo.jpg|209px|link=http://hci.rwth-aachen.de/fablab|Fab Lab Aachen]]<br />
|[[File:Aachen_Schwaneberg_Group.png|150px|link=http://www.biotec.rwth-aachen.de|Schwaneberg Group]]<br />
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<br />
=Attribution of Scientific and General Support=<br />
<span class="anchor" id="support"></span><br />
Great projects do not only depend on solid funding, but even more on the invaluable support by great people. We found great people not only in our 'home institute' the iAMB, but across many partner institutions.<br />
<br />
<br />
* '''RWTH Institute of Molecular Biotechnology''' (Biology VII)<br />
** Dr. Ulrich Commandeur ''for giving us access to essential resources of the bio7''<br />
** Christina Dickmeis M.Sc. ''who answered lots of questions''<br />
* '''RWTH Institute of Biotechnology''' (Biology VI)<br />
** David Schönauer and Alan Mertens M.Sc. ''who helped us to purify proteins''<br />
* '''Helmholtz Institute for Biomedical Engineering'''<br />
** Prof. Dr. Lothar Elling and Sophia Böcker, M.Sc. ''for giving us access to their gal-3 expression plasmid''<br />
* '''RWTH Institute of Applied Microbiology''' (iAMB/Biology IV)<br />
** Prof. Dr. Miriam Agler-Rosenbaum ''for scientific information on quorum sensing''<br />
** Dr. Martin Zimmermann ''who counseled us on balancing iGEM with our studies''<br />
** Dr. Andrea Germer ''for teaching us Gibson cloning''<br />
** Erick Bosire Maosa M.Sc. ''who advised us on cultivation of Pseudomonas''<br />
** Gisela Beissel, Annette Schreer and Kalle Hüser ''for their great patience with our entropy generation''<br />
* '''Forschungszentrum Jülich'''<br />
** Marianne Heß ''who was and is essential for travel organization''<br />
** Dr. Hanno Scharr ''for advice on image analysis''<br />
* '''Fab Lab Aachen'''<br />
** René Bohne, Jan Zimmermann and Jan Thar ''who generously gave us access to the lasercutter and 3D printer''<br />
* '''Others'''<br />
** Dr. Helen Rosenkranz, ABBt, ''for helping us with hundreds of administrative paperwork''<br />
** Dr. Heinz-Albert Becker, NEAnderLab, ''for his help with the organization of the school project''<br />
** Ulrike Eisel, Gymnasium am Neandertal, ''for her help with the organization of the school project''<br />
** Fachschaft Biowissenschaften ''for food supplies and access to their rooms''<br />
** Dr. Joachim Fröhlingsdorf, MakeLight, ''who inspired us to expand our OD/F project''<br />
** Univ.-Prof. Dr. Wolfgang Dott, Uniklinik RWTH Aachen, ''for helpful feedback regarding fields of application for our biosensor''<br />
<br />
Finally, we want to thank '''Simon&nbsp;Unthan''', '''Michael&nbsp;Limberg''', '''Henrik&nbsp;Cordes''', '''Sven&nbsp;Jager''' and '''Team&nbsp;Bielefeld''' for their advice guiding us through our first iGEM participation! <br />
<br />
Special thanks to '''Dr.-Ing. Suresh Sudarsan''' for a final check for typos on our wiki!<br />
<br />
<br />
{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/AttributionsTeam:Aachen/Attributions2014-10-18T03:50:27Z<p>Nbailly: /* Members */</p>
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# General Support<br />
# Fundraising help and advice<br />
# Partners<br />
# Project support and advice<br />
# Lab support<br />
# Difficult technique support<br />
# Project advisor support<br />
# Wiki support<br />
# Presentation coaching<br />
# Policy & Practices support<br />
# Thanks and acknowledgements for all other people involved in helping make a successful iGEM team.<br />
--><br />
=Members=<br />
<span class="anchor" id="members"></span><br />
<br />
A core concept of iGEM is the collaboration within an interdisciplinary student team. Accordingly, students of different age, gender and field of study came together to found our team in Aachen, in order to realize our project with joint forces and enthusiasm for the overall goal. Working closely together, we therefore not only have the chance to learn from each other, but also to excel ourselves. Using our collective creativity and common responsibility, we will create something special in order to make our contribution to synthetic biology.<br />
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<a href="https://2014.igem.org/User:Mosthege" style="color:black"><br />
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<b> Michael Osthege </b><br />
<br/><br/><br />
<i> Molecular and Applied Biotechnology </i><br />
<br/><br/><br />
Man for Everything<br />
<br/><br/><br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/3/35/Aachen_team_member_Michael_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
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<a href="https://2014.igem.org/User:Fgohr" style="color:black"><br />
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<b> Florian Gohr </b><br />
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<i> Molecular and Applied Biotechnology </i><br />
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King Gibson<br />
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<a href="https://2014.igem.org/User:AZimmermann" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> Arne Zimmermann </b><br />
<br/><br/><br />
<i> Molecular and Applied Biotechnology </i><br />
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The Patience itself<br />
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<!-- click for more information --><br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/8/88/Aachen_team_member_Arne_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:Nbailly" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> Nina Bailly </b><br />
<br/><br/><br />
<i> Molecular and Applied Biotechnology </i><br />
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Flask Prep Princess<br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/4/48/Aachen_team_member_Nina_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:VeraA" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> Vera Alexandrova </b><br />
<br/><br/><br />
<i> Biology </i><br />
<br/><br/><br />
Goddess of Plasmid Prep<br />
<br/><br/><br />
<!-- click for more information --><br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/8/81/Aachen_team_member_Vera_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
</li><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:Pdemling" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> Philipp Demling </b><br />
<br/><br/><br />
<i> Molecular and Applied Biotechnology </i><br />
<br/><br/><br />
Prince of bad Puns<br />
<br/><br/><br />
<!-- click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/7/77/Aachen_team_member_Philipp_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
</li><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:R.hanke" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> René Hanke </b><br />
<br/><br/><br />
<i> Molecular and Applied Biotechnology </i><br />
<br/><br/><br />
Creative Mind and Cook<br />
<br/><br/><br />
<!-- click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/b/b7/Aachen_team_member_Rene_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
</li><br />
<br />
<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:PatrickO" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> Patrick Opdenstein </b><br />
<br/><br/><br />
<i> Molecular and Applied Biotechnology </i><br />
<br/><br/><br />
Graphics God<br />
<br/><br/><br />
<!-- click for more information --><br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/1/1a/Aachen_team_member_Patrick_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
</li><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:Bpeeters" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> Björn Peeters </b><br />
<br/><br/><br />
<i> Computational Engineering Science </i><br />
<br/><br/><br />
Hardware Master<br />
<br/><br/><br />
<!-- click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/8/8d/Aachen_team_member_Bjoern_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:Eshani.sood" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> Eshani Sood </b><br />
<br/><br/><br />
<i> Biomedical Engineering </i><br />
<br/><br/><br />
Sunshine<br />
<br/><br/><br />
<!-- click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/5/52/Aachen_team_member_Eshani_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
</li><br />
<br />
<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:Mjoppich" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> Markus Joppich </b><br />
<br/><br/><br />
<i> Computer Science </i><br />
<br/><br/><br />
Software Expert and Travel Assistant<br />
<br/><br/><br />
<!-- click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/8/83/Aachen_team_member_Markus_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
</li><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:Ansgar" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> Ansgar Niemöller </b><br />
<br/><br/><br />
<i> Computational Engineering Science </i><br />
<br/><br/><br />
GUI Master<br />
<!-- click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/4/4e/Aachen_team_member_Ansgar_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
</li><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:J.plum" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> Julia Plum </b><br />
<br/><br/><br />
<i> Biology and Business Administration </i><br />
<br/><br/><br />
Fundraising and Balancing<br />
<br/><br/><br />
<!-- click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/c/c9/Aachen_team_member_Julia_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
</li><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:StefanReinhold" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> Stefan Reinhold </b><br />
<br/><br/><br />
<i> Molecular and Applied Biotechnology </i><br />
<br/><br/><br />
Treasurer<br />
<br/><br/><br />
<!-- click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/0/0c/Aachen_team_member_Stefan_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
</li><br />
<br />
<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:Aschechtel" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> Anna Schechtel </b><br />
<br/><br/><br />
<i> Biology </i><br />
<br/><br/><br />
Chip Queen<br />
<br/><br/><br />
<!-- click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/a/a4/Aachen_team_member_Anna_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
</li><br />
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= Advisors =<br />
<br />
<html><img class="imgshadow" style="float: left;" src="https://static.igem.org/mediawiki/2014/5/57/Aachen_team_member_Suresh_01.jpg" width="200px" /></html><br />
<br />
'''Dr.-Ing. Suresh Sudarsan'''<br />
<br />
I'm working as a post-doc with Prof. Blank at RWTH Aachen University. In my Ph.D. I worked under the guidance of Prof. Andreas Schmid (2008-2012) at TU Dortmund, and Prof. Matthias Reuss (2009-2011) at the University of Stuttgart, with a focus on elucidating the link between the central and aromatic metabolism of ''P. putida'' using a systems biology approach.<br />
<br />
I'm fascinated about understanding the collective behavior of microorganisms and their metabolic potential in different niches. In my research, I use tools in ''metabolic engineering & systems biology'' such as metabolomics, fast sampling, kinetic/dynamic modeling and metabolic flux analysis. <br />
<br />
In iGEM, I enjoy working with a group of young scientists from Aachen to achieve our common goal of detecting ''Pseudomonas aeruginosa'' on hard surfaces. Besides science, I try to get myself involved in adventurous sports...but honestly... I just like to relax my day with a delicious meal and a good sci-fi movie!<br />
<br />
<br />
<br />
<html><img class="imgshadow" style="float: left;" src="https://static.igem.org/mediawiki/2014/c/c8/Aachen_team_member_Ljubica.jpg" width="200px" /></html><br />
<br />
'''Dr. rer. nat. Ljubica Vojcic'''<br />
<br />
I am working as a Subgroup Leader in Prof. Schwaneberg research group at the Institute of Biotechnology, RWTH Aachen. The research area of the Schwaneberg group focuses on directed protein evolution by developing novel methods for generating diversity at the gene level, analyzing consequences of mutational biases on the protein level and developing novel high-throughput screening systems that will lead to improved biocatalysts for prominent applications in industry. In particular, my core expertise is development of high throughput screening systems for different enzyme classes in order to redefine the screening step no longer as bottleneck in directed evolution. <br />
<br />
In iGEM, I enjoyed very much to work with highly motivated and ambitious young scientists from RWTH Aachen University. I truly believe that our collaboration has just started and that we will enjoy jointly solving the scientific challenges in the near future. <br />
<br />
<br />
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<br />
=Instructors=<br />
<span class="anchor" id="instructors"></span><br />
== Prof. Dr.-Ing. Lars M. Blank ==<br />
==== RWTH Institute for Applied Microbiology (iAMB) ====<br />
<html><p><img class="imgshadow" style="float: left;" src="https://static.igem.org/mediawiki/2014/4/42/Aachen_LarsBlank.jpg" width="150px" /></html> <br />
Prof. Blank focuses his research on fundamental and applied aspects of microbial metabolism. Of specific interest is the interaction between the metabolic network and the introduced genetic and environmental perturbations. The research on in silico/in vivo metabolic network operations is aimed at a deeper understanding of cell function, with the ultimate goal of rational cell engineering.<br />
<br />
In his teaching, Prof. Blank focuses on the integration of biological concepts with the tools from bioinformatics and engineering. He believes that a sound knowledge base in life sciences is the key for creative and thus successful work in the areas of Metabolic Engineering and Synthetic Biology. Read more about Prof. Blank's work on the [http://www.iamb.rwth-aachen.de/html/members.php?s=det&id=3 iAMB's website].<br />
<html><br><br><br></p></html><br />
<br />
== Prof. Dr. Wolfgang Wiechert ==<br />
==== Forschungszentrum Jülich, Institute of Bio- and Geosciences (IGB-1) ====<br />
<html><p><img class="imgshadow" style="float: left;" src="https://static.igem.org/mediawiki/2014/8/88/Aachen_WolfgangWiechert.jpg" width="150px" /></html> <br />
Prof. Wiechert's main area of work lies within the field of applied systems bio(techno)logy of microorganisms with a special focus on methodological developments for quantitative biology. Characteristics of his research work are a close integration of experimental and theoretical work within multi disciplinary projects. As the head of the Systems Biotechnology research division at Forschungszentrum Jülich, he is developing methods for quantitative metabolomics, fluxomics and proteomics including model based mathematical methods for experimental design, parameter estimation, and process optimization in biotechnological systems. Future work will also incorporate micro fluidic methods for single cell analysis. In general, all research results are used to drive forward the process of gaining knowledge in the course of an iterative improvement of industrial production systems. This proceeds in close cooperation of all working groups at the IBG-1. Together with industrial partners also diverse examples from industry are investigated and further developed. Read more about Prof. Wiechert's work on the [http://www.fz-juelich.de/SharedDocs/Personen/IBG/IBG-1/EN/Research_groups/general/wiechert.html/ IBG-1 website].<br />
<html><br><br><br></p></html><br />
<br />
== Prof. Dr. Ulrich Schwaneberg ==<br />
==== RWTH Institute for Biotechnology, Leibniz Institute for Interactive Materials (DWI) ====<br />
<html><p><img class="imgshadow" style="float: left;" src="https://static.igem.org/mediawiki/2014/f/fe/Aachen_UlrichSchwaneberg.jpg" width="150px" /></html> <br />
The Schwaneberg Group seeks to be at the research frontier in the interdisciplinary field of directed protein evolution by developing novel methods for generating diversity at the gene level, analyzing consequence of mutational biases on the protein level and developing novel high-throughput screening systems that will ultimately lead to tailored-biocatalysts for significant applications in industry. They train students in the cutting edge technologies of laboratory evolution, biocatalyst engineering and high throughput screening methodologies. The Schwaneberg Group believes in integrating fundamental principles of protein design with environmental awareness in their research and seeks to promote international scientific collaborations. Read more about Prof. Schwaneberg and his work on the [http://www.biotec.rwth-aachen.de/ Schwaneberg Group's website].<br />
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<br />
<br />
This project would not have been possible without the generous help and advice from many great people and organizations. We thank all the partners listed below for their essential contributions and financial support that covered registration fees, travel costs, the organization of the meetup in September, laboratory materials and expenses for services and materials.<br />
<br />
=Partners=<br />
<span class="anchor" id="partners"></span><br />
<div align="center"><br />
{|cellpadding="25"<br />
|[[File:Logo_Buergerstiftung-Aachen_hoch.jpg|159px|center|link=http://www.buergerstiftung-aachen.de|Bürgerstiftung Aachen]]<br />
|[[File:Aachen_Niersverband_Firmenlogo.jpg|318px|link=http://www.niersverband.de/|Niersverband]]<br />
|}<br />
{|cellpadding="13"<br />
|[[File:Aachen_Genscript_Logo.png|240px|link=http://www.genscript.com/|Genscript]]<br />
|[[File:Aachen_Eurofinsgenomics.png|193px|link=http://www.eurofinsgenomics.eu/|Eurofins Genomics]]<br />
|[[File:Aachen_Labomedic_Logo.jpg|256px|link=http://www.labomedic.de/|Labomedic]]<br />
|}<br />
<br />
{|cellpadding="35"<br />
|[[File:Aachen_bmbf.jpg|326px|link=http://www.bmbf.de/de/24140.php|BMBF]]<br />
|[[File:Aachen_idt_.png|217px|link=http://www.idt-biologika.de/|IDT]]<br />
|}<br />
<br />
{|cellpadding="25"<br />
|[[File:M2p_labs_logo.jpg|103px|link=http://www.m2p-labs.com/|m2p labs]]<br />
|[[File:Aachen_Roth_Logo.png|109px|link=http://www.carlroth.com/pages/index/COM/index.jsp?market=COM&lang=en-com|Carl Roth]]<br />
|[[File:Aachen_ProRWTH_logo.png|121px|link=http://www.prorwth.de/|pro RWTH]]<br />
|}<br />
{|cellpadding="25"<br />
|[[File:Aachen_Logo_iAMB.png|229px|link=http://www.iamb.rwth-aachen.de|Institute of Applied Microbiology - iAMB]]<br />
|[[File:Aachen_Logo_bio7.png|229px|link=http://www.molbiotech.rwth-aachen.de|Institute for Molecular Biotechnology]]<br />
|[[File:Aachen_Logo_HISynBio.png|246px|link=http://www.helmholtz.de/en/about_us/initiating_and_networking/assuring_excellence/synthetic_biology|Helmholtz Association - Initiative on Synthetic Biology]]<br />
|}<br />
{|cellpadding="35"<br />
|[[File:Aachen_Logo_ABBt.png|209px|link=http://www.biologie.rwth-aachen.de|Aachen Biology and Biotechnology - ABBt]]<br />
|[[File:Aachen_juelich.png|239px|link=http://www.fz-juelich.de/|Forschungszentrum Jülich]]<br />
|[[File:Aachen_FabLabAachenLogo.jpg|209px|link=http://hci.rwth-aachen.de/fablab|Fab Lab Aachen]]<br />
|[[File:Aachen_Schwaneberg_Group.png|150px|link=http://www.biotec.rwth-aachen.de|Schwaneberg Group]]<br />
|}<br />
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<br />
=Attribution of Scientific and General Support=<br />
<span class="anchor" id="support"></span><br />
Great projects do not only depend on solid funding, but even more on the invaluable support by great people. We found great people not only in our 'home institute' the iAMB, but across many partner institutions.<br />
<br />
<br />
* '''RWTH Institute of Molecular Biotechnology''' (Biology VII)<br />
** Dr. Ulrich Commandeur ''for giving us access to essential resources of the bio7''<br />
** Christina Dickmeis M.Sc. ''who answered lots of questions''<br />
* '''RWTH Institute of Biotechnology''' (Biology VI)<br />
** David Schönauer and Alan Mertens M.Sc. ''who helped us to purify proteins''<br />
* '''Helmholtz Institute for Biomedical Engineering'''<br />
** Prof. Dr. Lothar Elling and Sophia Böcker, M.Sc. ''for giving us access to their gal-3 expression plasmid''<br />
* '''RWTH Institute of Applied Microbiology''' (iAMB/Biology IV)<br />
** Prof. Dr. Miriam Agler-Rosenbaum ''for scientific information on quorum sensing''<br />
** Dr. Martin Zimmermann ''who counseled us on balancing iGEM with our studies''<br />
** Dr. Andrea Germer ''for teaching us Gibson cloning''<br />
** Erick Bosire Maosa M.Sc. ''who advised us on cultivation of Pseudomonas''<br />
** Gisela Beissel, Annette Schreer and Kalle Hüser ''for their great patience with our entropy generation''<br />
* '''Forschungszentrum Jülich'''<br />
** Marianne Heß ''who was and is essential for travel organization''<br />
** Dr. Hanno Scharr ''for advice on image analysis''<br />
* '''Fab Lab Aachen'''<br />
** René Bohne, Jan Zimmermann and Jan Thar ''who generously gave us access to the lasercutter and 3D printer''<br />
* '''Others'''<br />
** Dr. Helen Rosenkranz, ABBt, ''for helping us with hundreds of administrative paperwork''<br />
** Dr. Heinz-Albert Becker, NEAnderLab, ''for his help with the organization of the school project''<br />
** Ulrike Eisel, Gymnasium am Neandertal, ''for her help with the organization of the school project''<br />
** Fachschaft Biowissenschaften ''for food supplies and access to their rooms''<br />
** Dr. Joachim Fröhlingsdorf, MakeLight, ''who inspired us to expand our OD/F project''<br />
** Univ.-Prof. Dr. Wolfgang Dott, Uniklinik RWTH Aachen, ''for helpful feedback regarding fields of application for our biosensor''<br />
<br />
Finally, we want to thank '''Simon&nbsp;Unthan''', '''Michael&nbsp;Limberg''', '''Henrik&nbsp;Cordes''', '''Sven&nbsp;Jager''' and '''Team&nbsp;Bielefeld''' for their advice guiding us through our first iGEM participation! <br />
<br />
Special thanks to '''Dr.-Ing. Suresh Sudarsan''' for a final check for typos on our wiki!<br />
<br />
<br />
{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/AttributionsTeam:Aachen/Attributions2014-10-18T03:47:23Z<p>Nbailly: </p>
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# General Support<br />
# Fundraising help and advice<br />
# Partners<br />
# Project support and advice<br />
# Lab support<br />
# Difficult technique support<br />
# Project advisor support<br />
# Wiki support<br />
# Presentation coaching<br />
# Policy & Practices support<br />
# Thanks and acknowledgements for all other people involved in helping make a successful iGEM team.<br />
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=Members=<br />
<span class="anchor" id="members"></span><br />
<br />
A core concept of iGEM is the collaboration within an interdisciplinary student team. Accordingly, students of different age, gender and field of study came together to found our team in Aachen, in order to realize our project with joint forces and enthusiasm for the overall goal. Working closely together, we therefore not only have the chance to learn from each other, but also to excel ourselves. Using our collective creativity and common responsibility, we will create something special in order to make our contribution to synthetic biology.<br />
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<a href="https://2014.igem.org/User:Mosthege" style="color:black"><br />
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<b> Michael Osthege </b><br />
<br/><br/><br />
<i> Molecular and Applied Biotechnology </i><br />
<br/><br/><br />
Man for Everything<br />
<br/><br/><br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/3/35/Aachen_team_member_Michael_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
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<a href="https://2014.igem.org/User:Fgohr" style="color:black"><br />
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<b> Florian Gohr </b><br />
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<i> Molecular and Applied Biotechnology </i><br />
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King Gibson<br />
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<a href="https://2014.igem.org/User:AZimmermann" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> Arne Zimmermann </b><br />
<br/><br/><br />
<i> Molecular and Applied Biotechnology </i><br />
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The Patience itself<br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/8/88/Aachen_team_member_Arne_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:Nbailly" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> Nina Bailly </b><br />
<br/><br/><br />
<i> Molecular and Applied Biotechnology </i><br />
<br/><br/><br />
Shake Flask Prep Princess<br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/4/48/Aachen_team_member_Nina_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:VeraA" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> Vera Alexandrova </b><br />
<br/><br/><br />
<i> Biology </i><br />
<br/><br/><br />
Goddess of Plasmid Prep<br />
<br/><br/><br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/8/81/Aachen_team_member_Vera_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
</li><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:Pdemling" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> Philipp Demling </b><br />
<br/><br/><br />
<i> Molecular and Applied Biotechnology </i><br />
<br/><br/><br />
Prince of bad Puns<br />
<br/><br/><br />
<!-- click for more information --><br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/7/77/Aachen_team_member_Philipp_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
</li><br />
<br />
<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:R.hanke" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> René Hanke </b><br />
<br/><br/><br />
<i> Molecular and Applied Biotechnology </i><br />
<br/><br/><br />
Creative Mind and Cook<br />
<br/><br/><br />
<!-- click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/b/b7/Aachen_team_member_Rene_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
</li><br />
<br />
<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:PatrickO" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> Patrick Opdenstein </b><br />
<br/><br/><br />
<i> Molecular and Applied Biotechnology </i><br />
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Graphics God<br />
<br/><br/><br />
<!-- click for more information --><br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/1/1a/Aachen_team_member_Patrick_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
</li><br />
<br />
<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:Bpeeters" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> Björn Peeters </b><br />
<br/><br/><br />
<i> Computational Engineering Science </i><br />
<br/><br/><br />
Hardware Master<br />
<br/><br/><br />
<!-- click for more information --><br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/8/8d/Aachen_team_member_Bjoern_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:Eshani.sood" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> Eshani Sood </b><br />
<br/><br/><br />
<i> Biomedical Engineering </i><br />
<br/><br/><br />
Sunshine<br />
<br/><br/><br />
<!-- click for more information --><br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/5/52/Aachen_team_member_Eshani_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
</li><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:Mjoppich" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> Markus Joppich </b><br />
<br/><br/><br />
<i> Computer Science </i><br />
<br/><br/><br />
Software Expert and Travel Assistant<br />
<br/><br/><br />
<!-- click for more information --><br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/8/83/Aachen_team_member_Markus_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
</li><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:Ansgar" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> Ansgar Niemöller </b><br />
<br/><br/><br />
<i> Computational Engineering Science </i><br />
<br/><br/><br />
GUI Master<br />
<!-- click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/4/4e/Aachen_team_member_Ansgar_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
</li><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:J.plum" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> Julia Plum </b><br />
<br/><br/><br />
<i> Biology and Business Administration </i><br />
<br/><br/><br />
Fundraising and Balancing<br />
<br/><br/><br />
<!-- click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/c/c9/Aachen_team_member_Julia_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
</li><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:StefanReinhold" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
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<b> Stefan Reinhold </b><br />
<br/><br/><br />
<i> Molecular and Applied Biotechnology </i><br />
<br/><br/><br />
Treasurer<br />
<br/><br/><br />
<!-- click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/0/0c/Aachen_team_member_Stefan_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
</li><br />
<br />
<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a href="https://2014.igem.org/User:Aschechtel" style="color:black"><br />
<div class="team-item team-info" style="height: 180px; width: 180px;"><br />
<br/><br/><br />
<b> Anna Schechtel </b><br />
<br/><br/><br />
<i> Biology </i><br />
<br/><br/><br />
Chip Queen<br />
<br/><br/><br />
<!-- click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/a/a4/Aachen_team_member_Anna_01.jpg); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"> </div></a><br />
</li><br />
</ul><br />
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<br />
= Advisors =<br />
<br />
<html><img class="imgshadow" style="float: left;" src="https://static.igem.org/mediawiki/2014/5/57/Aachen_team_member_Suresh_01.jpg" width="200px" /></html><br />
<br />
'''Dr.-Ing. Suresh Sudarsan'''<br />
<br />
I'm working as a post-doc with Prof. Blank at RWTH Aachen University. In my Ph.D. I worked under the guidance of Prof. Andreas Schmid (2008-2012) at TU Dortmund, and Prof. Matthias Reuss (2009-2011) at the University of Stuttgart, with a focus on elucidating the link between the central and aromatic metabolism of ''P. putida'' using a systems biology approach.<br />
<br />
I'm fascinated about understanding the collective behavior of microorganisms and their metabolic potential in different niches. In my research, I use tools in ''metabolic engineering & systems biology'' such as metabolomics, fast sampling, kinetic/dynamic modeling and metabolic flux analysis. <br />
<br />
In iGEM, I enjoy working with a group of young scientists from Aachen to achieve our common goal of detecting ''Pseudomonas aeruginosa'' on hard surfaces. Besides science, I try to get myself involved in adventurous sports...but honestly... I just like to relax my day with a delicious meal and a good sci-fi movie!<br />
<br />
<br />
<br />
<html><img class="imgshadow" style="float: left;" src="https://static.igem.org/mediawiki/2014/c/c8/Aachen_team_member_Ljubica.jpg" width="200px" /></html><br />
<br />
'''Dr. rer. nat. Ljubica Vojcic'''<br />
<br />
I am working as a Subgroup Leader in Prof. Schwaneberg research group at the Institute of Biotechnology, RWTH Aachen. The research area of the Schwaneberg group focuses on directed protein evolution by developing novel methods for generating diversity at the gene level, analyzing consequences of mutational biases on the protein level and developing novel high-throughput screening systems that will lead to improved biocatalysts for prominent applications in industry. In particular, my core expertise is development of high throughput screening systems for different enzyme classes in order to redefine the screening step no longer as bottleneck in directed evolution. <br />
<br />
In iGEM, I enjoyed very much to work with highly motivated and ambitious young scientists from RWTH Aachen University. I truly believe that our collaboration has just started and that we will enjoy jointly solving the scientific challenges in the near future. <br />
<br />
<br />
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<br />
=Instructors=<br />
<span class="anchor" id="instructors"></span><br />
== Prof. Dr.-Ing. Lars M. Blank ==<br />
==== RWTH Institute for Applied Microbiology (iAMB) ====<br />
<html><p><img class="imgshadow" style="float: left;" src="https://static.igem.org/mediawiki/2014/4/42/Aachen_LarsBlank.jpg" width="150px" /></html> <br />
Prof. Blank focuses his research on fundamental and applied aspects of microbial metabolism. Of specific interest is the interaction between the metabolic network and the introduced genetic and environmental perturbations. The research on in silico/in vivo metabolic network operations is aimed at a deeper understanding of cell function, with the ultimate goal of rational cell engineering.<br />
<br />
In his teaching, Prof. Blank focuses on the integration of biological concepts with the tools from bioinformatics and engineering. He believes that a sound knowledge base in life sciences is the key for creative and thus successful work in the areas of Metabolic Engineering and Synthetic Biology. Read more about Prof. Blank's work on the [http://www.iamb.rwth-aachen.de/html/members.php?s=det&id=3 iAMB's website].<br />
<html><br><br><br></p></html><br />
<br />
== Prof. Dr. Wolfgang Wiechert ==<br />
==== Forschungszentrum Jülich, Institute of Bio- and Geosciences (IGB-1) ====<br />
<html><p><img class="imgshadow" style="float: left;" src="https://static.igem.org/mediawiki/2014/8/88/Aachen_WolfgangWiechert.jpg" width="150px" /></html> <br />
Prof. Wiechert's main area of work lies within the field of applied systems bio(techno)logy of microorganisms with a special focus on methodological developments for quantitative biology. Characteristics of his research work are a close integration of experimental and theoretical work within multi disciplinary projects. As the head of the Systems Biotechnology research division at Forschungszentrum Jülich, he is developing methods for quantitative metabolomics, fluxomics and proteomics including model based mathematical methods for experimental design, parameter estimation, and process optimization in biotechnological systems. Future work will also incorporate micro fluidic methods for single cell analysis. In general, all research results are used to drive forward the process of gaining knowledge in the course of an iterative improvement of industrial production systems. This proceeds in close cooperation of all working groups at the IBG-1. Together with industrial partners also diverse examples from industry are investigated and further developed. Read more about Prof. Wiechert's work on the [http://www.fz-juelich.de/SharedDocs/Personen/IBG/IBG-1/EN/Research_groups/general/wiechert.html/ IBG-1 website].<br />
<html><br><br><br></p></html><br />
<br />
== Prof. Dr. Ulrich Schwaneberg ==<br />
==== RWTH Institute for Biotechnology, Leibniz Institute for Interactive Materials (DWI) ====<br />
<html><p><img class="imgshadow" style="float: left;" src="https://static.igem.org/mediawiki/2014/f/fe/Aachen_UlrichSchwaneberg.jpg" width="150px" /></html> <br />
The Schwaneberg Group seeks to be at the research frontier in the interdisciplinary field of directed protein evolution by developing novel methods for generating diversity at the gene level, analyzing consequence of mutational biases on the protein level and developing novel high-throughput screening systems that will ultimately lead to tailored-biocatalysts for significant applications in industry. They train students in the cutting edge technologies of laboratory evolution, biocatalyst engineering and high throughput screening methodologies. The Schwaneberg Group believes in integrating fundamental principles of protein design with environmental awareness in their research and seeks to promote international scientific collaborations. Read more about Prof. Schwaneberg and his work on the [http://www.biotec.rwth-aachen.de/ Schwaneberg Group's website].<br />
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<br />
This project would not have been possible without the generous help and advice from many great people and organizations. We thank all the partners listed below for their essential contributions and financial support that covered registration fees, travel costs, the organization of the meetup in September, laboratory materials and expenses for services and materials.<br />
<br />
=Partners=<br />
<span class="anchor" id="partners"></span><br />
<div align="center"><br />
{|cellpadding="25"<br />
|[[File:Logo_Buergerstiftung-Aachen_hoch.jpg|159px|center|link=http://www.buergerstiftung-aachen.de|Bürgerstiftung Aachen]]<br />
|[[File:Aachen_Niersverband_Firmenlogo.jpg|318px|link=http://www.niersverband.de/|Niersverband]]<br />
|}<br />
{|cellpadding="13"<br />
|[[File:Aachen_Genscript_Logo.png|240px|link=http://www.genscript.com/|Genscript]]<br />
|[[File:Aachen_Eurofinsgenomics.png|193px|link=http://www.eurofinsgenomics.eu/|Eurofins Genomics]]<br />
|[[File:Aachen_Labomedic_Logo.jpg|256px|link=http://www.labomedic.de/|Labomedic]]<br />
|}<br />
<br />
{|cellpadding="35"<br />
|[[File:Aachen_bmbf.jpg|326px|link=http://www.bmbf.de/de/24140.php|BMBF]]<br />
|[[File:Aachen_idt_.png|217px|link=http://www.idt-biologika.de/|IDT]]<br />
|}<br />
<br />
{|cellpadding="25"<br />
|[[File:M2p_labs_logo.jpg|103px|link=http://www.m2p-labs.com/|m2p labs]]<br />
|[[File:Aachen_Roth_Logo.png|109px|link=http://www.carlroth.com/pages/index/COM/index.jsp?market=COM&lang=en-com|Carl Roth]]<br />
|[[File:Aachen_ProRWTH_logo.png|121px|link=http://www.prorwth.de/|pro RWTH]]<br />
|}<br />
{|cellpadding="25"<br />
|[[File:Aachen_Logo_iAMB.png|229px|link=http://www.iamb.rwth-aachen.de|Institute of Applied Microbiology - iAMB]]<br />
|[[File:Aachen_Logo_bio7.png|229px|link=http://www.molbiotech.rwth-aachen.de|Institute for Molecular Biotechnology]]<br />
|[[File:Aachen_Logo_HISynBio.png|246px|link=http://www.helmholtz.de/en/about_us/initiating_and_networking/assuring_excellence/synthetic_biology|Helmholtz Association - Initiative on Synthetic Biology]]<br />
|}<br />
{|cellpadding="35"<br />
|[[File:Aachen_Logo_ABBt.png|209px|link=http://www.biologie.rwth-aachen.de|Aachen Biology and Biotechnology - ABBt]]<br />
|[[File:Aachen_juelich.png|239px|link=http://www.fz-juelich.de/|Forschungszentrum Jülich]]<br />
|[[File:Aachen_FabLabAachenLogo.jpg|209px|link=http://hci.rwth-aachen.de/fablab|Fab Lab Aachen]]<br />
|[[File:Aachen_Schwaneberg_Group.png|150px|link=http://www.biotec.rwth-aachen.de|Schwaneberg Group]]<br />
|}<br />
<div><br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
=Attribution of Scientific and General Support=<br />
<span class="anchor" id="support"></span><br />
Great projects do not only depend on solid funding, but even more on the invaluable support by great people. We found great people not only in our 'home institute' the iAMB, but across many partner institutions.<br />
<br />
<br />
* '''RWTH Institute of Molecular Biotechnology''' (Biology VII)<br />
** Dr. Ulrich Commandeur ''for giving us access to essential resources of the bio7''<br />
** Christina Dickmeis M.Sc. ''who answered lots of questions''<br />
* '''RWTH Institute of Biotechnology''' (Biology VI)<br />
** David Schönauer and Alan Mertens M.Sc. ''who helped us to purify proteins''<br />
* '''Helmholtz Institute for Biomedical Engineering'''<br />
** Prof. Dr. Lothar Elling and Sophia Böcker, M.Sc. ''for giving us access to their gal-3 expression plasmid''<br />
* '''RWTH Institute of Applied Microbiology''' (iAMB/Biology IV)<br />
** Prof. Dr. Miriam Agler-Rosenbaum ''for scientific information on quorum sensing''<br />
** Dr. Martin Zimmermann ''who counseled us on balancing iGEM with our studies''<br />
** Dr. Andrea Germer ''for teaching us Gibson cloning''<br />
** Erick Bosire Maosa M.Sc. ''who advised us on cultivation of Pseudomonas''<br />
** Gisela Beissel, Annette Schreer and Kalle Hüser ''for their great patience with our entropy generation''<br />
* '''Forschungszentrum Jülich'''<br />
** Marianne Heß ''who was and is essential for travel organization''<br />
** Dr. Hanno Scharr ''for advice on image analysis''<br />
* '''Fab Lab Aachen'''<br />
** René Bohne, Jan Zimmermann and Jan Thar ''who generously gave us access to the lasercutter and 3D printer''<br />
* '''Others'''<br />
** Dr. Helen Rosenkranz, ABBt, ''for helping us with hundreds of administrative paperwork''<br />
** Dr. Heinz-Albert Becker, NEAnderLab, ''for his help with the organization of the school project''<br />
** Ulrike Eisel, Gymnasium am Neandertal, ''for her help with the organization of the school project''<br />
** Fachschaft Biowissenschaften ''for food supplies and access to their rooms''<br />
** Dr. Joachim Fröhlingsdorf, MakeLight, ''who inspired us to expand our OD/F project''<br />
** Univ.-Prof. Dr. Wolfgang Dott, Uniklinik RWTH Aachen, ''for helpful feedback regarding fields of application for our biosensor''<br />
<br />
Finally, we want to thank '''Simon&nbsp;Unthan''', '''Michael&nbsp;Limberg''', '''Henrik&nbsp;Cordes''', '''Sven&nbsp;Jager''' and '''Team&nbsp;Bielefeld''' for their advice guiding us through our first iGEM participation! <br />
<br />
Special thanks to '''Dr.-Ing. Suresh Sudarsan''' for a final check for typos on our wiki!<br />
<br />
<br />
{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/Notebook/Software/TimeseriesLabellerTeam:Aachen/Notebook/Software/TimeseriesLabeller2014-10-18T03:46:36Z<p>Nbailly: /* Timeseries Labeller */</p>
<hr />
<div>__NOTOC__<br />
<br />
{{Team:Aachen/Header}}<br />
<br />
=Timeseries Labeller=<br />
A common task in the processing of visual, two-dimensional data, such as pictures of our [[Team:Aachen/Project/2D_Biosensor|2D Biosensor]] is the assembly of time series.<br />
<br />
To efficiently process the pictures taken by our [[Team:Aachen/Project/Measurement_Device|''WatsOn'']] device, we programmed this software to automatically recognize timestamps from filenames. <br />
<br />
{{Team:Aachen/FigureFloat|Aachen_TimeseriesLabeller1.png|title=Using the Timeseries Labeller|subtitle=After files have been loaded into the software, the user can define a label that will appear on all pictures. The automatically recognized timestamps can be manually edited and the unit. The desired size of each frame can be adjusted before the images are processed.|width=697px}}<br />
<br />
After the files have been processed, they can easily be used to assemble a movie, or animated GIF with readily available tools.<br />
<br />
Here, we provide both the executable and the sources as a single ZIP-file:<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2014/e/ed/Aachen_TimeseriesLabeller.zip" style="font-size:160%; font-weight:bold;">Get it!</a></html><br />
<br />
<br />
''If you have questions on the Timeseries&nbsp;Labeller, please contact [mailto:thecakedev@hotmail.com Michael].''<br />
<br />
<br />
<br />
{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/Notebook/Software/MeasurartyTeam:Aachen/Notebook/Software/Measurarty2014-10-18T03:46:03Z<p>Nbailly: </p>
<hr />
<div>__NOTOC__<br />
{{CSS/Main}}<br />
{{Team:Aachen/Stylesheet}}<br />
{{Team:Aachen/Header}}<br />
<br />
<html><br />
<link rel="stylesheet" href="https://2014.igem.org/Team:Aachen/Scripts/highlight.pack.default.css?action=raw&ctype=text/css"><br />
<script src="https://2014.igem.org/Team:Aachen/Scripts/highlight.pack.js?action=raw&ctype=text/javascript"></script><br />
<script>hljs.initHighlightingOnLoad();</script><br />
</html><br />
<br />
= ''Measurarty'' =<br />
<br />
''Measurarty'' is the evil player in the game of ''Cellock Holmes'' and ''WatsOn''.<br />
''Measurarty'' is the pathogen detection logic behind our project.<br />
Using our ''Measurarty'' algorithm, we want to automatically detect pathogens from the chip photos delivered by WatsOn, without human interaction.<br />
Besides reducing the risk of human errors, this makes our device usable by almost everyone.<br />
<br />
<html><br />
<center><br />
<ul class="menusmall-grid"><br />
<!-- Overview --><br />
<br />
<li style="width: 156px;margin-left: 8px;margin-right: 8px;"><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Software/Measurarty#intro" style="color:black"><br />
<div class="menusmall-item menusmall-info" ><div class="menukachel">Intro</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/1/1b/Aachen_Measurarty_Intro_button.png); norepeat scroll 0% 0% transparent; background-size:100%"><br />
</div><br />
</a><br />
</li><br />
<br />
<li style="width: 156px;margin-left: 8px;margin-right: 8px;"><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Software/Measurarty#SRM" style="color:black"><br />
<div class="menusmall-item menusmall-info" ><div class="menukachel">SRM!</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/2/2d/Aachen_Puzzels_button.png); norepeat scroll 0% 0% transparent; background-size:100%"><br />
</div><br />
</a><br />
</li><br />
<br />
<li style="width: 156px;margin-left: 8px;margin-right: 8px;"><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Software/Measurarty#segment" style="color:black"><br />
<div class="menusmall-item menusmall-info" ><div class="menukachel">Segment!</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/e/e1/Aachen_SEgment_button.png); norepeat scroll 0% 0% transparent; background-size:100%"><br />
</div><br />
</a><br />
</li><br />
<br />
<li style="width: 156px;margin-left: 8px;margin-right: 8px;"><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Software/Measurarty#classification" style="color:black"><br />
<div class="menusmall-item menusmall-info" ><div class="menukachel">Classify!</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/f/f9/Aachen_Classify_button.png); norepeat scroll 0% 0% transparent; background-size:100%"><br />
</div><br />
</a><br />
</li><br />
<br />
<li style="width: 156px;margin-left: 8px;margin-right: 8px;"><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Software/Measurarty#measurartyachievements" style="color:black"><br />
<div class="menusmall-item menusmall-info" ><div class="menukachel">Achieve-<br/>ments</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/e/ef/Aachen_14-10-15_Medal_Cellocks_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%"><br />
</div><br />
</a><br />
</li><br />
<br />
</ul><br />
</center><br />
</html><br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_Measurarty_Intro_button.png|right|150px]]<br />
<br />
== ''Measurarty'' - An Introduction ==<br />
<span class="anchor" id="intro"></span><br />
<br />
Our [https://2014.igem.org/Team:Aachen/Notebook/Engineering/WatsOn#watsonsoftware device control software] is able to take images of incubated chips inside WatsOn. Yet, that does not bring the user closer to the answer of the question:<br />
<br />
<center>'''What's on the chip?'''</center><br />
<br />
In fact, answering this question seems trivial for a human: Just check whether a colony grown has grown on the chip and you're done. This task is even easier with our chip system, because these show fluorescence wherever a pathogen has been detected.<br />
<br />
But is this an easy task for a computer? Actually not. The task of automatic detection is tried by several disciplines in computer science, from pattern recognition over machine learning to by medical imaging chairs.<br />
<br />
Here, we would like to present a pipeline for this task that makes use of '''easy segmentation and classification algorithms'''.<br />
First, ''Measurarty'' segments the target image using '''Statistical Region Merging (SRM)''' in order to find regions of similar properties. After this step, we can segment the picture using '''histogram thresholding''' in [http://en.wikipedia.org/wiki/HSL_and_HSV HSV color space] to find candidate regions for pathogens.<br />
Finally, a classification algorithm can detect the pathogen on our chips.<br />
<br />
To demonstrate the algorithm, the following sample image will be discussed.<br />
<br />
{{Team:Aachen/Figure|align=center|Aachen_meas_test.jpg|title=Image taken from WatsOn to be analyzed by ''Measurarty'' algorithm|width=700px}}<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_Puzzels_button.png|right|150px]]<br />
<br />
== Statistical Region Merging (SRM) ==<br />
<span class="anchor" id="SRM"></span><br />
<br />
Before briefly introducing Statistical Region Merging (SRM), we would like to explain why we need this step, and why this algorithm is an ideal choice.<br />
<br />
Compared to other clustering algorithms, SRM is quite leight weight, yet delivers ''deterministic'' results and is not dependent on a certain seed (like ''k''-means, for example).<br />
<br />
On the other hand, it can create as many refinements as one wants and is thus flexible enough for the our purposes. Finally, there's already been knowledge about this algorithm in the group.<br />
<br />
Statistical Region Merging (SRM) (Nook and Nielson, 2004) is a clustering technique also used directly for image segmentation.<br />
A region $R$ is a set of pixels and the cardinality $\lvert R \rvert$ determines how many pixels are in one region.<br />
Starting with a sorted set of connected regions (w. r. t. some distance function $f$), two regions $R$ and $R'$ are merged if the qualification criteria $\vert \overline{R'}-\overline{R} \vert \leq \sqrt{b^2(R)+b^2(R')}$ with $b(R) = g \cdot \sqrt{\frac{\ln \frac{\mathcal{R}_{\lvert R \rvert}}{\delta}}{2Q\lvert R \rvert}}$ is fulfilled.<br />
Therefore, $\mathcal{R}_{\lvert R \rvert}$ is the set of regions with $\lvert R \rvert$ pixels.<br />
Typically $Q$ is chosen as $Q \in \lbrack 256, 1\rbrack$ and $\delta = \frac{1}{\lvert I \rvert^2}$.<br />
<br />
The $Q$ parameter mainly influences the merging process. For an example, see the figure ''SRM Regions'' below. The lower the chosen value for $Q$, more coarse the regions become. Using a union-find structure, the segmentation does not need to be recalculated for each $Q$ level. For the step from $q$ to $\frac{q}{2}$, just the qualification criteria needs to be applied to the regions from the $q$ result. A MATLAB implementation is also available (Boltz, 2009).<br />
<br />
{{Team:Aachen/FigureDual|Aachen_srm_regions_3.PNG|Aachen_srm_regions_2.PNG|title1=SRM regions in random colors|title2=SRM regions (average color)|subtitle1=Different regions from an SRM run starting at $Q=256$ (top left) and going to $Q=1$ (bottom right). Each region is assigned a random color.|subtitle2=Different regions from an SRM run starting at $Q=256$ (top left) and going to $Q=1$ (bottom right). Each region is assigned the average color of that region.|width=425px}} <br />
<br />
=== SRM Clustering ===<br />
<br />
In our project, we used Statistical Region Merging for clustering. In contrast to other algorithms, such as ''k-means'', this approach is highly deterministic.<br />
For our purposes we only have one SRM run for $Q=256$.<br />
<br />
In MATLAB, we use the previously mentioned code from MATLAB Fileexchange (Boltz, 2009).<br />
For our Qt-based GUI we implemented the SRM method ourselves.<br />
<br />
The SRM clustering reduces the amount of different colors in the image and hence eases the recognition of parts belonging together.<br />
<br />
<html><br />
<div class="codediv"><br />
<pre><code class="matlab"><br />
Qlevel = 256;<br />
[maps,images]=singlesrm(double(image),Qlevel);<br />
</code></pre><br />
</div><br />
</html><br />
<br />
Finally, if applied to our test-image, regions are created and homogenoues regions form:<br />
<br />
{{Team:Aachen/Figure|align=center|Aachen_meas_srmed.png|title=Regions created with SRM clustering|width=700px}}<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_SEgment_button.png|right|150px]]<br />
<br />
== Segmentation ==<br />
<span class="anchor" id="segment"></span><br />
<br />
In the segmentation stage all background regions are removed. This task is quite crucial. If one removes too few, the final stage of finding pathogens might get irritated.<br />
On the other hand, if one removes too many regions, positive hits might get removed early before detection. This surely must be avoided.<br />
<br />
We opted for a simple thresholding step because it showed that while being easy, it is an effective weapon against the uniform background. In fact, the good image quality we wanted to reach with our device allows now less sophisticated methods.<br />
Also the less computational intensive the steps are, the better they might even run directly on the Raspberry Pi in our device!<br />
<br />
The HSV thresholding is performed on each component seperately. For more information on the HSV color space we refer to [http://en.wikipedia.org/wiki/HSL_and_HSV Wikipedia]. The first component is the hue which we select to be inbetween $0.462$ and $0.520$ to select any blue-greenish color. We will not see bright green due to the filter selection in our device.<br />
The saturation value must be high, between $0.99$ and $1.0$.<br />
Moreover, the value component of the HSV image has to lie between $0.25$ and $0.32$, which assumes a relatively dark color.<br />
<br />
Indeed, these values are not problem specific, but specific for each setup and therefore have to be determined empirically.<br />
<br />
The remainder of this stage creates a mask of pixels that fulfill the conditions.<br />
<br />
<html><br />
<div class="codediv"><br />
<pre><code class="matlab"><br />
% Auto-generated by colorThresholder app on 15-Oct-2014<br />
%-------------------------------------------------------<br />
function [maskedRGBImage] = createMask(srmimg)<br />
RGB = srmimg;<br />
<br />
% Convert RGB image to chosen color space<br />
I = rgb2hsv(RGB);<br />
<br />
% Define thresholds for channel 1 based on histogram settings<br />
channel1Min = 0.462;<br />
channel1Max = 0.520;<br />
<br />
% Define thresholds for channel 2 based on histogram settings<br />
channel2Min = 0.99;<br />
channel2Max = 1.000;<br />
<br />
% Define thresholds for channel 3 based on histogram settings<br />
channel3Min = 0.25;<br />
channel3Max = 0.32;<br />
<br />
% Create mask based on chosen histogram thresholds<br />
BW = (I(:,:,1) >= channel1Min ) & (I(:,:,1) <= channel1Max) & ...<br />
(I(:,:,2) >= channel2Min ) & (I(:,:,2) <= channel2Max) & ...<br />
(I(:,:,3) >= channel3Min ) & (I(:,:,3) <= channel3Max);<br />
<br />
% Initialize output masked image based on input image.<br />
maskedRGBImage = RGB;<br />
<br />
% Set background pixels where BW is false to zero.<br />
maskedRGBImage(repmat(~BW,[1 1 3])) = 0;<br />
<br />
end<br />
</code></pre><br />
</div><br />
</html><br />
<br />
If you apply this HSV masking code to the SRMed test image, the following is created:<br />
<br />
{{Team:Aachen/Figure|align=center|Aachen_meas_masked.png|title=Masked image|width=700px}}<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_Classify_button.png|right|150px]]<br />
<br />
== Classification ==<br />
<span class="anchor" id="classification"></span><br />
<br />
=== Smoothness Index ===<br />
<br />
For position prediction in virtual environments, jitter or noise in the output signal is not wanted though often present.<br />
Since discovering smooth areas is a similar problem to jitter detection, a simple method for determining jitter can be used to measure non-jitter, smoothness (Joppich, Rausch and Kuhlen, 2013).<br />
It is assumed that jitter-free areas of a position signal do not differ in velocity.<br />
<br />
Smooth areas do not differ in intensity, and therefore only low changes in velocity (intensity change) can be recorded.<br />
For the reduction of noise, this operation is performed on the smoothed input image.<br />
Then the smoothness $s$ of a pixel $p$ in its k-neighbourhood $\mathcal{N}_k$ can be determined as:<br />
\begin{equation}<br />
s(p) = \sum\limits_{p' \in \mathcal{N}_k} \nabla(p') / \arg\max\limits_{p} s(p)<br />
\end{equation}<br />
<br />
Using thresholding, $TS_l \leq s(p) \leq TS_u \wedge TI_l \leq I \leq TI_u$, different areas, such as background or pathogen, can be selected.<br />
<br />
For the empirical choice of thresholds, it can be argued that these are tailored to the specific case.<br />
While this surely is true to a certain extent, the here presented method has been successfully tested on images from a completely different domain, and no changes to the thresholds have been made to make it work.<br />
A proper theoretical evaluation is emphasized, however, is probably not the aim of the iGEM competition.<br />
<br />
Finally, selecting for the red region, this delivers the location of possible pathogens.<br />
Since the size of the agar chips is variable but fixed a quantitative analysis can be performed by counting pixels for instance.<br />
<br />
=== Empirical Evaluation ===<br />
<br />
Using our MATLAB code, we found the lower threshold for the smoothness index to be $TS_l = 0.85$ and the upper threshold $TS_u = \infty$.<br />
Similarly, for $TI_l = 235$ and $TI_u = \infty$.<br />
<br />
Using these settings, we can find a response already in images taken after 42&nbsp;minutes.<br />
<br />
Ideally, one would rate the quality of the image segmentation using some ground truth, such as manual delineations. This still has to be implemented for our method.<br />
However, from visual observations, our method is showing promising results.<br />
<br />
* image of smoothness index<br />
<br />
=== Automatic Classification ===<br />
<br />
<br />
<html><br />
<div class="codediv"><br />
<pre><code class="matlab"><br />
function [mask, seg] = automaticseeds(im)<br />
<br />
imc = im;<br />
<br />
%% to grayscale and filtering<br />
Z = double(rgb2gray(im));<br />
Z = 255 * Z / max(max(Z));<br />
<br />
filtertype = 'disk';<br />
Z = filter2(fspecial(filtertype), Z);<br />
Z = filter2(fspecial(filtertype), filter2(fspecial(filtertype), Z));<br />
Z = 255 * Z / max(max(Z)); <br />
<br />
%% calculating similarity score/smoothness index<br />
k=4;<br />
sSI = similarity(Z,k);<br />
sSI = sSI / max(max(sSI)); <br />
<br />
%% classify<br />
pathogene = ((sSI > 0.85) == 1) & ((Z > 235) == 1); <br />
<br />
mask = ones( size(imc) );<br />
seg = zeros( size(imc) );<br />
<br />
<br />
%% output<br />
for i=1:size(im,1)<br />
for j=1:size(im,2)<br />
<br />
if (pathogene(i,j) == 1)<br />
seg(i,j,1:3) = [255 0 0];<br />
mask(i, j, 1:3) = [0 0 0];<br />
end<br />
end<br />
end<br />
end<br />
</code></pre><br />
</div><br />
</html><br />
<br />
This code actually creates two intermediate images from which the similarity index is calculated.<br />
First the smoothed (disk-filter) input image is created and stored:<br />
<br />
{{Team:Aachen/Figure|align=center|Aachen_meas_smoothed.png|title=Smoothed image|width=700px}}<br />
<br />
Only white regions are candidate regions.<br />
After smoothing, the similarity index is calculated. As expected, edges are detected and limit the area from which the target region can be selected.<br />
<br />
{{Team:Aachen/Figure|align=center|Aachen_meas_smiliarity.png|title=Smoothness Index|width=700px}}<br />
<br />
Finally the selected pathogen region is selected by the black area in the following picture:<br />
<br />
{{Team:Aachen/Figure|align=center|Aachen_meas_mask.png|title=Selected region|width=700px}}<br />
<br />
Combined with the input image, the final segmentation is received:<br />
<br />
{{Team:Aachen/Figure|align=center|Aachen_meas_final.png|title=Final the analyzed image|width=700px}}<br />
<br />
[[File:Aachen_14-10-15_Medal_Cellocks_iNB.png|right|150px]]<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
== Achievements ==<br />
<span class="anchor" id="measurartyachievements"></span><br />
<br />
''Measurarty'' is the image analysis logic behind our project.<br />
It is comprised of simple constructs put together into a pipeline, that is clearly laid out, easily maintainable and - if needed - easily adaptable.<br />
For example, changing from green to red fluorescence, only means to change the ''createMask'' function to select another target area.<br />
<br />
Overall the results are convincing. We have not yet performed a comparison to a manual delineation, however, by eye the results look promising and have a low error.<br />
<br />
Talking about computational complexity, the MATLAB code of course performs better than our own C++ implementation, which must be regarded as a proof-of-principle.<br />
<br />
Space-wise, the code depends heavily on the image size $O( x \cdot y)$ (width $x$, height $y$, which also limits the number of edges in SRM between regions, as each pixel is one region to start with. However, it cannot take less memory, as the image is stored in an uncompressed format.<br />
<br />
On the computational side, the thresholding, image conversion and gradient steps are linear in the number of pixels, and are thus in $O(x \cdot y)$.<br />
Unfortunately, the summation of the gradient for the smoothness index adds a heavy factor to it (k-neighbourhood for smoothness index).<br />
Due to the merging step in our C++-SRM algorithm implementation, our code has to do $O(x^2 \cdot y^2)$ comparisons, which then finally results in a runtime complexity of $O( x^2 \cdot y^2)$.<br />
<br />
{{Team:Aachen/Figure|align=center|Aachen_meas_sizes.png|title=Pixel count of the detected pathogenic region versus time after induction.|width=700px}}<br />
<br />
From the above figure it can also be seen that the detected amount of pathogenic-area correlates with time after induction.<br />
The lag-phase can be explained first by the lag-phase of the cells, which first need to generate a response to the pathogen, and on the other hand, by too low fluorescence which is not detectable.<br />
The pixel count also meets the expectation when looking at the sample files by eye.<br />
<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|960px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/f/fc/Aachen_Measurarty_combined_slow.gif" width="960px"></html><br />
|-<br />
|'''{{{title|Detecting ''P. aeroginosa'' with K131026}}}'''<br />{{{subtitle|The left half shows the original images from the device and the right half shows the same pictures with the detected pathogenic region analyzed by ''Measurarty''.}}}<br />
|}<br />
</div><br />
</center><br />
<br />
It can be concluded that the ''Measurarty'' pipeline defines a robustly working chip-analysis algorithm which can detect pathogens from images supplied by ''WatsOn''.<br />
Therefore, this algorithm closes the gap between our biology, detection hardware and the user who wants easy-to-interpret results.<br />
<br />
For future prospects, it would be interesting to do a proper performance analysis on our code, to find hotspots and optimize the code. Many ''for''-loops leave plenty of room for vectorization and loop-unrolling. Parallelization, specifically with respect to embedded hardware such as the Raspberry Pi or Odroid U3, is limited to the extend that the overhead created would probably eliminate the improvements.<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
== Source Code ==<br />
<span class="anchor" id="source"></span><br />
<br />
''Measuarty'' is the image analysis logic behind our project. It has been prototyped and developed in [http://www.mathworks.de/academia/student-competitions/igem/ MATLAB], and only later been ported into our ''WatsOn'' GUI.<br />
<br />
We are happy to provide you with a zip-ped download of our MATLAB code here, as well as on the iGEM software repository on [https://github.com/orgs/igemsoftware/teams/aachen2014 github].<br />
<br />
* [https://static.igem.org/mediawiki/2014/6/6e/Aachen_measurarty.zip MATLAB code]<br />
* link [https://github.com/igemsoftware/AachenSoftProject2014/tree/master/measurarty github]<br />
<br />
For the C++ conversion please see [https://2014.igem.org/Team:Aachen/Notebook/Engineering/WatsOn#watsonsoftware our ''WatsOn'' Software] section.<br />
<br />
=== Using the MATLAB Code ===<br />
<br />
In general, please follow the included ''README.MD'' file. Our package comes with a set of test files from one of our experiments.<br />
After installing the Statistical Region Merging code (see readme), you can simply run ''igem_srm_demo.m''. Select your current folder, and MATLAB will automatically segment and classify the included jpg-images.<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
== References ==<br />
<span class="anchor" id="measurartyrefs"></span><br />
<br />
* Boltz, S. (2009, October 20). Image segmentation using statistical region merging - File Exchange - MATLAB Central. Image segmentation using statistical region merging. Retrieved December 12, 2013, from http://www.mathworks.com/matlabcentral/fileexchange/25619-image-segmentation-using-statistical-region-merging<br />
<br />
* Joppich, M., Rausch, D., & Kuhlen, T. (2013). Adaptive human motion prediction using multiple model approaches.. Virtuelle und erweiterte Realität (p. 169–180). 10. Workshop der GI-Fachgruppe VR/AR: Shaker.<br />
<br />
* Nock, R., & Nielsen, F. (2004). Statistical region merging. IEEE Transactions on Pattern Analysis and Machine Intelligence, 26(11), 1452-1458.<br />
<br />
<br />
<br />
{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/Notebook/Engineering/ODFTeam:Aachen/Notebook/Engineering/ODF2014-10-18T03:45:02Z<p>Nbailly: /* Saccharomyces cerevisiae */</p>
<hr />
<div>__NOTOC__<br />
{{Team:Aachen/Header}}<br />
{{Team:Aachen/Stylesheet}}<br />
= OD/F Device =<br />
<br />
On this page we present the technical details of our OD/F Device. You can skip to specific chapters by clicking on the panels below:<br />
<center><br />
<html><ul class="team-grid" style="width:1064px;"><br />
<!-- Overview --><br />
<br />
<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;"><br />
<a href="https://2014.igem.org/Team:Aachen/Notebook/Engineering/ODF#dev" style="color:black"><br />
<div class="team-item team-info" style="width:214px;height:214px;font-size: large;line-height:1.5em;"><br />
<br/><br><br />
<b>General Considerations</b><br />
<br/><br/><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/0/0f/Aachen_14-10-10_ODF_Button_ipo.png); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a><br />
</li><br />
<br />
<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;"><br />
<a href="https://2014.igem.org/Team:Aachen/Notebook/Engineering/ODF#od" style="color:black"><br />
<div class="team-item team-info" style="width:214px;height:214px;font-size: large;line-height:1.5em;"><br />
<br/><br/><br />
<b>OD Device</b><br />
<br/><br/><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/0/04/Aachen_Cuvette_button_v1_ipo.png); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a><br />
</li><br />
<br />
<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;"><br />
<a href="https://2014.igem.org/Team:Aachen/Notebook/Engineering/ODF#f" style="color:black"><br />
<div class="team-item team-info" style="width:214px;height:214px;font-size: large;line-height:1.5em;"><br />
<br/><br/><br />
<b>F Device</b><br />
<br/><br/><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/5/55/Aachen_17-10-14_Glowing_cuvette-ipo.png); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a><br />
</li><br />
<br />
<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;"><br />
<a href="https://2014.igem.org/Team:Aachen/Notebook/Engineering/ODF#diy" style="color:black"><br />
<div class="team-item team-info" style="width:214px;height:214px;font-size: large;line-height:1.5em;"><br />
<br/><br/><br />
<b>DIY</b><br />
<br/><br/><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/9/9e/Aachen_14-10-15_DIY_Cellocks_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a><br />
</li><br />
</ul><br />
</center><br />
</html><br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_14-10-10_ODF_Button_ipo.png|right|150px]]<br />
<br />
= General Considerations =<br />
<span class="anchor" id="dev"></span><br />
<br />
Building the OD/F Device has been an interesting task. While this device has been developed mainly by the IT division of our team, we got an immense support from the biologists suffering from color-blindness, yet eager to help selecting the best color filters for the LEDs.<br />
<br />
The measuring principle and guidelines for this project have already been presented in the [https://2014.igem.org/Team:Aachen/OD/F_device project] section.<br />
Here, details about selecting filters, code and a construction manual are presented.<br />
<br />
=== Cuvette Holder ===<br />
The essential part of this device is the '''cuvette holder'''. In short, we had to overcome a dilemma created by the need for an optimal height for the sensor:<br />
* A too low sensor position bears problems with sedimentation as well as light diffraction from the bottom of the cuvette.<br />
* The sensor has to be as close as possible to the bottom so that enough light shines through for the fluorescence measurement.<br />
<br />
As a compromise, we place the sensor at a height of 0.75&nbsp;cm. It is comparable to one of the standard heights (0.2&nbsp;cm, 0.8&nbsp;cm, 1.2&nbsp;cm) of OD meters. It is important to note that our device works with filling volumens of just 1&nbsp;mL, which in fact comes close to reality in the lab.<br />
<br />
The final cuvette holder design is rendered from a [https://2014.igem.org/Team:Aachen/Notebook/Engineering/Cuvette3D?action=raw stl-file] shown below:<br />
<br />
<html><br />
<center> <br />
<iframe src="https://2014.igem.org/Team:Aachen/Notebook/Engineering/Cuvette3D?action=render<br />
" width=500px height=500px frameBorder="0"></iframe><br />
<br/><br />
<b>Cuvette Holder developed for our OD/F Device.</b><br />
</center><br />
</html><br />
<br />
=== Light Filters ===<br />
'''Finding the right and optimal filters is a tough challenge'''.<br />
The main goal throughout our project has been to choose easily available parts which are also inexpensive. Thus choosing Schott glasses as filters could not be considered. Alternatively, filters used for illumination of theaters were found to be an ideal solution.<br />
<br />
We tested serveral filters and the optimal configuration of filters used is listed below.<br />
<br />
{| class="wikitable"<br />
! Mode<br />
! Fluorescence Protein<br />
! Filter Name<br />
! Filter<br />
! Peak Excitation<br />
! Peak Emission<br />
|-<br />
| Fluorescence<br />
| [http://parts.igem.org/Part:BBa_E0040 GFPmut3b]<br />
| [http://leefilters.com/lighting/colour-details.html#736 Twickenham Green]<br />
| [[File:Aachen_Filter_736.png|200px]]<br />
| 501nm<br />
| 511nm<br />
|-<br />
| Optical Density<br />
| --<br />
| [http://leefilters.com/lighting/colour-details.html#019 Fire]<br />
| [[File:Aachen_Filter_019.png|200px]]<br />
| 600nm<br />
| 600nm<br />
|}<br />
<br />
The fluorescence protein [http://parts.igem.org/Part:BBa_E0040 GFPmut3b] has a peak excitation at 501&nbsp;nm and a peak emission at 511&nbsp;nm - too close together for our low-cost filters to block the excitation light but transmit the emitted light. Thus, we chose to excite at around 485&nbsp;nm reduce false positive results below 500&nbsp;nm. However, no adequate filter for these settings could be found.<br />
Eventually, using the dark greenish [http://leefilters.com/lighting/colour-details.html#736 Twickenham Green] filter only little amounts of light shorter than 500&nbsp;nm gets through, reducing any bias from excitation illumination significantly. Unfortunately, the transmission rate of this filter is quite bad, 20% only, for the target emission wavelength of 511&nbsp;nm.<br />
<br />
== Linearity of the Hardware Light Sensor ==<br />
<span class="anchor" id="lin"></span><br />
It is crucial that the selected hardware is mapping reality into the digital world of our $\mu$-Controller.<br />
In order to sense reality our setup uses a light to frequency sensor, [https://www.sparkfun.com/datasheets/Sensors/Imaging/TSL235R-LF.pdf TSL235R-LF].<br />
The light to frequency sensor resembles the most to a photo transistor and thus is less sensible to temperature than a light dependant resistor.<br />
Additionally counting a frequency using interrupts seems to be easier and more accurate than using the analog to digital converter.<br />
<br />
Using a dilution series of purified [https://2011.igem.org/Team:Glasgow/LOV2 iLOV] we could determine the characteristic curve for the light sensor. Finally we can conclude that the sensor is linear as expected and shown in the [https://www.sparkfun.com/datasheets/Sensors/Imaging/TSL235R-LF.pdf datasheet].<br />
<br />
{{Team:Aachen/Figure|align=center|Aachen 15-10-14 Linearity iFG.PNG|title=Linearity of TSL235R-LF sensor|subtitle=Dilution series of GFP expressing ''E. coli'' showing linearity between fluorescence count and dilution. The linearity of the chosen sensor can be determined.|width=700px}}<br />
<br />
We are measuring optical density using our in-house developed cuvette holder.<br />
Particularly for optical density measurement, the amount of light shining through the sample is crucial.<br />
If there is too few light, there will be not enough light registered at the sensor, and the resolution of the measurement shrinks. This should be prevented.<br />
The chosen light to frequency sensor is reported to be very sensitive on the amount of light shining on it.<br />
There are reports of the sensor breaking when put into [https://www.sparkfun.com/products/9768 sunlight on a nice day], and not being sensitive at both high light or low light [http://kesslerarduino.wordpress.com/author/kevinmkessler/page/2/ conditions].<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
[[File:Aachen_Cuvette_button_v1_ipo.png|right|150px]]<br />
<br />
= Evaluation of the Optical Density Measurement =<br />
<span class="anchor" id="od"></span><br />
<br />
=== From Transmittance to True Optical Density ===<br />
At very low levels, uncorrected photometric determinations of cell densities show a decreasing proportionaility to actual cell density.<br />
<br />
This can also be observed using our device.<br />
<br />
In general, photometric determination of bacterial concentrations depends primarily on light scattering, rather than light absorption. Therefore, often not absorption is measured, but transmittance. For this, the relationship between optical density (OD) and transmitted light $\frac{I_0}{I}$ exists as:<br />
<br />
$$ OD = \frac{I_0}{I} = \kappa \cdot c$$<br />
<br />
where $I_0$ is the intensity of incoming light and $I$ the amount of the light passing through.<br />
<br />
However, this equation is linear only in a certain range.<br />
While one can tackle this non-linearity by using dilutions of the culture, correcting the error systematically is another way to overcome this limitation.<br />
<br />
For our OD device we needed to correlate the transmittance measured by our sensor to an optical density anyway.<br />
Our team members from the deterministic sciences emphasized on the correction method, which was conducted according to Lawrence and Maier (2006):<br />
<br />
* The relative density ($RD$) of each sample in a dilution series is calculated using $\frac{min(dilution)}{dilution}$.<br />
* The uncorrected optical density is derived from the transmission T [%]: $OD = 2 - \log T$<br />
* Finally, the unit optical density is calculated as $\frac{OD}{TD}$.<br />
* The average of the stable unit optical densities is used to calculate the true optical density $ OD_{unit} \cdot RD $.<br />
This way, the correlation between transmission and true optical density can be computed.<br />
The derived function allows the conversion from transmission to optical density on our device and therefore calibrates our device.<br />
<br />
<br />
In our experiments, we find in accordance to Lawrence and Maier that the correction majorly depends on the technical equipment used, especially the LED, sensor and cuvettes.<br />
While this at first sight looks disappointing, it is also expected:<br />
Transmittance is the fraction of light not absorbed by some medium relative to the cell-free and clear medium.<br />
However, the transmittance is not only dependent on the amount of cells in the way of the light's beam, but also how much light shines through the cuvette in which fashion, and in which fraction is received by the sensor in which angles.<br />
<br />
Using the above formula we performed this experiment for ''Pseudomonas putida'' and ''Saccharomyces cerevisiae'' and asked team [https://2014.igem.org/Team:Aachen/Collaborations/Freiburg Freiburg] to perform the same experiment using mamallian cells.<br />
<br />
=== Experiments ===<br />
<br />
We performed several experiments during the development of the device.<br />
Finally, we can relate the measured transmittance to the true Optical Density (true OD), and further, we can relate the true OD to the true OD of the photospectrometer in our lab.<br />
By doing so, we can calibrate our device to meaningful values.<br />
<br />
We have done this according to the previous section for ''Pseudomonas putida'' and ''Saccharomyces cerevisiae''.<br />
<br />
The final function for calculating the OD from the transmission calculated by our device can be calculated as<br />
<br />
$$ OD(T) = f(T) \circ g(device) $$<br />
<br />
where $f$ transforms transforms transmittance $T$ to true optical density for our device, and $g$ transforms true optical density of our device into the true optical density of the photospectrometer. This way our device is calibrated according to the photospectrometer.<br />
<br />
==== ''Pseudomonas putida'' ====<br />
<br />
{{Team:Aachen/Figure|align=center|Aachen 15-10-14 Pputida iFG.PNG|title=True Optical Density to Transmittance plot for ''P. putida''|subtitle=|width=700px}}<br />
</center><br />
<br />
<center><br />
{{Team:Aachen/Figure|align=center|Aachen 15-10-14 Pputida OD iFG.PNG|title=Corrolating the true optical density of the OD/F Device to true optical density of the photospectrometer for ''P. putida''|subtitle=|width=700px}}<br />
</center><br />
<br />
==== ''Saccharomyces cerevisiae'' ====<br />
<br />
<center><br />
{{Team:Aachen/Figure|align=center|Aachen 15-10-14 Scer iFG.PNG|title=True Optical Density to Transmittance plot for ''S. cerivisiae''|subtitle=|width=700px}}<br />
</center><br />
<br />
<center><br />
{{Team:Aachen/Figure|align=center|Aachen 15-10-14 Scer OD iFG.PNG|title=Corrolating the true optical density of the OD/F Device to true optical density of the photospectrometer for ''S. cerivisiae''|subtitle=|width=700px}}<br />
</center><br />
<br />
From these plots, it can be seen that our device delivers robust and reproducible results for both procaryotes and eucaryotes.<br />
Also the function from transmittance to true OD follows a clear pattern, making its calculation possible on a low-end device like a microcontroller.<br />
<br />
It is interesting to observer that function $g$, mapping the true OD of our device to the true OD of the photospectrometer, are close together for both ''P. putida'' and ''S. cerivisae'', as seen by the regression coefficient.<br />
In fact, 3.416 and 3.461 are such close together, that the minor deviation could be just measuring inaccuracy.<br />
Therefore, we fix the regression coefficient for converting true OD of our device to true OD of the photospectrometer to an average of 3.432 .<br />
<br />
Additionally, the function $f$ for mapping transmittance to true OD for our device is similar for all cell types, as seen in the following figure. Therefore the exponential regression curve for all cell types specifies this function.<br />
<br />
<center><br />
{{Team:Aachen/Figure|Aachen_ODallstrains1.png|title=Transmission of different cell types at OD-values from 0.001-1|subtitle=The transmittance data of NIH&nbsp;3T3 cells (mouse fibroblasts) align with the transmittance of ''P.&nbsp;putida'' and ''S.&nbsp;cerevisiae'' strains, even though the measured optical densities are lower by 1-2 orders of magnitude.|width=800px}}<br />
</center><br />
Finally, we have empirically determined our $OD(T)$ function by finding $f$ and $g$, such that we can convert true OD to the optical density of the photospectrometer.<br />
<br />
By this evaluation, we have shown that our self-build OD/F Device can compete with commercial systems.<br />
It is easy to calibrate by just calculating the true optical density.<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_17-10-14_Glowing_cuvette-ipo.png|right|150px]]<br />
<br />
= Evaluation of the Fluorescence Measurement =<br />
<span class="anchor" id="f"></span><br />
<br />
For the evaluation of the fluorescence measurement, we performed a dilution series of a constitutively GFPmut3b expressing ''E. coli''.<br />
<br />
The below figure shows the absolute measurements for both the platereader and our OD/F Device. The abrupt jump at 50% concentration can be explained by a second dilution step and is prevalent in both devices.<br />
It can be seen that the platereader show a much higher difference between the GFP and non-GFP cell culture at a higher standard deviation.<br />
Another interesting metric is the difference between the GFP and non-GFP, which can be seen as the normalized fluorescence measure.<br />
<br />
<center><br />
{{Team:Aachen/Figure|align=center|Aachn 15-10-14 F platereader ODF iFG.PNG|title=Fluorescence Measurement comparison OD/F Device and Platereader (absolute values)|subtitle=Comparison of a fluorescence measurement of our device and the platereader. Our OD/F Device shows no significant linearity.|width=700px}}<br />
</center><br />
<br />
If one compares the results there, as in the below figure with normalized fluorescence values, interesting observations can be made.<br />
First, both platereader and OD/F Device show very similar results.<br />
The regression curves differ only in a linear factor.<br />
Most interestingly the general fit of the OD/F Device to a linear function seems to be better than the platereader.<br />
Overall the linearity which has been observed earlier (in testing the general setup) could be verified.<br />
Therefore our do-it-yourself OD/F Device can be used to determine fluorescence.<br />
<br />
<center><br />
{{Team:Aachen/Figure|align=center|Aachen 15-10-14 F platereader ODF2 iFG.PNG|title=Fluorescence Measurement comparison OD/F Device and platereader using normalized fluorescence values|subtitle=Comparison of a fluorescence measurement of our device and the platereader. Our OD/F Device shows no significant. A non-GFP expressing ''E. coli'' dilution has been used to normalize the GFP dilution series. A linear correlation can be seen.|width=700px}}<br />
</center><br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_14-10-15_DIY_Cellocks_iNB.png|right|150px]]<br />
<br />
= DIY: How to Build Your Own Device =<br />
<span class="anchor" id="diy"></span><br />
== Technical Components ==<br />
<br />
While the casing and the cuvette holder are custom made, most of the parts are pre-made and can be readily bought. The previous section lists all needed parts.<br />
To get all these parts for creating your own OD/F Device is easy using the internet. Thus all '''potential customers have a market access''' to the used components. <br />
<br />
Please find our custom parts for download below. Despite being custom parts, these are quite inenxpensive - so feel free to give our OD/F Device a test! For printing the 3D model of our cuvette holder and laser cutting our case, we want to express many thanks to [http://hci.rwth-aachen.de/fablab Fablab Aachen].<span class="anchor" id="ref2"></span><br />
<br />
* cuvette holder [https://2014.igem.org/Template:Team:Aachen/cuvette.stl?action=raw STL file]<br />
* casing single device [https://static.igem.org/mediawiki/2014/b/bb/Aachen_ODF_device_casing.svg.zip SVG file]<br />
* lid combined device [https://static.igem.org/mediawiki/2014/4/46/Aachen_Odf_device_combined_lid.svg.zip SVG file]<br />
* Arduino Code for single device [https://static.igem.org/mediawiki/2014/2/2f/Arduino_od_f_single.zip Sketchbook (.ino)]<br />
* Arduino Code for combined device [https://static.igem.org/mediawiki/2014/e/e8/Arduino_odf_combined.zip Sketchbook (.ino)]<br />
<br />
You will need a special [http://www.exp-tech.de/images/product_images/description%20images/YWRobot/1602/LiquidCrystal_I2C1602V2.rar library] for the display, which can not be uploaded for legal reasons.<br />
<br />
<br />
<center><br />
'''All needed components their quantities and prices for creating your own OD/F Device'''<br />
{| class="wikitable"<br />
! align="center" |'''OD/F Device'''<br />
!! align="center" | <br />
!! align="center" |''' 1€='''<br />
!! align="center" |''' $1.27'''<br />
!! align="center" |''' on 14/10/2014'''<br />
!! align="center" | <br />
|-<br />
! Quantity!!Component!! Costs [€] !! Costs [$] !! Final [€] !! Final [$] <br />
|-<br />
| 1||[http://www.dx.com/p/uno-r3-development-board-microcontroller-mega328p-atmega16u2-compat-for-arduino-blue-black-215600#.VDzwV9ysWBp Arduino UNO R3]||9.17||11.65||9.17||11.65 <br />
|-<br />
| 1||[http://www.mouser.de/ProductDetail/ams/TSL235R-LF/?qs=14HO7rLZPQsjmBHaoYCzkA%3D%3D TSL 235R]||2.47||3.14||2.47||3.14 <br />
|-<br />
| 1||[http://www.dx.com/p/16-x-2-character-lcd-display-module-with-blue-backlight-121356 Display 16x2]||2.58||3.28||2.58||3.28 <br />
|-<br />
| 1||[http://www.dx.com/p/lcd1602-adapter-board-w-iic-i2c-interface-black-works-with-official-arduino-boards-216865#.VDzxHNysWBp LCD Display to I2C]||1.57||1.99||1.57||1.99 <br />
|-<br />
| 1||[http://www.newark.com/multicomp/mcpas6b1m1ce3/switch-pushbutton-spst-400ma-125v/dp/12P7696?ost=1638329 Pushbutton]||2.90||3.69||2.90||3.69 <br />
|-<br />
| 1||[http://shop.leefiltersusa.com/Swatch-Book-Designers-Edition-SWB.htm filter leaflet]||1.57||2.00||1.57||2.00 <br />
|-<br />
| 20||[http://www.dx.com/p/diy-male-to-female-dupont-breadboard-jumper-wires-black-multi-color-40-pcs-10cm-339078#.VDzxSdysWBp jumper wire cables]||0.09||0.11||1.80||2.28 <br />
|-<br />
| 1||[http://www.dx.com/p/syb-170-mini-breadboard-for-diy-project-red-140101#.VDzyudysWBo small breadboard]||1.98||2.51||1.98||2.51 <br />
|-<br />
| 1||[http://www.dx.com/p/universal-ac-charger-w-dual-usb-output-for-iphone-ipad-ipod-white-us-plug-244893 power supply]||2.20||2.80||2.20||2.80<br />
|-<br />
| 1||cuvette holder (3D print service of your choice)||6.44||8.18||6.44||8.18<br />
|-<br />
| 1||3&nbsp;mm acrylic glas (black)||7.98||10.14||7.98||10.14<br />
|-<br />
| 1||[http://www.dx.com/p/prototype-universal-printed-circuit-board-breadboard-golden-10-piece-pack-143913 Prototype Universal Printed Circuit Board]||2.27||2.88||2.27||2.88<br />
|-<br />
| 1||[http://www.dx.com/p/2-54mm-1x40-pin-breakaway-straight-male-header-10-piece-pack-144191#.VEGRF2d_tGg Male Headers]||2.14||2.72||2.14||2.72<br />
|- style="border-top: 2px solid #808080;"<br />
| 1||[http://www.mouser.de/ProductDetail/Dialight/550-2505F/?qs=0KZIkTEbAAvqMAW7suDOXg== LED 600&nbsp;nm]||0.94||1.19||0.94||1.19<br />
|-<br />
! -!!Total OD!!-!!-!! 46.01 !! 58.45 <br />
|-<br />
| 1||[http://www.leds.de/Low-Mid-Power-LEDs/SuperFlux-LEDs/Nichia-Superflux-LED-blau-3lm-100-NSPBR70BSS.html LED 480&nbsp;nm]||0.99||1.26||0.99||1.26<br />
|-<br />
! -!!Total F!!-!!-!! 46.06 !! 58.52 <br />
|- style="border-top: 2px solid #808080;;"<br />
| 1||[http://www.mouser.de/ProductDetail/Dialight/550-2505F/?qs=0KZIkTEbAAvqMAW7suDOXg== LED 600nm]||0.94||1.19||0.94||1.19 <br />
|-<br />
| 1||[http://www.leds.de/Low-Mid-Power-LEDs/SuperFlux-LEDs/Nichia-Superflux-LED-blau-3lm-100-NSPBR70BSS.html LED 480&nbsp;nm]||0.99||1.26||0.99||1.26<br />
|-<br />
| 1||cuvette holder (3D print service of your choice)||6.44||8.18||6.44||8.18<br />
|-<br />
! -!!Total OD/F!!-!!-!! 53.44 !! 67.89 <br />
|-<br />
|}<br />
</center><br />
<br />
For more detailed economical information about the OD/F project visit our [https://2014.igem.org/Team:Aachen/PolicyPractices/Economics Economical View] page.<br />
<br />
== Breadboards ==<br />
<br />
=== Optical Density ===<br />
<br />
{{Team:Aachen/Figure|Aachen ODdevice Steckplatine.png|align=center|title=Breadboard of our OD device|subtitle=To build your own OD device, connect the parts as shown in this diagram.|width=900px}}<br />
<br />
If you want to build our OD device, make sure to use the following secret ingredients:<br />
* Filter: [http://www.leefilters.com/lighting/colour-details.html#019 Fire 019]<br />
* LED: [http://www.mouser.de/ProductDetail/Dialight/550-2505F/?qs=0KZIkTEbAAvqMAW7suDOXg== 600 nm] Dialight 550-2505F<br />
<br />
=== Fluorescence ===<br />
<br />
{{Team:Aachen/Figure|Aachen_Fdevice_Steckplatine.png|align=center|title=Our novel biosensor approach|subtitle=Expression of the TEV protease is induced by HSL. The protease cleaves the GFP-REACh fusion protein to elecit a fluorescence response.|width=900px}}<br />
If you want to build the OD device, make sure to use the following secret ingredients:<br />
* Filter: [http://www.leefilters.com/lighting/colour-details.html#736 Twickenham Green 736]<br />
* LED: [http://www.leds.de/Low-Mid-Power-LEDs/SuperFlux-LEDs/Nichia-Superflux-LED-blau-3lm-100-NSPBR70BSS.html 480 nm] NSPBR70BSS LED<br />
<br />
=== Combined Device ===<br />
Even though evaluation of the measurements have been performed in two separate device, it is fairly well possible to put everything into one casing.<br />
All you need to do is choosing another lid, and connect a second light to frequency sensor to your Arduino.<br />
Right at the bottom we present you the differences in wiring things up.<br />
Building the combined device is straight forward and very similar to the single device. You will need a slightly larger connector, a different lid for your case, and some more cables. The corresponding fritzing-layout is presented below.<br />
<br />
{{Team:Aachen/Figure|Aachen_ODF_combined_Steckplatine.png|align=center|title=Our novel biosensor approach|subtitle=Expression of the TEV protease is induced by HSL. The protease cleaves the GFP-REACh fusion protein to elecit a fluorescence response.|width=900px}}<br />
<br />
== Construction Steps ==<br />
{| class="wikitable centered"<br />
|-<br />
| [[File:Aachen_ODF_9.JPG|300px]] || First we want to assemble the casing. Once you have all the cut parts, you can start to assemble them. For cutting, we really recommend using a laser cutter.<br />
|-<br />
| [[File:Aachen_ODF_8.JPG|300px]] || Attach the cuvette-holder holders such that the cuvette holder is placed directly under the opening hole.<br />
|-<br />
| [[File:Aachen_ODF_4.JPG|300px]] || Next build the lid of the device. At this stage you can already mount the button. We recommend to glue any parts.<br />
|-<br />
| [[File:Aachen_ODF_3.JPG|300px]] || Your lid finally should look like this.<br />
|-<br />
| [[File:Aachen_ODF_11.JPG|150px]][[File:Aachen_ODF_10.JPG|150px]] || Next we want to assemble the cuvette holders. On the side with the square hole attach the light-to-frequency sensor with glue. For the OD case place the orange LED opposite, or for fluorescence, the LED in the hole in the bottom. Make sure to close any remaining open hole! Please attach a piece of filter foil (approx. $7 \times 7 mm^2$) from the inside in front of the light to frequency converter. Forceps is highly recommended.<br />
|-<br />
| [[File:Aachen_ODF_12.JPG|300px]] || Your final assembly should then look like this. Now place the correct filter into the cuvette holder, directly in front of the sensor. Make sure that the filter does not degrade due to the glue!<br />
|-<br />
| [[File:Aachen_ODF_14.JPG|300px]] || As the case can be used for both, fluorescence and OD measurement, we use a combined plug. Just three header rows (7 pins, 9pins for combined) and connect them as we did.<br />
|-<br />
| [[File:Aachen_ODF_1.JPG|300px]] || Now we're doing the wiring. Connect the Arduino 5V and GND such that you have one 5V and one GND line on your breadboard.<br />
|-<br />
| [[File:Aachen_ODF_2.JPG|300px]] || Then connect the button to 5V on the one side, and to GND via a resistor on the other side. Connect this side also to port __ on your Arduino. This will sense the blank. Next connect the display to the Arduino and our connector. See the Fritzing diagram at the bottom for a detailed information.<br />
|-<br />
| [[File:Aachen_ODF_13.JPG|300px]] || Now put everything into the case and ...<br />
|-<br />
| [[File:Aachen_ODF_6.JPG|300px]] || ... also place the cuvette holder into the device. Attach the display to the device lid and close the casing.<br />
|-<br />
| [[File:Aachen_ODF_7.JPG|300px]] || Congratulations! You have finished constructing your own OD/F Device!<br />
|-<br />
| [[File:Aachen_zwei_Kuevetten.jpg|300px]] || ... or even the combined device!<br />
|}<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
== References ==<br />
Lawrence, J. V., & Maier, S. (1977). Correction for the inherent error in optical density readings. Applied and Environmental Microbiology, 33(2), 482–484. Available at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=170707&tool=pmcentrez&rendertype=abstract.<br />
<br />
{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/Notebook/Engineering/ODFTeam:Aachen/Notebook/Engineering/ODF2014-10-18T03:44:49Z<p>Nbailly: /* Saccharomyces cerevisiae */</p>
<hr />
<div>__NOTOC__<br />
{{Team:Aachen/Header}}<br />
{{Team:Aachen/Stylesheet}}<br />
= OD/F Device =<br />
<br />
On this page we present the technical details of our OD/F Device. You can skip to specific chapters by clicking on the panels below:<br />
<center><br />
<html><ul class="team-grid" style="width:1064px;"><br />
<!-- Overview --><br />
<br />
<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;"><br />
<a href="https://2014.igem.org/Team:Aachen/Notebook/Engineering/ODF#dev" style="color:black"><br />
<div class="team-item team-info" style="width:214px;height:214px;font-size: large;line-height:1.5em;"><br />
<br/><br><br />
<b>General Considerations</b><br />
<br/><br/><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/0/0f/Aachen_14-10-10_ODF_Button_ipo.png); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a><br />
</li><br />
<br />
<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;"><br />
<a href="https://2014.igem.org/Team:Aachen/Notebook/Engineering/ODF#od" style="color:black"><br />
<div class="team-item team-info" style="width:214px;height:214px;font-size: large;line-height:1.5em;"><br />
<br/><br/><br />
<b>OD Device</b><br />
<br/><br/><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/0/04/Aachen_Cuvette_button_v1_ipo.png); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a><br />
</li><br />
<br />
<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;"><br />
<a href="https://2014.igem.org/Team:Aachen/Notebook/Engineering/ODF#f" style="color:black"><br />
<div class="team-item team-info" style="width:214px;height:214px;font-size: large;line-height:1.5em;"><br />
<br/><br/><br />
<b>F Device</b><br />
<br/><br/><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/5/55/Aachen_17-10-14_Glowing_cuvette-ipo.png); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a><br />
</li><br />
<br />
<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;"><br />
<a href="https://2014.igem.org/Team:Aachen/Notebook/Engineering/ODF#diy" style="color:black"><br />
<div class="team-item team-info" style="width:214px;height:214px;font-size: large;line-height:1.5em;"><br />
<br/><br/><br />
<b>DIY</b><br />
<br/><br/><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/9/9e/Aachen_14-10-15_DIY_Cellocks_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a><br />
</li><br />
</ul><br />
</center><br />
</html><br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_14-10-10_ODF_Button_ipo.png|right|150px]]<br />
<br />
= General Considerations =<br />
<span class="anchor" id="dev"></span><br />
<br />
Building the OD/F Device has been an interesting task. While this device has been developed mainly by the IT division of our team, we got an immense support from the biologists suffering from color-blindness, yet eager to help selecting the best color filters for the LEDs.<br />
<br />
The measuring principle and guidelines for this project have already been presented in the [https://2014.igem.org/Team:Aachen/OD/F_device project] section.<br />
Here, details about selecting filters, code and a construction manual are presented.<br />
<br />
=== Cuvette Holder ===<br />
The essential part of this device is the '''cuvette holder'''. In short, we had to overcome a dilemma created by the need for an optimal height for the sensor:<br />
* A too low sensor position bears problems with sedimentation as well as light diffraction from the bottom of the cuvette.<br />
* The sensor has to be as close as possible to the bottom so that enough light shines through for the fluorescence measurement.<br />
<br />
As a compromise, we place the sensor at a height of 0.75&nbsp;cm. It is comparable to one of the standard heights (0.2&nbsp;cm, 0.8&nbsp;cm, 1.2&nbsp;cm) of OD meters. It is important to note that our device works with filling volumens of just 1&nbsp;mL, which in fact comes close to reality in the lab.<br />
<br />
The final cuvette holder design is rendered from a [https://2014.igem.org/Team:Aachen/Notebook/Engineering/Cuvette3D?action=raw stl-file] shown below:<br />
<br />
<html><br />
<center> <br />
<iframe src="https://2014.igem.org/Team:Aachen/Notebook/Engineering/Cuvette3D?action=render<br />
" width=500px height=500px frameBorder="0"></iframe><br />
<br/><br />
<b>Cuvette Holder developed for our OD/F Device.</b><br />
</center><br />
</html><br />
<br />
=== Light Filters ===<br />
'''Finding the right and optimal filters is a tough challenge'''.<br />
The main goal throughout our project has been to choose easily available parts which are also inexpensive. Thus choosing Schott glasses as filters could not be considered. Alternatively, filters used for illumination of theaters were found to be an ideal solution.<br />
<br />
We tested serveral filters and the optimal configuration of filters used is listed below.<br />
<br />
{| class="wikitable"<br />
! Mode<br />
! Fluorescence Protein<br />
! Filter Name<br />
! Filter<br />
! Peak Excitation<br />
! Peak Emission<br />
|-<br />
| Fluorescence<br />
| [http://parts.igem.org/Part:BBa_E0040 GFPmut3b]<br />
| [http://leefilters.com/lighting/colour-details.html#736 Twickenham Green]<br />
| [[File:Aachen_Filter_736.png|200px]]<br />
| 501nm<br />
| 511nm<br />
|-<br />
| Optical Density<br />
| --<br />
| [http://leefilters.com/lighting/colour-details.html#019 Fire]<br />
| [[File:Aachen_Filter_019.png|200px]]<br />
| 600nm<br />
| 600nm<br />
|}<br />
<br />
The fluorescence protein [http://parts.igem.org/Part:BBa_E0040 GFPmut3b] has a peak excitation at 501&nbsp;nm and a peak emission at 511&nbsp;nm - too close together for our low-cost filters to block the excitation light but transmit the emitted light. Thus, we chose to excite at around 485&nbsp;nm reduce false positive results below 500&nbsp;nm. However, no adequate filter for these settings could be found.<br />
Eventually, using the dark greenish [http://leefilters.com/lighting/colour-details.html#736 Twickenham Green] filter only little amounts of light shorter than 500&nbsp;nm gets through, reducing any bias from excitation illumination significantly. Unfortunately, the transmission rate of this filter is quite bad, 20% only, for the target emission wavelength of 511&nbsp;nm.<br />
<br />
== Linearity of the Hardware Light Sensor ==<br />
<span class="anchor" id="lin"></span><br />
It is crucial that the selected hardware is mapping reality into the digital world of our $\mu$-Controller.<br />
In order to sense reality our setup uses a light to frequency sensor, [https://www.sparkfun.com/datasheets/Sensors/Imaging/TSL235R-LF.pdf TSL235R-LF].<br />
The light to frequency sensor resembles the most to a photo transistor and thus is less sensible to temperature than a light dependant resistor.<br />
Additionally counting a frequency using interrupts seems to be easier and more accurate than using the analog to digital converter.<br />
<br />
Using a dilution series of purified [https://2011.igem.org/Team:Glasgow/LOV2 iLOV] we could determine the characteristic curve for the light sensor. Finally we can conclude that the sensor is linear as expected and shown in the [https://www.sparkfun.com/datasheets/Sensors/Imaging/TSL235R-LF.pdf datasheet].<br />
<br />
{{Team:Aachen/Figure|align=center|Aachen 15-10-14 Linearity iFG.PNG|title=Linearity of TSL235R-LF sensor|subtitle=Dilution series of GFP expressing ''E. coli'' showing linearity between fluorescence count and dilution. The linearity of the chosen sensor can be determined.|width=700px}}<br />
<br />
We are measuring optical density using our in-house developed cuvette holder.<br />
Particularly for optical density measurement, the amount of light shining through the sample is crucial.<br />
If there is too few light, there will be not enough light registered at the sensor, and the resolution of the measurement shrinks. This should be prevented.<br />
The chosen light to frequency sensor is reported to be very sensitive on the amount of light shining on it.<br />
There are reports of the sensor breaking when put into [https://www.sparkfun.com/products/9768 sunlight on a nice day], and not being sensitive at both high light or low light [http://kesslerarduino.wordpress.com/author/kevinmkessler/page/2/ conditions].<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
[[File:Aachen_Cuvette_button_v1_ipo.png|right|150px]]<br />
<br />
= Evaluation of the Optical Density Measurement =<br />
<span class="anchor" id="od"></span><br />
<br />
=== From Transmittance to True Optical Density ===<br />
At very low levels, uncorrected photometric determinations of cell densities show a decreasing proportionaility to actual cell density.<br />
<br />
This can also be observed using our device.<br />
<br />
In general, photometric determination of bacterial concentrations depends primarily on light scattering, rather than light absorption. Therefore, often not absorption is measured, but transmittance. For this, the relationship between optical density (OD) and transmitted light $\frac{I_0}{I}$ exists as:<br />
<br />
$$ OD = \frac{I_0}{I} = \kappa \cdot c$$<br />
<br />
where $I_0$ is the intensity of incoming light and $I$ the amount of the light passing through.<br />
<br />
However, this equation is linear only in a certain range.<br />
While one can tackle this non-linearity by using dilutions of the culture, correcting the error systematically is another way to overcome this limitation.<br />
<br />
For our OD device we needed to correlate the transmittance measured by our sensor to an optical density anyway.<br />
Our team members from the deterministic sciences emphasized on the correction method, which was conducted according to Lawrence and Maier (2006):<br />
<br />
* The relative density ($RD$) of each sample in a dilution series is calculated using $\frac{min(dilution)}{dilution}$.<br />
* The uncorrected optical density is derived from the transmission T [%]: $OD = 2 - \log T$<br />
* Finally, the unit optical density is calculated as $\frac{OD}{TD}$.<br />
* The average of the stable unit optical densities is used to calculate the true optical density $ OD_{unit} \cdot RD $.<br />
This way, the correlation between transmission and true optical density can be computed.<br />
The derived function allows the conversion from transmission to optical density on our device and therefore calibrates our device.<br />
<br />
<br />
In our experiments, we find in accordance to Lawrence and Maier that the correction majorly depends on the technical equipment used, especially the LED, sensor and cuvettes.<br />
While this at first sight looks disappointing, it is also expected:<br />
Transmittance is the fraction of light not absorbed by some medium relative to the cell-free and clear medium.<br />
However, the transmittance is not only dependent on the amount of cells in the way of the light's beam, but also how much light shines through the cuvette in which fashion, and in which fraction is received by the sensor in which angles.<br />
<br />
Using the above formula we performed this experiment for ''Pseudomonas putida'' and ''Saccharomyces cerevisiae'' and asked team [https://2014.igem.org/Team:Aachen/Collaborations/Freiburg Freiburg] to perform the same experiment using mamallian cells.<br />
<br />
=== Experiments ===<br />
<br />
We performed several experiments during the development of the device.<br />
Finally, we can relate the measured transmittance to the true Optical Density (true OD), and further, we can relate the true OD to the true OD of the photospectrometer in our lab.<br />
By doing so, we can calibrate our device to meaningful values.<br />
<br />
We have done this according to the previous section for ''Pseudomonas putida'' and ''Saccharomyces cerevisiae''.<br />
<br />
The final function for calculating the OD from the transmission calculated by our device can be calculated as<br />
<br />
$$ OD(T) = f(T) \circ g(device) $$<br />
<br />
where $f$ transforms transforms transmittance $T$ to true optical density for our device, and $g$ transforms true optical density of our device into the true optical density of the photospectrometer. This way our device is calibrated according to the photospectrometer.<br />
<br />
==== ''Pseudomonas putida'' ====<br />
<br />
{{Team:Aachen/Figure|align=center|Aachen 15-10-14 Pputida iFG.PNG|title=True Optical Density to Transmittance plot for ''P. putida''|subtitle=|width=700px}}<br />
</center><br />
<br />
<center><br />
{{Team:Aachen/Figure|align=center|Aachen 15-10-14 Pputida OD iFG.PNG|title=Corrolating the true optical density of the OD/F Device to true optical density of the photospectrometer for ''P. putida''|subtitle=|width=700px}}<br />
</center><br />
<br />
==== ''Saccharomyces cerevisiae'' ====<br />
<br />
<center><br />
{{Team:Aachen/Figure|align=center|Aachen 15-10-14 Scer iFG.PNG|title=True Optical Density to Transmittance plot for ''S. cerivisiae''|subtitle=|width=700px}}<br />
</center><br />
<br />
<center><br />
{{Team:Aachen/Figure|align=center|Aachen 15-10-14 Scer OD iFG.PNG|title=Corrolating the true optical density of the OD/F Device to true optical density of the photospectrometer for ''S. cerivisiae''|subtitle=|width=700px}}<br />
</center><br />
<br />
From these plots, it can be seen that our device delivers robust and reproducible results for both procaryotes and eucaryotes.<br />
Also the function from transmittance to true OD follows a clear pattern, making its calculation possible on a low-end device like a microcontroller.<br />
<br />
It is interesting to observer that function $g$, mapping the true OD of our device to the true OD of the photospectrometer, are close together for both ''P. putida'' and ''S. cerivisae'', as seen by the regression coefficient.<br />
In fact, 3.416 and 3.461 are such close together, that the minor deviation could be just measuring inaccuracy.<br />
Therefore, we fix the regression coefficient for converting true OD of our device to true OD of the photospectrometer to an average of 3.432 .<br />
<br />
Additionally, the function $f$ for mapping transmittance to true OD for our device is similar for all cell types, as seen in the following figure. Therefore the exponential regression curve for all cell types specifies this function.<br />
<br />
<center><br />
{{Team:Aachen/Figure|Aachen_ODallstrains1.png|title=Transmission of different cell types at OD-values from 0.001-1|subtitle=The transmittance data of NIH&nbsp;3T3 cells (mouse fibroblasts) align with the transmittance of ''P.&nbsp;putida'' and ''S.&nbsp;cerevisiae'' strains, even though the measured optical densities are lower by 1-2 orders of magnitude.|width=800px}}<br />
</center><br />
Finally, we have empirically determined our $OD(T)$ function by finding $f$ and $g$, such that we can convert true OD to the optical density of the photospectrometer.<br />
<br />
By this evaluation, we have shown that our self-build OD/F Device can compete with commercial systems.<br />
It is easy to calibrate by just calculating the true optical density.<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_17-10-14_Glowing_cuvette-ipo.png|right|150px]]<br />
<br />
= Evaluation of the Fluorescence Measurement =<br />
<span class="anchor" id="f"></span><br />
<br />
For the evaluation of the fluorescence measurement, we performed a dilution series of a constitutively GFPmut3b expressing ''E. coli''.<br />
<br />
The below figure shows the absolute measurements for both the platereader and our OD/F Device. The abrupt jump at 50% concentration can be explained by a second dilution step and is prevalent in both devices.<br />
It can be seen that the platereader show a much higher difference between the GFP and non-GFP cell culture at a higher standard deviation.<br />
Another interesting metric is the difference between the GFP and non-GFP, which can be seen as the normalized fluorescence measure.<br />
<br />
<center><br />
{{Team:Aachen/Figure|align=center|Aachn 15-10-14 F platereader ODF iFG.PNG|title=Fluorescence Measurement comparison OD/F Device and Platereader (absolute values)|subtitle=Comparison of a fluorescence measurement of our device and the platereader. Our OD/F Device shows no significant linearity.|width=700px}}<br />
</center><br />
<br />
If one compares the results there, as in the below figure with normalized fluorescence values, interesting observations can be made.<br />
First, both platereader and OD/F Device show very similar results.<br />
The regression curves differ only in a linear factor.<br />
Most interestingly the general fit of the OD/F Device to a linear function seems to be better than the platereader.<br />
Overall the linearity which has been observed earlier (in testing the general setup) could be verified.<br />
Therefore our do-it-yourself OD/F Device can be used to determine fluorescence.<br />
<br />
<center><br />
{{Team:Aachen/Figure|align=center|Aachen 15-10-14 F platereader ODF2 iFG.PNG|title=Fluorescence Measurement comparison OD/F Device and platereader using normalized fluorescence values|subtitle=Comparison of a fluorescence measurement of our device and the platereader. Our OD/F Device shows no significant. A non-GFP expressing ''E. coli'' dilution has been used to normalize the GFP dilution series. A linear correlation can be seen.|width=700px}}<br />
</center><br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_14-10-15_DIY_Cellocks_iNB.png|right|150px]]<br />
<br />
= DIY: How to Build Your Own Device =<br />
<span class="anchor" id="diy"></span><br />
== Technical Components ==<br />
<br />
While the casing and the cuvette holder are custom made, most of the parts are pre-made and can be readily bought. The previous section lists all needed parts.<br />
To get all these parts for creating your own OD/F Device is easy using the internet. Thus all '''potential customers have a market access''' to the used components. <br />
<br />
Please find our custom parts for download below. Despite being custom parts, these are quite inenxpensive - so feel free to give our OD/F Device a test! For printing the 3D model of our cuvette holder and laser cutting our case, we want to express many thanks to [http://hci.rwth-aachen.de/fablab Fablab Aachen].<span class="anchor" id="ref2"></span><br />
<br />
* cuvette holder [https://2014.igem.org/Template:Team:Aachen/cuvette.stl?action=raw STL file]<br />
* casing single device [https://static.igem.org/mediawiki/2014/b/bb/Aachen_ODF_device_casing.svg.zip SVG file]<br />
* lid combined device [https://static.igem.org/mediawiki/2014/4/46/Aachen_Odf_device_combined_lid.svg.zip SVG file]<br />
* Arduino Code for single device [https://static.igem.org/mediawiki/2014/2/2f/Arduino_od_f_single.zip Sketchbook (.ino)]<br />
* Arduino Code for combined device [https://static.igem.org/mediawiki/2014/e/e8/Arduino_odf_combined.zip Sketchbook (.ino)]<br />
<br />
You will need a special [http://www.exp-tech.de/images/product_images/description%20images/YWRobot/1602/LiquidCrystal_I2C1602V2.rar library] for the display, which can not be uploaded for legal reasons.<br />
<br />
<br />
<center><br />
'''All needed components their quantities and prices for creating your own OD/F Device'''<br />
{| class="wikitable"<br />
! align="center" |'''OD/F Device'''<br />
!! align="center" | <br />
!! align="center" |''' 1€='''<br />
!! align="center" |''' $1.27'''<br />
!! align="center" |''' on 14/10/2014'''<br />
!! align="center" | <br />
|-<br />
! Quantity!!Component!! Costs [€] !! Costs [$] !! Final [€] !! Final [$] <br />
|-<br />
| 1||[http://www.dx.com/p/uno-r3-development-board-microcontroller-mega328p-atmega16u2-compat-for-arduino-blue-black-215600#.VDzwV9ysWBp Arduino UNO R3]||9.17||11.65||9.17||11.65 <br />
|-<br />
| 1||[http://www.mouser.de/ProductDetail/ams/TSL235R-LF/?qs=14HO7rLZPQsjmBHaoYCzkA%3D%3D TSL 235R]||2.47||3.14||2.47||3.14 <br />
|-<br />
| 1||[http://www.dx.com/p/16-x-2-character-lcd-display-module-with-blue-backlight-121356 Display 16x2]||2.58||3.28||2.58||3.28 <br />
|-<br />
| 1||[http://www.dx.com/p/lcd1602-adapter-board-w-iic-i2c-interface-black-works-with-official-arduino-boards-216865#.VDzxHNysWBp LCD Display to I2C]||1.57||1.99||1.57||1.99 <br />
|-<br />
| 1||[http://www.newark.com/multicomp/mcpas6b1m1ce3/switch-pushbutton-spst-400ma-125v/dp/12P7696?ost=1638329 Pushbutton]||2.90||3.69||2.90||3.69 <br />
|-<br />
| 1||[http://shop.leefiltersusa.com/Swatch-Book-Designers-Edition-SWB.htm filter leaflet]||1.57||2.00||1.57||2.00 <br />
|-<br />
| 20||[http://www.dx.com/p/diy-male-to-female-dupont-breadboard-jumper-wires-black-multi-color-40-pcs-10cm-339078#.VDzxSdysWBp jumper wire cables]||0.09||0.11||1.80||2.28 <br />
|-<br />
| 1||[http://www.dx.com/p/syb-170-mini-breadboard-for-diy-project-red-140101#.VDzyudysWBo small breadboard]||1.98||2.51||1.98||2.51 <br />
|-<br />
| 1||[http://www.dx.com/p/universal-ac-charger-w-dual-usb-output-for-iphone-ipad-ipod-white-us-plug-244893 power supply]||2.20||2.80||2.20||2.80<br />
|-<br />
| 1||cuvette holder (3D print service of your choice)||6.44||8.18||6.44||8.18<br />
|-<br />
| 1||3&nbsp;mm acrylic glas (black)||7.98||10.14||7.98||10.14<br />
|-<br />
| 1||[http://www.dx.com/p/prototype-universal-printed-circuit-board-breadboard-golden-10-piece-pack-143913 Prototype Universal Printed Circuit Board]||2.27||2.88||2.27||2.88<br />
|-<br />
| 1||[http://www.dx.com/p/2-54mm-1x40-pin-breakaway-straight-male-header-10-piece-pack-144191#.VEGRF2d_tGg Male Headers]||2.14||2.72||2.14||2.72<br />
|- style="border-top: 2px solid #808080;"<br />
| 1||[http://www.mouser.de/ProductDetail/Dialight/550-2505F/?qs=0KZIkTEbAAvqMAW7suDOXg== LED 600&nbsp;nm]||0.94||1.19||0.94||1.19<br />
|-<br />
! -!!Total OD!!-!!-!! 46.01 !! 58.45 <br />
|-<br />
| 1||[http://www.leds.de/Low-Mid-Power-LEDs/SuperFlux-LEDs/Nichia-Superflux-LED-blau-3lm-100-NSPBR70BSS.html LED 480&nbsp;nm]||0.99||1.26||0.99||1.26<br />
|-<br />
! -!!Total F!!-!!-!! 46.06 !! 58.52 <br />
|- style="border-top: 2px solid #808080;;"<br />
| 1||[http://www.mouser.de/ProductDetail/Dialight/550-2505F/?qs=0KZIkTEbAAvqMAW7suDOXg== LED 600nm]||0.94||1.19||0.94||1.19 <br />
|-<br />
| 1||[http://www.leds.de/Low-Mid-Power-LEDs/SuperFlux-LEDs/Nichia-Superflux-LED-blau-3lm-100-NSPBR70BSS.html LED 480&nbsp;nm]||0.99||1.26||0.99||1.26<br />
|-<br />
| 1||cuvette holder (3D print service of your choice)||6.44||8.18||6.44||8.18<br />
|-<br />
! -!!Total OD/F!!-!!-!! 53.44 !! 67.89 <br />
|-<br />
|}<br />
</center><br />
<br />
For more detailed economical information about the OD/F project visit our [https://2014.igem.org/Team:Aachen/PolicyPractices/Economics Economical View] page.<br />
<br />
== Breadboards ==<br />
<br />
=== Optical Density ===<br />
<br />
{{Team:Aachen/Figure|Aachen ODdevice Steckplatine.png|align=center|title=Breadboard of our OD device|subtitle=To build your own OD device, connect the parts as shown in this diagram.|width=900px}}<br />
<br />
If you want to build our OD device, make sure to use the following secret ingredients:<br />
* Filter: [http://www.leefilters.com/lighting/colour-details.html#019 Fire 019]<br />
* LED: [http://www.mouser.de/ProductDetail/Dialight/550-2505F/?qs=0KZIkTEbAAvqMAW7suDOXg== 600 nm] Dialight 550-2505F<br />
<br />
=== Fluorescence ===<br />
<br />
{{Team:Aachen/Figure|Aachen_Fdevice_Steckplatine.png|align=center|title=Our novel biosensor approach|subtitle=Expression of the TEV protease is induced by HSL. The protease cleaves the GFP-REACh fusion protein to elecit a fluorescence response.|width=900px}}<br />
If you want to build the OD device, make sure to use the following secret ingredients:<br />
* Filter: [http://www.leefilters.com/lighting/colour-details.html#736 Twickenham Green 736]<br />
* LED: [http://www.leds.de/Low-Mid-Power-LEDs/SuperFlux-LEDs/Nichia-Superflux-LED-blau-3lm-100-NSPBR70BSS.html 480 nm] NSPBR70BSS LED<br />
<br />
=== Combined Device ===<br />
Even though evaluation of the measurements have been performed in two separate device, it is fairly well possible to put everything into one casing.<br />
All you need to do is choosing another lid, and connect a second light to frequency sensor to your Arduino.<br />
Right at the bottom we present you the differences in wiring things up.<br />
Building the combined device is straight forward and very similar to the single device. You will need a slightly larger connector, a different lid for your case, and some more cables. The corresponding fritzing-layout is presented below.<br />
<br />
{{Team:Aachen/Figure|Aachen_ODF_combined_Steckplatine.png|align=center|title=Our novel biosensor approach|subtitle=Expression of the TEV protease is induced by HSL. The protease cleaves the GFP-REACh fusion protein to elecit a fluorescence response.|width=900px}}<br />
<br />
== Construction Steps ==<br />
{| class="wikitable centered"<br />
|-<br />
| [[File:Aachen_ODF_9.JPG|300px]] || First we want to assemble the casing. Once you have all the cut parts, you can start to assemble them. For cutting, we really recommend using a laser cutter.<br />
|-<br />
| [[File:Aachen_ODF_8.JPG|300px]] || Attach the cuvette-holder holders such that the cuvette holder is placed directly under the opening hole.<br />
|-<br />
| [[File:Aachen_ODF_4.JPG|300px]] || Next build the lid of the device. At this stage you can already mount the button. We recommend to glue any parts.<br />
|-<br />
| [[File:Aachen_ODF_3.JPG|300px]] || Your lid finally should look like this.<br />
|-<br />
| [[File:Aachen_ODF_11.JPG|150px]][[File:Aachen_ODF_10.JPG|150px]] || Next we want to assemble the cuvette holders. On the side with the square hole attach the light-to-frequency sensor with glue. For the OD case place the orange LED opposite, or for fluorescence, the LED in the hole in the bottom. Make sure to close any remaining open hole! Please attach a piece of filter foil (approx. $7 \times 7 mm^2$) from the inside in front of the light to frequency converter. Forceps is highly recommended.<br />
|-<br />
| [[File:Aachen_ODF_12.JPG|300px]] || Your final assembly should then look like this. Now place the correct filter into the cuvette holder, directly in front of the sensor. Make sure that the filter does not degrade due to the glue!<br />
|-<br />
| [[File:Aachen_ODF_14.JPG|300px]] || As the case can be used for both, fluorescence and OD measurement, we use a combined plug. Just three header rows (7 pins, 9pins for combined) and connect them as we did.<br />
|-<br />
| [[File:Aachen_ODF_1.JPG|300px]] || Now we're doing the wiring. Connect the Arduino 5V and GND such that you have one 5V and one GND line on your breadboard.<br />
|-<br />
| [[File:Aachen_ODF_2.JPG|300px]] || Then connect the button to 5V on the one side, and to GND via a resistor on the other side. Connect this side also to port __ on your Arduino. This will sense the blank. Next connect the display to the Arduino and our connector. See the Fritzing diagram at the bottom for a detailed information.<br />
|-<br />
| [[File:Aachen_ODF_13.JPG|300px]] || Now put everything into the case and ...<br />
|-<br />
| [[File:Aachen_ODF_6.JPG|300px]] || ... also place the cuvette holder into the device. Attach the display to the device lid and close the casing.<br />
|-<br />
| [[File:Aachen_ODF_7.JPG|300px]] || Congratulations! You have finished constructing your own OD/F Device!<br />
|-<br />
| [[File:Aachen_zwei_Kuevetten.jpg|300px]] || ... or even the combined device!<br />
|}<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
== References ==<br />
Lawrence, J. V., & Maier, S. (1977). Correction for the inherent error in optical density readings. Applied and Environmental Microbiology, 33(2), 482–484. Available at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=170707&tool=pmcentrez&rendertype=abstract.<br />
<br />
{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/Notebook/Engineering/ODFTeam:Aachen/Notebook/Engineering/ODF2014-10-18T03:44:20Z<p>Nbailly: /* Pseudomonas putida */</p>
<hr />
<div>__NOTOC__<br />
{{Team:Aachen/Header}}<br />
{{Team:Aachen/Stylesheet}}<br />
= OD/F Device =<br />
<br />
On this page we present the technical details of our OD/F Device. You can skip to specific chapters by clicking on the panels below:<br />
<center><br />
<html><ul class="team-grid" style="width:1064px;"><br />
<!-- Overview --><br />
<br />
<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;"><br />
<a href="https://2014.igem.org/Team:Aachen/Notebook/Engineering/ODF#dev" style="color:black"><br />
<div class="team-item team-info" style="width:214px;height:214px;font-size: large;line-height:1.5em;"><br />
<br/><br><br />
<b>General Considerations</b><br />
<br/><br/><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/0/0f/Aachen_14-10-10_ODF_Button_ipo.png); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a><br />
</li><br />
<br />
<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;"><br />
<a href="https://2014.igem.org/Team:Aachen/Notebook/Engineering/ODF#od" style="color:black"><br />
<div class="team-item team-info" style="width:214px;height:214px;font-size: large;line-height:1.5em;"><br />
<br/><br/><br />
<b>OD Device</b><br />
<br/><br/><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/0/04/Aachen_Cuvette_button_v1_ipo.png); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a><br />
</li><br />
<br />
<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;"><br />
<a href="https://2014.igem.org/Team:Aachen/Notebook/Engineering/ODF#f" style="color:black"><br />
<div class="team-item team-info" style="width:214px;height:214px;font-size: large;line-height:1.5em;"><br />
<br/><br/><br />
<b>F Device</b><br />
<br/><br/><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/5/55/Aachen_17-10-14_Glowing_cuvette-ipo.png); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a><br />
</li><br />
<br />
<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;"><br />
<a href="https://2014.igem.org/Team:Aachen/Notebook/Engineering/ODF#diy" style="color:black"><br />
<div class="team-item team-info" style="width:214px;height:214px;font-size: large;line-height:1.5em;"><br />
<br/><br/><br />
<b>DIY</b><br />
<br/><br/><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/9/9e/Aachen_14-10-15_DIY_Cellocks_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a><br />
</li><br />
</ul><br />
</center><br />
</html><br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_14-10-10_ODF_Button_ipo.png|right|150px]]<br />
<br />
= General Considerations =<br />
<span class="anchor" id="dev"></span><br />
<br />
Building the OD/F Device has been an interesting task. While this device has been developed mainly by the IT division of our team, we got an immense support from the biologists suffering from color-blindness, yet eager to help selecting the best color filters for the LEDs.<br />
<br />
The measuring principle and guidelines for this project have already been presented in the [https://2014.igem.org/Team:Aachen/OD/F_device project] section.<br />
Here, details about selecting filters, code and a construction manual are presented.<br />
<br />
=== Cuvette Holder ===<br />
The essential part of this device is the '''cuvette holder'''. In short, we had to overcome a dilemma created by the need for an optimal height for the sensor:<br />
* A too low sensor position bears problems with sedimentation as well as light diffraction from the bottom of the cuvette.<br />
* The sensor has to be as close as possible to the bottom so that enough light shines through for the fluorescence measurement.<br />
<br />
As a compromise, we place the sensor at a height of 0.75&nbsp;cm. It is comparable to one of the standard heights (0.2&nbsp;cm, 0.8&nbsp;cm, 1.2&nbsp;cm) of OD meters. It is important to note that our device works with filling volumens of just 1&nbsp;mL, which in fact comes close to reality in the lab.<br />
<br />
The final cuvette holder design is rendered from a [https://2014.igem.org/Team:Aachen/Notebook/Engineering/Cuvette3D?action=raw stl-file] shown below:<br />
<br />
<html><br />
<center> <br />
<iframe src="https://2014.igem.org/Team:Aachen/Notebook/Engineering/Cuvette3D?action=render<br />
" width=500px height=500px frameBorder="0"></iframe><br />
<br/><br />
<b>Cuvette Holder developed for our OD/F Device.</b><br />
</center><br />
</html><br />
<br />
=== Light Filters ===<br />
'''Finding the right and optimal filters is a tough challenge'''.<br />
The main goal throughout our project has been to choose easily available parts which are also inexpensive. Thus choosing Schott glasses as filters could not be considered. Alternatively, filters used for illumination of theaters were found to be an ideal solution.<br />
<br />
We tested serveral filters and the optimal configuration of filters used is listed below.<br />
<br />
{| class="wikitable"<br />
! Mode<br />
! Fluorescence Protein<br />
! Filter Name<br />
! Filter<br />
! Peak Excitation<br />
! Peak Emission<br />
|-<br />
| Fluorescence<br />
| [http://parts.igem.org/Part:BBa_E0040 GFPmut3b]<br />
| [http://leefilters.com/lighting/colour-details.html#736 Twickenham Green]<br />
| [[File:Aachen_Filter_736.png|200px]]<br />
| 501nm<br />
| 511nm<br />
|-<br />
| Optical Density<br />
| --<br />
| [http://leefilters.com/lighting/colour-details.html#019 Fire]<br />
| [[File:Aachen_Filter_019.png|200px]]<br />
| 600nm<br />
| 600nm<br />
|}<br />
<br />
The fluorescence protein [http://parts.igem.org/Part:BBa_E0040 GFPmut3b] has a peak excitation at 501&nbsp;nm and a peak emission at 511&nbsp;nm - too close together for our low-cost filters to block the excitation light but transmit the emitted light. Thus, we chose to excite at around 485&nbsp;nm reduce false positive results below 500&nbsp;nm. However, no adequate filter for these settings could be found.<br />
Eventually, using the dark greenish [http://leefilters.com/lighting/colour-details.html#736 Twickenham Green] filter only little amounts of light shorter than 500&nbsp;nm gets through, reducing any bias from excitation illumination significantly. Unfortunately, the transmission rate of this filter is quite bad, 20% only, for the target emission wavelength of 511&nbsp;nm.<br />
<br />
== Linearity of the Hardware Light Sensor ==<br />
<span class="anchor" id="lin"></span><br />
It is crucial that the selected hardware is mapping reality into the digital world of our $\mu$-Controller.<br />
In order to sense reality our setup uses a light to frequency sensor, [https://www.sparkfun.com/datasheets/Sensors/Imaging/TSL235R-LF.pdf TSL235R-LF].<br />
The light to frequency sensor resembles the most to a photo transistor and thus is less sensible to temperature than a light dependant resistor.<br />
Additionally counting a frequency using interrupts seems to be easier and more accurate than using the analog to digital converter.<br />
<br />
Using a dilution series of purified [https://2011.igem.org/Team:Glasgow/LOV2 iLOV] we could determine the characteristic curve for the light sensor. Finally we can conclude that the sensor is linear as expected and shown in the [https://www.sparkfun.com/datasheets/Sensors/Imaging/TSL235R-LF.pdf datasheet].<br />
<br />
{{Team:Aachen/Figure|align=center|Aachen 15-10-14 Linearity iFG.PNG|title=Linearity of TSL235R-LF sensor|subtitle=Dilution series of GFP expressing ''E. coli'' showing linearity between fluorescence count and dilution. The linearity of the chosen sensor can be determined.|width=700px}}<br />
<br />
We are measuring optical density using our in-house developed cuvette holder.<br />
Particularly for optical density measurement, the amount of light shining through the sample is crucial.<br />
If there is too few light, there will be not enough light registered at the sensor, and the resolution of the measurement shrinks. This should be prevented.<br />
The chosen light to frequency sensor is reported to be very sensitive on the amount of light shining on it.<br />
There are reports of the sensor breaking when put into [https://www.sparkfun.com/products/9768 sunlight on a nice day], and not being sensitive at both high light or low light [http://kesslerarduino.wordpress.com/author/kevinmkessler/page/2/ conditions].<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
[[File:Aachen_Cuvette_button_v1_ipo.png|right|150px]]<br />
<br />
= Evaluation of the Optical Density Measurement =<br />
<span class="anchor" id="od"></span><br />
<br />
=== From Transmittance to True Optical Density ===<br />
At very low levels, uncorrected photometric determinations of cell densities show a decreasing proportionaility to actual cell density.<br />
<br />
This can also be observed using our device.<br />
<br />
In general, photometric determination of bacterial concentrations depends primarily on light scattering, rather than light absorption. Therefore, often not absorption is measured, but transmittance. For this, the relationship between optical density (OD) and transmitted light $\frac{I_0}{I}$ exists as:<br />
<br />
$$ OD = \frac{I_0}{I} = \kappa \cdot c$$<br />
<br />
where $I_0$ is the intensity of incoming light and $I$ the amount of the light passing through.<br />
<br />
However, this equation is linear only in a certain range.<br />
While one can tackle this non-linearity by using dilutions of the culture, correcting the error systematically is another way to overcome this limitation.<br />
<br />
For our OD device we needed to correlate the transmittance measured by our sensor to an optical density anyway.<br />
Our team members from the deterministic sciences emphasized on the correction method, which was conducted according to Lawrence and Maier (2006):<br />
<br />
* The relative density ($RD$) of each sample in a dilution series is calculated using $\frac{min(dilution)}{dilution}$.<br />
* The uncorrected optical density is derived from the transmission T [%]: $OD = 2 - \log T$<br />
* Finally, the unit optical density is calculated as $\frac{OD}{TD}$.<br />
* The average of the stable unit optical densities is used to calculate the true optical density $ OD_{unit} \cdot RD $.<br />
This way, the correlation between transmission and true optical density can be computed.<br />
The derived function allows the conversion from transmission to optical density on our device and therefore calibrates our device.<br />
<br />
<br />
In our experiments, we find in accordance to Lawrence and Maier that the correction majorly depends on the technical equipment used, especially the LED, sensor and cuvettes.<br />
While this at first sight looks disappointing, it is also expected:<br />
Transmittance is the fraction of light not absorbed by some medium relative to the cell-free and clear medium.<br />
However, the transmittance is not only dependent on the amount of cells in the way of the light's beam, but also how much light shines through the cuvette in which fashion, and in which fraction is received by the sensor in which angles.<br />
<br />
Using the above formula we performed this experiment for ''Pseudomonas putida'' and ''Saccharomyces cerevisiae'' and asked team [https://2014.igem.org/Team:Aachen/Collaborations/Freiburg Freiburg] to perform the same experiment using mamallian cells.<br />
<br />
=== Experiments ===<br />
<br />
We performed several experiments during the development of the device.<br />
Finally, we can relate the measured transmittance to the true Optical Density (true OD), and further, we can relate the true OD to the true OD of the photospectrometer in our lab.<br />
By doing so, we can calibrate our device to meaningful values.<br />
<br />
We have done this according to the previous section for ''Pseudomonas putida'' and ''Saccharomyces cerevisiae''.<br />
<br />
The final function for calculating the OD from the transmission calculated by our device can be calculated as<br />
<br />
$$ OD(T) = f(T) \circ g(device) $$<br />
<br />
where $f$ transforms transforms transmittance $T$ to true optical density for our device, and $g$ transforms true optical density of our device into the true optical density of the photospectrometer. This way our device is calibrated according to the photospectrometer.<br />
<br />
==== ''Pseudomonas putida'' ====<br />
<br />
{{Team:Aachen/Figure|align=center|Aachen 15-10-14 Pputida iFG.PNG|title=True Optical Density to Transmittance plot for ''P. putida''|subtitle=|width=700px}}<br />
</center><br />
<br />
<center><br />
{{Team:Aachen/Figure|align=center|Aachen 15-10-14 Pputida OD iFG.PNG|title=Corrolating the true optical density of the OD/F Device to true optical density of the photospectrometer for ''P. putida''|subtitle=|width=700px}}<br />
</center><br />
<br />
==== ''Saccharomyces cerevisiae'' ====<br />
<br />
<center><br />
{{Team:Aachen/Figure|align=center|Aachen 15-10-14 Scer iFG.PNG|title=True Optical Density to Transmittance plot for ''S. cerivisiae''|subtitle=|width=700px}}<br />
</center><br />
<br />
<center><br />
{{Team:Aachen/Figure|align=center|Aachen 15-10-14 Scer OD iFG.PNG|title=Corrolating the true optical density of the OD/F Device to true optical density of the photospectrometer for ''S. cerivisiae''|subtitle=|width=700px}}<br />
</center><br />
<br />
From these plots, it can be seen that our device delivers robust and reproducible results for both procaryotes and eucaryotes.<br />
Also the function from transmittance to true OD follows a clear pattern, making its calculation possible on a low-end device like a microcontroller.<br />
<br />
It is interesting to observer that function $g$, mapping the true OD of our device to the true OD of the photospectrometer, are close together for both ''P. putida'' and ''S. cerivisae'', as seen by the regression coefficient.<br />
In fact, 3.416 and 3.461 are such close together, that the minor deviation could be just measuring inaccuracy.<br />
Therefore, we fix the regression coefficient for converting true OD of our device to true OD of the photospectrometer to an average of 3.432 .<br />
<br />
Additionally the function $f$ for mapping transmittance to true OD for our device is similar for all cell types, as seen in the following figure. Therefore the exponential regression curve for all cell types specifies this function.<br />
<br />
<center><br />
{{Team:Aachen/Figure|Aachen_ODallstrains1.png|title=Transmission of different cell types at OD-values from 0.001-1|subtitle=The transmittance data of NIH&nbsp;3T3 cells (mouse fibroblasts) align with the transmittance of ''P.&nbsp;putida'' and ''S.&nbsp;cerevisiae'' strains, even though the measured optical densities are lower by 1-2 orders of magnitude.|width=800px}}<br />
</center><br />
Finally, we have empirically determined our $OD(T)$ function by finding $f$ and $g$, such that we can convert true OD to the optical density of the photospectrometer.<br />
<br />
By this evaluation, we have shown that our self-build OD/F Device can compete with commercial systems.<br />
It is easy to calibrate by just calculating the true optical density.<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_17-10-14_Glowing_cuvette-ipo.png|right|150px]]<br />
<br />
= Evaluation of the Fluorescence Measurement =<br />
<span class="anchor" id="f"></span><br />
<br />
For the evaluation of the fluorescence measurement, we performed a dilution series of a constitutively GFPmut3b expressing ''E. coli''.<br />
<br />
The below figure shows the absolute measurements for both the platereader and our OD/F Device. The abrupt jump at 50% concentration can be explained by a second dilution step and is prevalent in both devices.<br />
It can be seen that the platereader show a much higher difference between the GFP and non-GFP cell culture at a higher standard deviation.<br />
Another interesting metric is the difference between the GFP and non-GFP, which can be seen as the normalized fluorescence measure.<br />
<br />
<center><br />
{{Team:Aachen/Figure|align=center|Aachn 15-10-14 F platereader ODF iFG.PNG|title=Fluorescence Measurement comparison OD/F Device and Platereader (absolute values)|subtitle=Comparison of a fluorescence measurement of our device and the platereader. Our OD/F Device shows no significant linearity.|width=700px}}<br />
</center><br />
<br />
If one compares the results there, as in the below figure with normalized fluorescence values, interesting observations can be made.<br />
First, both platereader and OD/F Device show very similar results.<br />
The regression curves differ only in a linear factor.<br />
Most interestingly the general fit of the OD/F Device to a linear function seems to be better than the platereader.<br />
Overall the linearity which has been observed earlier (in testing the general setup) could be verified.<br />
Therefore our do-it-yourself OD/F Device can be used to determine fluorescence.<br />
<br />
<center><br />
{{Team:Aachen/Figure|align=center|Aachen 15-10-14 F platereader ODF2 iFG.PNG|title=Fluorescence Measurement comparison OD/F Device and platereader using normalized fluorescence values|subtitle=Comparison of a fluorescence measurement of our device and the platereader. Our OD/F Device shows no significant. A non-GFP expressing ''E. coli'' dilution has been used to normalize the GFP dilution series. A linear correlation can be seen.|width=700px}}<br />
</center><br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_14-10-15_DIY_Cellocks_iNB.png|right|150px]]<br />
<br />
= DIY: How to Build Your Own Device =<br />
<span class="anchor" id="diy"></span><br />
== Technical Components ==<br />
<br />
While the casing and the cuvette holder are custom made, most of the parts are pre-made and can be readily bought. The previous section lists all needed parts.<br />
To get all these parts for creating your own OD/F Device is easy using the internet. Thus all '''potential customers have a market access''' to the used components. <br />
<br />
Please find our custom parts for download below. Despite being custom parts, these are quite inenxpensive - so feel free to give our OD/F Device a test! For printing the 3D model of our cuvette holder and laser cutting our case, we want to express many thanks to [http://hci.rwth-aachen.de/fablab Fablab Aachen].<span class="anchor" id="ref2"></span><br />
<br />
* cuvette holder [https://2014.igem.org/Template:Team:Aachen/cuvette.stl?action=raw STL file]<br />
* casing single device [https://static.igem.org/mediawiki/2014/b/bb/Aachen_ODF_device_casing.svg.zip SVG file]<br />
* lid combined device [https://static.igem.org/mediawiki/2014/4/46/Aachen_Odf_device_combined_lid.svg.zip SVG file]<br />
* Arduino Code for single device [https://static.igem.org/mediawiki/2014/2/2f/Arduino_od_f_single.zip Sketchbook (.ino)]<br />
* Arduino Code for combined device [https://static.igem.org/mediawiki/2014/e/e8/Arduino_odf_combined.zip Sketchbook (.ino)]<br />
<br />
You will need a special [http://www.exp-tech.de/images/product_images/description%20images/YWRobot/1602/LiquidCrystal_I2C1602V2.rar library] for the display, which can not be uploaded for legal reasons.<br />
<br />
<br />
<center><br />
'''All needed components their quantities and prices for creating your own OD/F Device'''<br />
{| class="wikitable"<br />
! align="center" |'''OD/F Device'''<br />
!! align="center" | <br />
!! align="center" |''' 1€='''<br />
!! align="center" |''' $1.27'''<br />
!! align="center" |''' on 14/10/2014'''<br />
!! align="center" | <br />
|-<br />
! Quantity!!Component!! Costs [€] !! Costs [$] !! Final [€] !! Final [$] <br />
|-<br />
| 1||[http://www.dx.com/p/uno-r3-development-board-microcontroller-mega328p-atmega16u2-compat-for-arduino-blue-black-215600#.VDzwV9ysWBp Arduino UNO R3]||9.17||11.65||9.17||11.65 <br />
|-<br />
| 1||[http://www.mouser.de/ProductDetail/ams/TSL235R-LF/?qs=14HO7rLZPQsjmBHaoYCzkA%3D%3D TSL 235R]||2.47||3.14||2.47||3.14 <br />
|-<br />
| 1||[http://www.dx.com/p/16-x-2-character-lcd-display-module-with-blue-backlight-121356 Display 16x2]||2.58||3.28||2.58||3.28 <br />
|-<br />
| 1||[http://www.dx.com/p/lcd1602-adapter-board-w-iic-i2c-interface-black-works-with-official-arduino-boards-216865#.VDzxHNysWBp LCD Display to I2C]||1.57||1.99||1.57||1.99 <br />
|-<br />
| 1||[http://www.newark.com/multicomp/mcpas6b1m1ce3/switch-pushbutton-spst-400ma-125v/dp/12P7696?ost=1638329 Pushbutton]||2.90||3.69||2.90||3.69 <br />
|-<br />
| 1||[http://shop.leefiltersusa.com/Swatch-Book-Designers-Edition-SWB.htm filter leaflet]||1.57||2.00||1.57||2.00 <br />
|-<br />
| 20||[http://www.dx.com/p/diy-male-to-female-dupont-breadboard-jumper-wires-black-multi-color-40-pcs-10cm-339078#.VDzxSdysWBp jumper wire cables]||0.09||0.11||1.80||2.28 <br />
|-<br />
| 1||[http://www.dx.com/p/syb-170-mini-breadboard-for-diy-project-red-140101#.VDzyudysWBo small breadboard]||1.98||2.51||1.98||2.51 <br />
|-<br />
| 1||[http://www.dx.com/p/universal-ac-charger-w-dual-usb-output-for-iphone-ipad-ipod-white-us-plug-244893 power supply]||2.20||2.80||2.20||2.80<br />
|-<br />
| 1||cuvette holder (3D print service of your choice)||6.44||8.18||6.44||8.18<br />
|-<br />
| 1||3&nbsp;mm acrylic glas (black)||7.98||10.14||7.98||10.14<br />
|-<br />
| 1||[http://www.dx.com/p/prototype-universal-printed-circuit-board-breadboard-golden-10-piece-pack-143913 Prototype Universal Printed Circuit Board]||2.27||2.88||2.27||2.88<br />
|-<br />
| 1||[http://www.dx.com/p/2-54mm-1x40-pin-breakaway-straight-male-header-10-piece-pack-144191#.VEGRF2d_tGg Male Headers]||2.14||2.72||2.14||2.72<br />
|- style="border-top: 2px solid #808080;"<br />
| 1||[http://www.mouser.de/ProductDetail/Dialight/550-2505F/?qs=0KZIkTEbAAvqMAW7suDOXg== LED 600&nbsp;nm]||0.94||1.19||0.94||1.19<br />
|-<br />
! -!!Total OD!!-!!-!! 46.01 !! 58.45 <br />
|-<br />
| 1||[http://www.leds.de/Low-Mid-Power-LEDs/SuperFlux-LEDs/Nichia-Superflux-LED-blau-3lm-100-NSPBR70BSS.html LED 480&nbsp;nm]||0.99||1.26||0.99||1.26<br />
|-<br />
! -!!Total F!!-!!-!! 46.06 !! 58.52 <br />
|- style="border-top: 2px solid #808080;;"<br />
| 1||[http://www.mouser.de/ProductDetail/Dialight/550-2505F/?qs=0KZIkTEbAAvqMAW7suDOXg== LED 600nm]||0.94||1.19||0.94||1.19 <br />
|-<br />
| 1||[http://www.leds.de/Low-Mid-Power-LEDs/SuperFlux-LEDs/Nichia-Superflux-LED-blau-3lm-100-NSPBR70BSS.html LED 480&nbsp;nm]||0.99||1.26||0.99||1.26<br />
|-<br />
| 1||cuvette holder (3D print service of your choice)||6.44||8.18||6.44||8.18<br />
|-<br />
! -!!Total OD/F!!-!!-!! 53.44 !! 67.89 <br />
|-<br />
|}<br />
</center><br />
<br />
For more detailed economical information about the OD/F project visit our [https://2014.igem.org/Team:Aachen/PolicyPractices/Economics Economical View] page.<br />
<br />
== Breadboards ==<br />
<br />
=== Optical Density ===<br />
<br />
{{Team:Aachen/Figure|Aachen ODdevice Steckplatine.png|align=center|title=Breadboard of our OD device|subtitle=To build your own OD device, connect the parts as shown in this diagram.|width=900px}}<br />
<br />
If you want to build our OD device, make sure to use the following secret ingredients:<br />
* Filter: [http://www.leefilters.com/lighting/colour-details.html#019 Fire 019]<br />
* LED: [http://www.mouser.de/ProductDetail/Dialight/550-2505F/?qs=0KZIkTEbAAvqMAW7suDOXg== 600 nm] Dialight 550-2505F<br />
<br />
=== Fluorescence ===<br />
<br />
{{Team:Aachen/Figure|Aachen_Fdevice_Steckplatine.png|align=center|title=Our novel biosensor approach|subtitle=Expression of the TEV protease is induced by HSL. The protease cleaves the GFP-REACh fusion protein to elecit a fluorescence response.|width=900px}}<br />
If you want to build the OD device, make sure to use the following secret ingredients:<br />
* Filter: [http://www.leefilters.com/lighting/colour-details.html#736 Twickenham Green 736]<br />
* LED: [http://www.leds.de/Low-Mid-Power-LEDs/SuperFlux-LEDs/Nichia-Superflux-LED-blau-3lm-100-NSPBR70BSS.html 480 nm] NSPBR70BSS LED<br />
<br />
=== Combined Device ===<br />
Even though evaluation of the measurements have been performed in two separate device, it is fairly well possible to put everything into one casing.<br />
All you need to do is choosing another lid, and connect a second light to frequency sensor to your Arduino.<br />
Right at the bottom we present you the differences in wiring things up.<br />
Building the combined device is straight forward and very similar to the single device. You will need a slightly larger connector, a different lid for your case, and some more cables. The corresponding fritzing-layout is presented below.<br />
<br />
{{Team:Aachen/Figure|Aachen_ODF_combined_Steckplatine.png|align=center|title=Our novel biosensor approach|subtitle=Expression of the TEV protease is induced by HSL. The protease cleaves the GFP-REACh fusion protein to elecit a fluorescence response.|width=900px}}<br />
<br />
== Construction Steps ==<br />
{| class="wikitable centered"<br />
|-<br />
| [[File:Aachen_ODF_9.JPG|300px]] || First we want to assemble the casing. Once you have all the cut parts, you can start to assemble them. For cutting, we really recommend using a laser cutter.<br />
|-<br />
| [[File:Aachen_ODF_8.JPG|300px]] || Attach the cuvette-holder holders such that the cuvette holder is placed directly under the opening hole.<br />
|-<br />
| [[File:Aachen_ODF_4.JPG|300px]] || Next build the lid of the device. At this stage you can already mount the button. We recommend to glue any parts.<br />
|-<br />
| [[File:Aachen_ODF_3.JPG|300px]] || Your lid finally should look like this.<br />
|-<br />
| [[File:Aachen_ODF_11.JPG|150px]][[File:Aachen_ODF_10.JPG|150px]] || Next we want to assemble the cuvette holders. On the side with the square hole attach the light-to-frequency sensor with glue. For the OD case place the orange LED opposite, or for fluorescence, the LED in the hole in the bottom. Make sure to close any remaining open hole! Please attach a piece of filter foil (approx. $7 \times 7 mm^2$) from the inside in front of the light to frequency converter. Forceps is highly recommended.<br />
|-<br />
| [[File:Aachen_ODF_12.JPG|300px]] || Your final assembly should then look like this. Now place the correct filter into the cuvette holder, directly in front of the sensor. Make sure that the filter does not degrade due to the glue!<br />
|-<br />
| [[File:Aachen_ODF_14.JPG|300px]] || As the case can be used for both, fluorescence and OD measurement, we use a combined plug. Just three header rows (7 pins, 9pins for combined) and connect them as we did.<br />
|-<br />
| [[File:Aachen_ODF_1.JPG|300px]] || Now we're doing the wiring. Connect the Arduino 5V and GND such that you have one 5V and one GND line on your breadboard.<br />
|-<br />
| [[File:Aachen_ODF_2.JPG|300px]] || Then connect the button to 5V on the one side, and to GND via a resistor on the other side. Connect this side also to port __ on your Arduino. This will sense the blank. Next connect the display to the Arduino and our connector. See the Fritzing diagram at the bottom for a detailed information.<br />
|-<br />
| [[File:Aachen_ODF_13.JPG|300px]] || Now put everything into the case and ...<br />
|-<br />
| [[File:Aachen_ODF_6.JPG|300px]] || ... also place the cuvette holder into the device. Attach the display to the device lid and close the casing.<br />
|-<br />
| [[File:Aachen_ODF_7.JPG|300px]] || Congratulations! You have finished constructing your own OD/F Device!<br />
|-<br />
| [[File:Aachen_zwei_Kuevetten.jpg|300px]] || ... or even the combined device!<br />
|}<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
== References ==<br />
Lawrence, J. V., & Maier, S. (1977). Correction for the inherent error in optical density readings. Applied and Environmental Microbiology, 33(2), 482–484. Available at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=170707&tool=pmcentrez&rendertype=abstract.<br />
<br />
{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/Notebook/Engineering/ODFTeam:Aachen/Notebook/Engineering/ODF2014-10-18T03:43:55Z<p>Nbailly: /* Linearity of the Hardware Light Sensor */</p>
<hr />
<div>__NOTOC__<br />
{{Team:Aachen/Header}}<br />
{{Team:Aachen/Stylesheet}}<br />
= OD/F Device =<br />
<br />
On this page we present the technical details of our OD/F Device. You can skip to specific chapters by clicking on the panels below:<br />
<center><br />
<html><ul class="team-grid" style="width:1064px;"><br />
<!-- Overview --><br />
<br />
<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;"><br />
<a href="https://2014.igem.org/Team:Aachen/Notebook/Engineering/ODF#dev" style="color:black"><br />
<div class="team-item team-info" style="width:214px;height:214px;font-size: large;line-height:1.5em;"><br />
<br/><br><br />
<b>General Considerations</b><br />
<br/><br/><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/0/0f/Aachen_14-10-10_ODF_Button_ipo.png); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a><br />
</li><br />
<br />
<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;"><br />
<a href="https://2014.igem.org/Team:Aachen/Notebook/Engineering/ODF#od" style="color:black"><br />
<div class="team-item team-info" style="width:214px;height:214px;font-size: large;line-height:1.5em;"><br />
<br/><br/><br />
<b>OD Device</b><br />
<br/><br/><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/0/04/Aachen_Cuvette_button_v1_ipo.png); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a><br />
</li><br />
<br />
<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;"><br />
<a href="https://2014.igem.org/Team:Aachen/Notebook/Engineering/ODF#f" style="color:black"><br />
<div class="team-item team-info" style="width:214px;height:214px;font-size: large;line-height:1.5em;"><br />
<br/><br/><br />
<b>F Device</b><br />
<br/><br/><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/5/55/Aachen_17-10-14_Glowing_cuvette-ipo.png); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a><br />
</li><br />
<br />
<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;"><br />
<a href="https://2014.igem.org/Team:Aachen/Notebook/Engineering/ODF#diy" style="color:black"><br />
<div class="team-item team-info" style="width:214px;height:214px;font-size: large;line-height:1.5em;"><br />
<br/><br/><br />
<b>DIY</b><br />
<br/><br/><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/9/9e/Aachen_14-10-15_DIY_Cellocks_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a><br />
</li><br />
</ul><br />
</center><br />
</html><br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_14-10-10_ODF_Button_ipo.png|right|150px]]<br />
<br />
= General Considerations =<br />
<span class="anchor" id="dev"></span><br />
<br />
Building the OD/F Device has been an interesting task. While this device has been developed mainly by the IT division of our team, we got an immense support from the biologists suffering from color-blindness, yet eager to help selecting the best color filters for the LEDs.<br />
<br />
The measuring principle and guidelines for this project have already been presented in the [https://2014.igem.org/Team:Aachen/OD/F_device project] section.<br />
Here, details about selecting filters, code and a construction manual are presented.<br />
<br />
=== Cuvette Holder ===<br />
The essential part of this device is the '''cuvette holder'''. In short, we had to overcome a dilemma created by the need for an optimal height for the sensor:<br />
* A too low sensor position bears problems with sedimentation as well as light diffraction from the bottom of the cuvette.<br />
* The sensor has to be as close as possible to the bottom so that enough light shines through for the fluorescence measurement.<br />
<br />
As a compromise, we place the sensor at a height of 0.75&nbsp;cm. It is comparable to one of the standard heights (0.2&nbsp;cm, 0.8&nbsp;cm, 1.2&nbsp;cm) of OD meters. It is important to note that our device works with filling volumens of just 1&nbsp;mL, which in fact comes close to reality in the lab.<br />
<br />
The final cuvette holder design is rendered from a [https://2014.igem.org/Team:Aachen/Notebook/Engineering/Cuvette3D?action=raw stl-file] shown below:<br />
<br />
<html><br />
<center> <br />
<iframe src="https://2014.igem.org/Team:Aachen/Notebook/Engineering/Cuvette3D?action=render<br />
" width=500px height=500px frameBorder="0"></iframe><br />
<br/><br />
<b>Cuvette Holder developed for our OD/F Device.</b><br />
</center><br />
</html><br />
<br />
=== Light Filters ===<br />
'''Finding the right and optimal filters is a tough challenge'''.<br />
The main goal throughout our project has been to choose easily available parts which are also inexpensive. Thus choosing Schott glasses as filters could not be considered. Alternatively, filters used for illumination of theaters were found to be an ideal solution.<br />
<br />
We tested serveral filters and the optimal configuration of filters used is listed below.<br />
<br />
{| class="wikitable"<br />
! Mode<br />
! Fluorescence Protein<br />
! Filter Name<br />
! Filter<br />
! Peak Excitation<br />
! Peak Emission<br />
|-<br />
| Fluorescence<br />
| [http://parts.igem.org/Part:BBa_E0040 GFPmut3b]<br />
| [http://leefilters.com/lighting/colour-details.html#736 Twickenham Green]<br />
| [[File:Aachen_Filter_736.png|200px]]<br />
| 501nm<br />
| 511nm<br />
|-<br />
| Optical Density<br />
| --<br />
| [http://leefilters.com/lighting/colour-details.html#019 Fire]<br />
| [[File:Aachen_Filter_019.png|200px]]<br />
| 600nm<br />
| 600nm<br />
|}<br />
<br />
The fluorescence protein [http://parts.igem.org/Part:BBa_E0040 GFPmut3b] has a peak excitation at 501&nbsp;nm and a peak emission at 511&nbsp;nm - too close together for our low-cost filters to block the excitation light but transmit the emitted light. Thus, we chose to excite at around 485&nbsp;nm reduce false positive results below 500&nbsp;nm. However, no adequate filter for these settings could be found.<br />
Eventually, using the dark greenish [http://leefilters.com/lighting/colour-details.html#736 Twickenham Green] filter only little amounts of light shorter than 500&nbsp;nm gets through, reducing any bias from excitation illumination significantly. Unfortunately, the transmission rate of this filter is quite bad, 20% only, for the target emission wavelength of 511&nbsp;nm.<br />
<br />
== Linearity of the Hardware Light Sensor ==<br />
<span class="anchor" id="lin"></span><br />
It is crucial that the selected hardware is mapping reality into the digital world of our $\mu$-Controller.<br />
In order to sense reality our setup uses a light to frequency sensor, [https://www.sparkfun.com/datasheets/Sensors/Imaging/TSL235R-LF.pdf TSL235R-LF].<br />
The light to frequency sensor resembles the most to a photo transistor and thus is less sensible to temperature than a light dependant resistor.<br />
Additionally counting a frequency using interrupts seems to be easier and more accurate than using the analog to digital converter.<br />
<br />
Using a dilution series of purified [https://2011.igem.org/Team:Glasgow/LOV2 iLOV] we could determine the characteristic curve for the light sensor. Finally we can conclude that the sensor is linear as expected and shown in the [https://www.sparkfun.com/datasheets/Sensors/Imaging/TSL235R-LF.pdf datasheet].<br />
<br />
{{Team:Aachen/Figure|align=center|Aachen 15-10-14 Linearity iFG.PNG|title=Linearity of TSL235R-LF sensor|subtitle=Dilution series of GFP expressing ''E. coli'' showing linearity between fluorescence count and dilution. The linearity of the chosen sensor can be determined.|width=700px}}<br />
<br />
We are measuring optical density using our in-house developed cuvette holder.<br />
Particularly for optical density measurement, the amount of light shining through the sample is crucial.<br />
If there is too few light, there will be not enough light registered at the sensor, and the resolution of the measurement shrinks. This should be prevented.<br />
The chosen light to frequency sensor is reported to be very sensitive on the amount of light shining on it.<br />
There are reports of the sensor breaking when put into [https://www.sparkfun.com/products/9768 sunlight on a nice day], and not being sensitive at both high light or low light [http://kesslerarduino.wordpress.com/author/kevinmkessler/page/2/ conditions].<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
[[File:Aachen_Cuvette_button_v1_ipo.png|right|150px]]<br />
<br />
= Evaluation of the Optical Density Measurement =<br />
<span class="anchor" id="od"></span><br />
<br />
=== From Transmittance to True Optical Density ===<br />
At very low levels, uncorrected photometric determinations of cell densities show a decreasing proportionaility to actual cell density.<br />
<br />
This can also be observed using our device.<br />
<br />
In general, photometric determination of bacterial concentrations depends primarily on light scattering, rather than light absorption. Therefore, often not absorption is measured, but transmittance. For this, the relationship between optical density (OD) and transmitted light $\frac{I_0}{I}$ exists as:<br />
<br />
$$ OD = \frac{I_0}{I} = \kappa \cdot c$$<br />
<br />
where $I_0$ is the intensity of incoming light and $I$ the amount of the light passing through.<br />
<br />
However, this equation is linear only in a certain range.<br />
While one can tackle this non-linearity by using dilutions of the culture, correcting the error systematically is another way to overcome this limitation.<br />
<br />
For our OD device we needed to correlate the transmittance measured by our sensor to an optical density anyway.<br />
Our team members from the deterministic sciences emphasized on the correction method, which was conducted according to Lawrence and Maier (2006):<br />
<br />
* The relative density ($RD$) of each sample in a dilution series is calculated using $\frac{min(dilution)}{dilution}$.<br />
* The uncorrected optical density is derived from the transmission T [%]: $OD = 2 - \log T$<br />
* Finally, the unit optical density is calculated as $\frac{OD}{TD}$.<br />
* The average of the stable unit optical densities is used to calculate the true optical density $ OD_{unit} \cdot RD $.<br />
This way, the correlation between transmission and true optical density can be computed.<br />
The derived function allows the conversion from transmission to optical density on our device and therefore calibrates our device.<br />
<br />
<br />
In our experiments, we find in accordance to Lawrence and Maier that the correction majorly depends on the technical equipment used, especially the LED, sensor and cuvettes.<br />
While this at first sight looks disappointing, it is also expected:<br />
Transmittance is the fraction of light not absorbed by some medium relative to the cell-free and clear medium.<br />
However, the transmittance is not only dependent on the amount of cells in the way of the light's beam, but also how much light shines through the cuvette in which fashion, and in which fraction is received by the sensor in which angles.<br />
<br />
Using the above formula we performed this experiment for ''Pseudomonas putida'' and ''Saccharomyces cerevisiae'' and asked team [https://2014.igem.org/Team:Aachen/Collaborations/Freiburg Freiburg] to perform the same experiment using mamallian cells.<br />
<br />
=== Experiments ===<br />
<br />
We performed several experiments during the development of the device.<br />
Finally, we can relate the measured transmittance to the true Optical Density (true OD), and further, we can relate the true OD to the true OD of the photospectrometer in our lab.<br />
By doing so, we can calibrate our device to meaningful values.<br />
<br />
We have done this according to the previous section for ''Pseudomonas putida'' and ''Saccharomyces cerevisiae''.<br />
<br />
The final function for calculating the OD from the transmission calculated by our device can be calculated as<br />
<br />
$$ OD(T) = f(T) \circ g(device) $$<br />
<br />
where $f$ transforms transforms transmittance $T$ to true optical density for our device, and $g$ transforms true optical density of our device into the true optical density of the photospectrometer. This way our device is calibrated according to the photospectrometer.<br />
<br />
==== ''Pseudomonas putida'' ====<br />
<br />
<center><br />
{{Team:Aachen/Figure|align=center|Aachen 15-10-14 Pputida iFG.PNG|title=True Optical Density to Transmittance plot for ''P. putida''|subtitle=|width=700px}}<br />
</center><br />
<br />
<center><br />
{{Team:Aachen/Figure|align=center|Aachen 15-10-14 Pputida OD iFG.PNG|title=Corrolating the true optical density of the OD/F Device to true optical density of the photospectrometer for ''P. putida''|subtitle=|width=700px}}<br />
</center><br />
<br />
==== ''Saccharomyces cerevisiae'' ====<br />
<br />
<center><br />
{{Team:Aachen/Figure|align=center|Aachen 15-10-14 Scer iFG.PNG|title=True Optical Density to Transmittance plot for ''S. cerivisiae''|subtitle=|width=700px}}<br />
</center><br />
<br />
<center><br />
{{Team:Aachen/Figure|align=center|Aachen 15-10-14 Scer OD iFG.PNG|title=Corrolating the true optical density of the OD/F Device to true optical density of the photospectrometer for ''S. cerivisiae''|subtitle=|width=700px}}<br />
</center><br />
<br />
From these plots, it can be seen that our device delivers robust and reproducible results for both procaryotes and eucaryotes.<br />
Also the function from transmittance to true OD follows a clear pattern, making its calculation possible on a low-end device like a microcontroller.<br />
<br />
It is interesting to observer that function $g$, mapping the true OD of our device to the true OD of the photospectrometer, are close together for both ''P. putida'' and ''S. cerivisae'', as seen by the regression coefficient.<br />
In fact, 3.416 and 3.461 are such close together, that the minor deviation could be just measuring inaccuracy.<br />
Therefore, we fix the regression coefficient for converting true OD of our device to true OD of the photospectrometer to an average of 3.432 .<br />
<br />
Additionally the function $f$ for mapping transmittance to true OD for our device is similar for all cell types, as seen in the following figure. Therefore the exponential regression curve for all cell types specifies this function.<br />
<br />
<center><br />
{{Team:Aachen/Figure|Aachen_ODallstrains1.png|title=Transmission of different cell types at OD-values from 0.001-1|subtitle=The transmittance data of NIH&nbsp;3T3 cells (mouse fibroblasts) align with the transmittance of ''P.&nbsp;putida'' and ''S.&nbsp;cerevisiae'' strains, even though the measured optical densities are lower by 1-2 orders of magnitude.|width=800px}}<br />
</center><br />
Finally, we have empirically determined our $OD(T)$ function by finding $f$ and $g$, such that we can convert true OD to the optical density of the photospectrometer.<br />
<br />
By this evaluation, we have shown that our self-build OD/F Device can compete with commercial systems.<br />
It is easy to calibrate by just calculating the true optical density.<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_17-10-14_Glowing_cuvette-ipo.png|right|150px]]<br />
<br />
= Evaluation of the Fluorescence Measurement =<br />
<span class="anchor" id="f"></span><br />
<br />
For the evaluation of the fluorescence measurement, we performed a dilution series of a constitutively GFPmut3b expressing ''E. coli''.<br />
<br />
The below figure shows the absolute measurements for both the platereader and our OD/F Device. The abrupt jump at 50% concentration can be explained by a second dilution step and is prevalent in both devices.<br />
It can be seen that the platereader show a much higher difference between the GFP and non-GFP cell culture at a higher standard deviation.<br />
Another interesting metric is the difference between the GFP and non-GFP, which can be seen as the normalized fluorescence measure.<br />
<br />
<center><br />
{{Team:Aachen/Figure|align=center|Aachn 15-10-14 F platereader ODF iFG.PNG|title=Fluorescence Measurement comparison OD/F Device and Platereader (absolute values)|subtitle=Comparison of a fluorescence measurement of our device and the platereader. Our OD/F Device shows no significant linearity.|width=700px}}<br />
</center><br />
<br />
If one compares the results there, as in the below figure with normalized fluorescence values, interesting observations can be made.<br />
First, both platereader and OD/F Device show very similar results.<br />
The regression curves differ only in a linear factor.<br />
Most interestingly the general fit of the OD/F Device to a linear function seems to be better than the platereader.<br />
Overall the linearity which has been observed earlier (in testing the general setup) could be verified.<br />
Therefore our do-it-yourself OD/F Device can be used to determine fluorescence.<br />
<br />
<center><br />
{{Team:Aachen/Figure|align=center|Aachen 15-10-14 F platereader ODF2 iFG.PNG|title=Fluorescence Measurement comparison OD/F Device and platereader using normalized fluorescence values|subtitle=Comparison of a fluorescence measurement of our device and the platereader. Our OD/F Device shows no significant. A non-GFP expressing ''E. coli'' dilution has been used to normalize the GFP dilution series. A linear correlation can be seen.|width=700px}}<br />
</center><br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_14-10-15_DIY_Cellocks_iNB.png|right|150px]]<br />
<br />
= DIY: How to Build Your Own Device =<br />
<span class="anchor" id="diy"></span><br />
== Technical Components ==<br />
<br />
While the casing and the cuvette holder are custom made, most of the parts are pre-made and can be readily bought. The previous section lists all needed parts.<br />
To get all these parts for creating your own OD/F Device is easy using the internet. Thus all '''potential customers have a market access''' to the used components. <br />
<br />
Please find our custom parts for download below. Despite being custom parts, these are quite inenxpensive - so feel free to give our OD/F Device a test! For printing the 3D model of our cuvette holder and laser cutting our case, we want to express many thanks to [http://hci.rwth-aachen.de/fablab Fablab Aachen].<span class="anchor" id="ref2"></span><br />
<br />
* cuvette holder [https://2014.igem.org/Template:Team:Aachen/cuvette.stl?action=raw STL file]<br />
* casing single device [https://static.igem.org/mediawiki/2014/b/bb/Aachen_ODF_device_casing.svg.zip SVG file]<br />
* lid combined device [https://static.igem.org/mediawiki/2014/4/46/Aachen_Odf_device_combined_lid.svg.zip SVG file]<br />
* Arduino Code for single device [https://static.igem.org/mediawiki/2014/2/2f/Arduino_od_f_single.zip Sketchbook (.ino)]<br />
* Arduino Code for combined device [https://static.igem.org/mediawiki/2014/e/e8/Arduino_odf_combined.zip Sketchbook (.ino)]<br />
<br />
You will need a special [http://www.exp-tech.de/images/product_images/description%20images/YWRobot/1602/LiquidCrystal_I2C1602V2.rar library] for the display, which can not be uploaded for legal reasons.<br />
<br />
<br />
<center><br />
'''All needed components their quantities and prices for creating your own OD/F Device'''<br />
{| class="wikitable"<br />
! align="center" |'''OD/F Device'''<br />
!! align="center" | <br />
!! align="center" |''' 1€='''<br />
!! align="center" |''' $1.27'''<br />
!! align="center" |''' on 14/10/2014'''<br />
!! align="center" | <br />
|-<br />
! Quantity!!Component!! Costs [€] !! Costs [$] !! Final [€] !! Final [$] <br />
|-<br />
| 1||[http://www.dx.com/p/uno-r3-development-board-microcontroller-mega328p-atmega16u2-compat-for-arduino-blue-black-215600#.VDzwV9ysWBp Arduino UNO R3]||9.17||11.65||9.17||11.65 <br />
|-<br />
| 1||[http://www.mouser.de/ProductDetail/ams/TSL235R-LF/?qs=14HO7rLZPQsjmBHaoYCzkA%3D%3D TSL 235R]||2.47||3.14||2.47||3.14 <br />
|-<br />
| 1||[http://www.dx.com/p/16-x-2-character-lcd-display-module-with-blue-backlight-121356 Display 16x2]||2.58||3.28||2.58||3.28 <br />
|-<br />
| 1||[http://www.dx.com/p/lcd1602-adapter-board-w-iic-i2c-interface-black-works-with-official-arduino-boards-216865#.VDzxHNysWBp LCD Display to I2C]||1.57||1.99||1.57||1.99 <br />
|-<br />
| 1||[http://www.newark.com/multicomp/mcpas6b1m1ce3/switch-pushbutton-spst-400ma-125v/dp/12P7696?ost=1638329 Pushbutton]||2.90||3.69||2.90||3.69 <br />
|-<br />
| 1||[http://shop.leefiltersusa.com/Swatch-Book-Designers-Edition-SWB.htm filter leaflet]||1.57||2.00||1.57||2.00 <br />
|-<br />
| 20||[http://www.dx.com/p/diy-male-to-female-dupont-breadboard-jumper-wires-black-multi-color-40-pcs-10cm-339078#.VDzxSdysWBp jumper wire cables]||0.09||0.11||1.80||2.28 <br />
|-<br />
| 1||[http://www.dx.com/p/syb-170-mini-breadboard-for-diy-project-red-140101#.VDzyudysWBo small breadboard]||1.98||2.51||1.98||2.51 <br />
|-<br />
| 1||[http://www.dx.com/p/universal-ac-charger-w-dual-usb-output-for-iphone-ipad-ipod-white-us-plug-244893 power supply]||2.20||2.80||2.20||2.80<br />
|-<br />
| 1||cuvette holder (3D print service of your choice)||6.44||8.18||6.44||8.18<br />
|-<br />
| 1||3&nbsp;mm acrylic glas (black)||7.98||10.14||7.98||10.14<br />
|-<br />
| 1||[http://www.dx.com/p/prototype-universal-printed-circuit-board-breadboard-golden-10-piece-pack-143913 Prototype Universal Printed Circuit Board]||2.27||2.88||2.27||2.88<br />
|-<br />
| 1||[http://www.dx.com/p/2-54mm-1x40-pin-breakaway-straight-male-header-10-piece-pack-144191#.VEGRF2d_tGg Male Headers]||2.14||2.72||2.14||2.72<br />
|- style="border-top: 2px solid #808080;"<br />
| 1||[http://www.mouser.de/ProductDetail/Dialight/550-2505F/?qs=0KZIkTEbAAvqMAW7suDOXg== LED 600&nbsp;nm]||0.94||1.19||0.94||1.19<br />
|-<br />
! -!!Total OD!!-!!-!! 46.01 !! 58.45 <br />
|-<br />
| 1||[http://www.leds.de/Low-Mid-Power-LEDs/SuperFlux-LEDs/Nichia-Superflux-LED-blau-3lm-100-NSPBR70BSS.html LED 480&nbsp;nm]||0.99||1.26||0.99||1.26<br />
|-<br />
! -!!Total F!!-!!-!! 46.06 !! 58.52 <br />
|- style="border-top: 2px solid #808080;;"<br />
| 1||[http://www.mouser.de/ProductDetail/Dialight/550-2505F/?qs=0KZIkTEbAAvqMAW7suDOXg== LED 600nm]||0.94||1.19||0.94||1.19 <br />
|-<br />
| 1||[http://www.leds.de/Low-Mid-Power-LEDs/SuperFlux-LEDs/Nichia-Superflux-LED-blau-3lm-100-NSPBR70BSS.html LED 480&nbsp;nm]||0.99||1.26||0.99||1.26<br />
|-<br />
| 1||cuvette holder (3D print service of your choice)||6.44||8.18||6.44||8.18<br />
|-<br />
! -!!Total OD/F!!-!!-!! 53.44 !! 67.89 <br />
|-<br />
|}<br />
</center><br />
<br />
For more detailed economical information about the OD/F project visit our [https://2014.igem.org/Team:Aachen/PolicyPractices/Economics Economical View] page.<br />
<br />
== Breadboards ==<br />
<br />
=== Optical Density ===<br />
<br />
{{Team:Aachen/Figure|Aachen ODdevice Steckplatine.png|align=center|title=Breadboard of our OD device|subtitle=To build your own OD device, connect the parts as shown in this diagram.|width=900px}}<br />
<br />
If you want to build our OD device, make sure to use the following secret ingredients:<br />
* Filter: [http://www.leefilters.com/lighting/colour-details.html#019 Fire 019]<br />
* LED: [http://www.mouser.de/ProductDetail/Dialight/550-2505F/?qs=0KZIkTEbAAvqMAW7suDOXg== 600 nm] Dialight 550-2505F<br />
<br />
=== Fluorescence ===<br />
<br />
{{Team:Aachen/Figure|Aachen_Fdevice_Steckplatine.png|align=center|title=Our novel biosensor approach|subtitle=Expression of the TEV protease is induced by HSL. The protease cleaves the GFP-REACh fusion protein to elecit a fluorescence response.|width=900px}}<br />
If you want to build the OD device, make sure to use the following secret ingredients:<br />
* Filter: [http://www.leefilters.com/lighting/colour-details.html#736 Twickenham Green 736]<br />
* LED: [http://www.leds.de/Low-Mid-Power-LEDs/SuperFlux-LEDs/Nichia-Superflux-LED-blau-3lm-100-NSPBR70BSS.html 480 nm] NSPBR70BSS LED<br />
<br />
=== Combined Device ===<br />
Even though evaluation of the measurements have been performed in two separate device, it is fairly well possible to put everything into one casing.<br />
All you need to do is choosing another lid, and connect a second light to frequency sensor to your Arduino.<br />
Right at the bottom we present you the differences in wiring things up.<br />
Building the combined device is straight forward and very similar to the single device. You will need a slightly larger connector, a different lid for your case, and some more cables. The corresponding fritzing-layout is presented below.<br />
<br />
{{Team:Aachen/Figure|Aachen_ODF_combined_Steckplatine.png|align=center|title=Our novel biosensor approach|subtitle=Expression of the TEV protease is induced by HSL. The protease cleaves the GFP-REACh fusion protein to elecit a fluorescence response.|width=900px}}<br />
<br />
== Construction Steps ==<br />
{| class="wikitable centered"<br />
|-<br />
| [[File:Aachen_ODF_9.JPG|300px]] || First we want to assemble the casing. Once you have all the cut parts, you can start to assemble them. For cutting, we really recommend using a laser cutter.<br />
|-<br />
| [[File:Aachen_ODF_8.JPG|300px]] || Attach the cuvette-holder holders such that the cuvette holder is placed directly under the opening hole.<br />
|-<br />
| [[File:Aachen_ODF_4.JPG|300px]] || Next build the lid of the device. At this stage you can already mount the button. We recommend to glue any parts.<br />
|-<br />
| [[File:Aachen_ODF_3.JPG|300px]] || Your lid finally should look like this.<br />
|-<br />
| [[File:Aachen_ODF_11.JPG|150px]][[File:Aachen_ODF_10.JPG|150px]] || Next we want to assemble the cuvette holders. On the side with the square hole attach the light-to-frequency sensor with glue. For the OD case place the orange LED opposite, or for fluorescence, the LED in the hole in the bottom. Make sure to close any remaining open hole! Please attach a piece of filter foil (approx. $7 \times 7 mm^2$) from the inside in front of the light to frequency converter. Forceps is highly recommended.<br />
|-<br />
| [[File:Aachen_ODF_12.JPG|300px]] || Your final assembly should then look like this. Now place the correct filter into the cuvette holder, directly in front of the sensor. Make sure that the filter does not degrade due to the glue!<br />
|-<br />
| [[File:Aachen_ODF_14.JPG|300px]] || As the case can be used for both, fluorescence and OD measurement, we use a combined plug. Just three header rows (7 pins, 9pins for combined) and connect them as we did.<br />
|-<br />
| [[File:Aachen_ODF_1.JPG|300px]] || Now we're doing the wiring. Connect the Arduino 5V and GND such that you have one 5V and one GND line on your breadboard.<br />
|-<br />
| [[File:Aachen_ODF_2.JPG|300px]] || Then connect the button to 5V on the one side, and to GND via a resistor on the other side. Connect this side also to port __ on your Arduino. This will sense the blank. Next connect the display to the Arduino and our connector. See the Fritzing diagram at the bottom for a detailed information.<br />
|-<br />
| [[File:Aachen_ODF_13.JPG|300px]] || Now put everything into the case and ...<br />
|-<br />
| [[File:Aachen_ODF_6.JPG|300px]] || ... also place the cuvette holder into the device. Attach the display to the device lid and close the casing.<br />
|-<br />
| [[File:Aachen_ODF_7.JPG|300px]] || Congratulations! You have finished constructing your own OD/F Device!<br />
|-<br />
| [[File:Aachen_zwei_Kuevetten.jpg|300px]] || ... or even the combined device!<br />
|}<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
== References ==<br />
Lawrence, J. V., & Maier, S. (1977). Correction for the inherent error in optical density readings. Applied and Environmental Microbiology, 33(2), 482–484. Available at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=170707&tool=pmcentrez&rendertype=abstract.<br />
<br />
{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/Notebook/Engineering/WatsOnTeam:Aachen/Notebook/Engineering/WatsOn2014-10-18T03:43:34Z<p>Nbailly: /* Troubleshooting & Useful Links */</p>
<hr />
<div>__NOTOC__<br />
{{CSS/Main}}<br />
{{Team:Aachen/Stylesheet}}<br />
{{Team:Aachen/Header}}<br />
<br />
= ''WatsOn'' =<br />
<br />
This page contains technical details and construction manuals for our measurement device ''WatsOn'' as well as information on the software controlling the hardware. For more details, please click on the respective tile. For the image analysis software, please visit our [https://2014.igem.org/Team:Aachen/Notebook/Software/Measurarty ''Measurarty''] page.<br />
<br />
<html><br />
<center><br />
<ul class="team-grid" style="width:inherit;"><br />
<!-- Overview --><br />
<br />
<li style="margin-right:20px;margin-left:20px;"><br />
<a href="https://2014.igem.org/Team:Aachen/Notebook/Engineering/WatsOn#watsonhardware" style="color:black"><br />
<div class="team-item team-info" ><br />
<div class="menukachel">Hardware</div><br />
<!-- <br />
<br/><br/><br />
<b>Hardware</b><br />
<br/><br/><br />
click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/5/59/Aachen_14-10-16_Hardware_button_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%"> </div></a><br />
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<li style="margin-right:20px;margin-left:20px;"><br />
<a href="https://2014.igem.org/Team:Aachen/Notebook/Engineering/WatsOn#watsonsoftware" style="color:black"><br />
<div class="team-item team-info" ><br />
<div class="menukachel">Software</div><br />
<!-- <br/><br><br />
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click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/1/13/Aachen_14-10-16_Software_button_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%"> </div></a><br />
</li><br />
<br />
<li style="margin-right:20px;margin-left:20px;"><br />
<a href="https://2014.igem.org/Team:Aachen/Notebook/Engineering/WatsOn#watsondiy" style="color:black"><br />
<div class="team-item team-info" ><br />
<div class="menukachel">DIY</div><br />
<!-- <br/><br/><br />
<b>DIY</b><br />
<br/><br/><br />
click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/9/9e/Aachen_14-10-15_DIY_Cellocks_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%"> </div></a><br />
</li><br />
<br />
</ul><br />
</center><br />
</html><br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
= Hardware =<br />
<span class="anchor" id="watsonhardware"></span><br />
<br />
{{Team:Aachen/FigureFloat|Aachen_Device_11.jpg|title=''WatsOn''|subtitle= |width=200px}}<br />
<br />
Our hardware consists of the casing and the electronical components. The casing which can be seen on the left was built from laser cut acrylic glass. A detailed description of the assembly is described in the section [https://2014.igem.org/Team:Aachen/Notebook/Engineering/WatsOn#watsondiy Build your own ''WatsOn].<br />
<br />
The connection between the different electronical elements is visualized below.<br />
<br />
{{Team:Aachen/Figure|Aachen_Device_Hardware_Graphics.png|title=Interaction of electronical components||width=750px}}<br />
<br />
* '''Raspberry Pi''' : The [http://www.raspberrypi.org/ Raspberry Pi] is a small single-board computer which runs a Linux operating system from an inserted SD card. The steps which are required to set up a fully working system are described in the [https://2014.igem.org/Team:Aachen/Notebook/Engineering/WatsOn#pisetup DIY section] of this page. The main purpose of the Raspberry Pi is to run the software described above, to control the attached camera and to show the GUI on the display. The big advantage of this board is that it is very powerful, cheap and therefore perfectly fit for our needs.<br />
<br />
* '''Raspberry Pi camera''': The camera is directly connected to the Raspberry Pi board and takes the images of the chips.<br />
<br />
* '''Arduino''': The [http://www.arduino.cc/ Arduino] board is also a single-board computer with less computing power than the Raspberry Pi but with a greater focus on controlling electronical components. Therefore, it is used to control the LEDs and the Peltier heater.<br />
<br />
* '''Relay''': The 2-channel relay works like two light switches which are either turned on or off. They control the 450&nbsp;nm and 480&nbsp;nm LEDs. The channels are connected and turned on and off by the Arduino board.<br />
<br />
* '''Peltier element''': A Peltier component transforms an applied power into a temperature gradient which leads to a hot surface on one side of the element and a cooler one on the other side. The Peltier element connected to the aluminum block heats up the interior of the device to incubate the sensing cells at 37°C.<br />
<br />
* '''USB WiFi stick''': The USB WiFi stick connects the Raspberry Pi to a local network.<br />
<br />
* '''Display''': A 8-digit display is connected to the Arduino board and shows the current interior temperature<br />
<br />
{{Team:Aachen/FigureFloatRight|Aachen_Filter_010.png|title=010|subtitle=|width=70px}}<br />
{{Team:Aachen/FigureFloatRight|Aachen_Filter_505.png|title=505|subtitle=|width=70px}}<br />
<br />
* '''Filter slides''': To block the undesired wavelenghts emitted from the LEDs a filter slide is placed in front of the camera. This step is taken to get a clear fluorescence signal from the chips. The characteristic of the filter slide is selected depending on the frequency of the LEDs which are either 450&nbsp;nm or 480&nbsp;nm ones. We used '505 Sally Green' for the 450&nbsp;nm and '010 Medium Yellow' for the 480&nbsp;nm LEDs. The filters are shown on the right.<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
= Software =<br />
<span class="anchor" id="watsonsoftware"></span><br />
<br />
The software consists of several parts which provide an user interface and manage the connection to the hardware.<br />
<br />
===GUI (Graphical User Interface)===<br />
On the graphical interface, the user can take images and time lapses of the chips inside the device. The software is written in C++. It makes use of the [http://qt-project.org/ Qt-Library] to provide a clear interface and a comfortable way to manage various software aspects such as handling images and establishing network connections. An advantage resulting from the utilization of Qt-Library is the multi-platform support for Windows, MacOS and Linux. Additionally, Qt is available with an Open Source license which can be used for free. The software can be used locally on the Raspberry Pi or remotely from a device in the same network.<br />
<br />
The scheme below shows the different components of the software:<br />
<br />
[[File:Aachen_Device_GUI.png|center|800px]]<br />
<br />
Features of the GUI include:<br />
* Change settings [1]:<br />
** The user can specify the iso-value and the shutter speed of the camera.<br />
** Custom settings can be labeled and saved for future reference.<br />
** Existing settings can be updated or deleted unless they are default configurations.<br />
* Take image/s [2]: <br />
** The excitation wavelength of GFP (480&nbsp;nm) and iLOV (450&nbsp;nm) can be selected.<br />
** The GUI offers two possibilities to take images:<br />
*** Take a single image with the active camera settings.<br />
*** Take time lapse shootings with the active camera settings and the specified interval. When activated, the images are saved automatically to a user defined directory with ascending filenames.<br />
** The last image which was taken by the camera is shown in the GUI, information containing the time stamp and used camera settings are displayed next to the image [3]. Previous images can be selected with the arrow buttons.<br />
* Analyze image [4]:<br />
** The image is analyzed by an image segmentation algorithm and shows whether the pathogen ''Pseudomonas&nbsp;aeruginosa'' is present on the chip or not<br />
<br />
''Download the GUI sourcecode:'' [https://static.igem.org/mediawiki/2014/9/90/Aachen_WatsOn_GUI.zip Download]<br />
<br />
===Backend===<br />
The backend is a software that runs on the Raspberry Pi and is responsible for the connection between the GUI and the hardware. If the user interface is executed on another device, e.g. a notebook, it has to be in the same network as the Raspberry Pi. The backend works like a web server that receives commands and acts according to the submitted parameters. It can take images and returns them to the GUI.<br />
<br />
Before an image is taken, the backend turns on the specified LEDs by sending a command to the connected Arduino board. Subsequently, the LEDs are turned off using the same mechanism. These steps are repeated in the given interval for a time lapse shooting.<br />
<br />
''Download the backend sourcecode:'' [https://static.igem.org/mediawiki/2014/7/77/Aachen_Device_Backend.zip Download]<br />
<html><br></html><br />
<br />
{{Team:Aachen/Figure|Aachen_Device_SoftwareBackend.png|title=Sample connection between GUI and backend for taking an image|subtitle= |width=900px}}<br />
<br />
===Arduino===<br />
The software on the Arduino board sets the power and thus controls the temperature of the Peltier heater. The power is set by evaluating the received values from the temperature sensors for the interior of the device and the aluminum block. Additionally, the Arduino receives commands from the Raspberry Pi to turn the LEDs on and off.<br />
<br />
''Download the Arduino sourcecode:'' [https://static.igem.org/mediawiki/2014/c/cd/Aachen_WatsOn_arduino.zip Download] <br />
<br />
===Measurarty===<br />
We have developed our own image analysis pipeline ''Measurarty''. Please go to the [https://2014.igem.org/Team:Aachen/Notebook/Software/Measurarty ''Measurarty''] project page for further information.<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_14-10-15_DIY_Cellocks_iNB.png|right|150px]]<br />
<br />
= DIY: How To Build Your Own ''WatsOn'' =<br />
<span class="anchor" id="watsondiy"></span><br />
<br />
==Technical Components==<br />
If you want to create your own ''WatsOn'' first take a look at the following list of necessary components. All parts except the laser cut acrylic glass can be readily purchased and do not require further adjustments.<br />
<br />
''Download the laser cutting plan here: [https://static.igem.org/mediawiki/2014/f/fd/Aachen_WatsOn_laser_cut.svg.zip Download] (for acrylic glass with a height of 6&nbsp;mm)<br />
<br />
'''All needed components, their quantities and prices for creating your own ''WatsOn'''''<br />
{| class="wikitable sortable"<br />
! align="center" |'''''WatsOn'''''<br />
!! align="center" | <br />
!! align="center" |''' 1€='''<br />
!! align="center" |''' $1.27'''<br />
!! align="center" |''' on 14/10/2014'''<br />
!! align="center" | <br />
|- class="unsortable"<br />
!Quantity !! Component !! Costs [€]!! Costs [$]!! Final [€]!! Final [$]<br />
|-<br />
| 1|| [http://www.prolighting.de/Zubehoer/Farbfilter/Lee-Filter_HT/Lee-Filters_Musterheft_Designer_Edition_i174_3965_0.htm filter slides] (medium yellow 010, sally green 505)||1.57||2.00||1.57||2.00<br />
|-<br />
| 1|| [http://www.dx.com/p/uno-r3-development-board-microcontroller-mega328p-atmega16u2-compat-for-arduino-blue-black-215600 Arduino UNO R3]||9.17||11.65||9.17||11.65<br />
|-<br />
| 1|| [http://www.dx.com/p/arduino-2-channel-relay-shield-module-red-144140 2-channel relay shield]||2.72||3.46||2.72||3.46<br />
|-<br />
| 40||jumper-wire cable||2.35||2.99||2.35||2.99<br />
|-<br />
| 1|| [http://www.dx.com/p/2-54mm-1x40-pin-breakaway-straight-male-header-10-piece-pack-144191 40er male header (10-Piece Pack)]||2.14||2.72||2.14||2.72<br />
|-<br />
| 1|| [http://www.dx.com/p/jtron-2-54mm-40-pin-single-row-seat-single-row-female-header-black-10-pcs-278953 40er female header (10-Piece Pack)]||2.05||2.60||2.05||2.60<br />
|-<br />
| 1|| [http://www.dx.com/p/prototype-universal-printed-circuit-board-breadboard-brown-5-piece-pack-130926 circuit board]||2.35||2.99||2.35||2.99<br />
|-<br />
| 1|| [http://www.newark.com/pro-signal/rp006/audio-video-cable-hdmi-1m-black/dp/96T7446 HDMI cable]||1.47||1.87||1.47||1.87<br />
|-<br />
| 1|| [http://www.dx.com/p/hd-053-high-speed-usb-2-0-7-port-hub-black-174817 7 port USB hub]||5.28||6.71||5.28||6.71<br />
|-<br />
| 1||[http://www.dx.com/p/dx-original-ultra-mini-usb-2-0-802-11n-b-g-150mbps-wi-fi-wlan-wireless-network-adapter-black-252716 USB WiFi stick]||4.21||5.35||4.21||5.35<br />
|-<br />
| 1||USB mouse and keyboard||9.84||12.50||9.84||12.50<br />
|-<br />
| 1|| [http://corporate.evonik.com/en/products/pages/default.aspx case acrylic glass XT 6mm~0.5<sup>2</sup>]||39.88||50.65||39.88||50.65<br />
|-<br />
| 1|| black spray paint for acrylic glass||5.15||6.54||5.15||6.54<br />
|-<br />
| 1|| [http://www.newark.com/raspberry-pi/raspberry-modb-512m/raspberry-pi-model-b-board/dp/68X0155 Raspberry Pi model B board]||27.56||35.00||27.56||35.00<br />
|-<br />
| 1||[http://www.newark.com/raspberry-pi/rpi-camera-board/add-on-brd-camera-module-raspberry/dp/69W0689 Raspberry Pi camera module]||19.69||25.00||19.69||25.00<br />
|-<br />
| 1||[http://www.pollin.de/shop/dt/NzUwOTc4OTk-/ 7” display]||39.35||49.97||39.35||49.97<br />
|-<br />
| 1||[http://www.dx.com/p/diy8-x-seven-segment-displays-module-for-arduino-595-driver-250813 8-segment display]||3.04||3.86||3.04||3.86<br />
|-11.81<br />
| 2|| [http://www.dx.com/p/arduino-dht11-digital-temperature-humidity-sensor-138531 digital temperature sensor DHT-22]||5.91||7.50||11.82||15.00<br />
|-<br />
| 1 ||aluminum block 100x100x15 mm||11.20||14.23||11.20||14.23<br />
|-<br />
| 1|| [http://www.dx.com/p/tec1-12706-semiconductor-thermoelectric-cooler-peltier-white-157283 Peltier heater 12V 60W]||3.54||4.49||3.54||4.49<br />
|-<br />
| 1||power supply||25.90||32.89||25.90||32.89<br />
|-<br />
| 6|| [http://www.leds.de/Low-Mid-Power-LEDs/SuperFlux-LEDs/Nichia-Superflux-LED-blau-3lm-100-NSPBR70BSS.html superflux LED 480nm]||0.99||1.26||5.94||7.54<br />
|-<br />
| 16||LED 450nm||0.37||0.47||5.94||7.54<br />
|-<br />
| 2|| Resistor 40 Ohm||0.12||0.15||0.24||0.30<br />
|-<br />
| 4|| Resistor 100 Ohm||0.12||0.15||0.48||0.60<br />
|-<br />
| 1||cupboard button||0.98||1.24||0.98||1.24<br />
|- class="sortbottom" style="background:#cfe2f4; border-top:2px #808080 solid; font-weight:bold"<br />
| -||Total||-||-||243.88||309.70<br />
|}<br />
<br />
You can find more economical information about ''WatsOn'' and the project on our [https://2014.igem.org/Team:Aachen/PolicyPractices/Economics Economical View] page.<br />
<br />
<br />
For building our '''''WatsOn''''' we used some tools that are not included in the list of necessary components because we assume that they are already available. We used a soldering iron to solder the resistors to the LEDs and fix the headers on the mount of the LEDs. For building electrical circuits our multimeter was very helpful. Furthermore, we applied special glue for plastic to hold the acrylic glass in place. All other components were fixed with tape or hot glue which is versatile and can be removed quickly during alignment of components.<br />
<br />
==Breadboard==<br />
<br />
{{Team:Aachen/Figure|Aachen_Device_Fritzing.png|align=center|title=Wiring of our device||width=900px}}<br />
<br />
==Construction Manual==<br />
<br />
{| class="wikitable centered"<br />
|-<br />
| [[File:Aachen_Device_1.jpg|300px]] || Start building your own ''WatsOn'' by assembling the base plate, the sides and the interior wall.<br />
|-style="border-top: 2px #808080 solid;"<br />
| [[File:Aachen_Device_2_3.jpg|350px] [File:Aachen_Device_.3jpg|300px]] || Fix the Peltier heater on the back of the aluminum block and place it in the hole of the interior wall.<html><br/></html>Arrange the 4x4 450&nbsp;nm LEDs and the 2x3 480&nbsp;nm LEDs<br />
|-style="border-top: 2px #808080 solid;"<br />
| [[File:Aachen_Device_7.jpg|350px]] || Assemble the camera holder with the camera and the corresponding filter slide on the lower part. Above the camera, you can place the temperature sensor for measuring the indoor temperature. Finally, put the fan on the back of the camera holder. <br />
|-style="border-top: 2px #808080 solid;"<br />
| [[File:Aachen_Device_8.jpg|350px]] || Connect the electronic components on the outside and the inside according to the wiring diagramm.<br />
|-style="border-top: 2px #808080 solid;"<br />
| [[File:Aachen_Device_4.jpg|350px]] || Put together the drawer.<br />
|-style="border-top: 2px #808080 solid;"<br />
| [[File:Aachen_Device_9.jpg|350px]] || Position the front panel and insert the drawer.<br />
|-style="border-top: 2px #808080 solid;"<br />
| [[File:Aachen_Device_10.jpg|350px]] || Place the temperature sensor measuring the aluminum block temperature directly on the block and put the back panel in front of it.<br />
|-style="border-top: 2px #808080 solid;"<br />
| [[File:Aachen_Device_6.jpg|350px]] || Setup the power supply<sup>[https://2014.igem.org/Team:Aachen/Notebook/Engineering/WatsOn#fn1 1]</sup> and connect all devices to either 5&nbsp;V or 12&nbsp;V. For security reasons it has been placed into an aluminium casing. Plug the USB hub connector into the Raspberry. If you use the GUI locally on the device a mouse and a keyboard need to be attached to the USB hub to navigate on the user interface. Follow the steps described in the section [https://2014.igem.org/Team:Aachen/Notebook/Engineering/WatsOn#pisetup Raspberry Pi - Setup].<br />
|-style="border-top: 2px #808080 solid;"<br />
| [[File:Aachen_Device_11.jpg|350px]] || Mount the device on top of the power supply casing. Add the display and apply some stickers to enjoy your custom-made ''WatsOn''.<br />
|}<br />
<br />
<span class="anchor" id="fn1"></span><br />
<sup>1</sup>By German law only certified electricians may work on 230&nbsp;V electronics. Therefore, the electrical workshop at our institute created the power supply specifically for our design.<br />
<br />
== Raspberry Pi - Setup ==<br />
<span class="anchor" id="pisetup"></span><br />
<br />
In order to get a running linux system on the Raspberry Pi which includes all required components and configurations the following steps have to be considered:<br />
<br />
* The Raspberry Pi needs an SD card on which the operating system will be installed. Go to the [http://www.raspberrypi.org/downloads/ download page of the Raspberry Pi Foundation] and select an operating system of your choice - we used Raspbian - or just download the NOOBS package which offers all different operating systems during setup. <br />
* Follow the specific image installation guidelines to install the downloaded system onto your SD card.<br />
* Once finished, insert the SD card in the slot on the Raspberry Pi board, connect a monitor over HDMI, plug in a USB mouse and keyboard and start the Raspberry Pi by connecting it to the micro USB power supply. Follow the installation instructions; these should be straightforward. After the installation you will be shown the desktop of your new system.<br />
* To be able to use the Raspberry Pi camera you need activate it over a terminal. Search for a desktop icon labeled "LxTerminal", double click it and a terminal will appear where you can enter commands which will be executed after you press Return. Enter "raspi-config", press Return and activate the camera with the displayed corresponding option.<br />
* To check if the camera works, enter "raspistill -t 5000 -o camera_test.jpg" in the terminal. An overlay shows a 5 second preview from the camera on the screen, then an image is taken and saved as "camera_test.jpg" in the current directory.<br />
* An issue concerning the Raspberry Pi camera is that it supports just a fixed-focus which is per default set to infinity. This can be solved by removing the glue dots fixing the lense und unscrewing it until the required distance is focused.<br />
* Download the source files for the backend server and the graphical user interface (GUI). To be able to compile the GUI, you need to install the Qt5-libraries. Follow [http://qt-project.org/wiki/Native_Build_of_Qt5_on_a_Raspberry_Pi this guide] on how to get the Qt source code, compile it and setup your environment correctly. Make sure that your Raspberry Pi is constantly running, since this process takes some time and must not be interrupted.<br />
* With the Qt-libraries installed, open a terminal and change to the directory where you put the source for the GUI (command "cd [path to source]"). Call "qmake" followed by "make" and you will start compilation of the program. When finished, you can launch the GUI with the command "./igem_GUI".<br />
* The backend - that will establish the connection between hardware and the user interface - requires you to install additional packages for Python which is a high-level general-purpose programming language and an interpreter that will ship with your system. Open the README in the "Backend" directory and follow the instructions.<br />
* You now should be able to launch the backend by calling "python takeimageserver.py &" from the terminal.<br />
* Now start the GUI. An input dialog will show up asking you to provide the IP address of the backend server or the Raspberry Pi, respectively. Since you are running the GUI and the backend on the same device, just press Enter to select the default entry which is the IP of the local host. After a few seconds, when the connection to the backend server has been established, the user interface gets enabled and you can start to take images and time lapse shootings. If the image is not focused you need to adjust the lense in front of the camera by rotating it. For the full list of features refer to the [https://2014.igem.org/Team:Aachen/Notebook/Engineering/WatsOn#watsonsoftware Software section] of this page.<br />
<br />
In case you want to run the GUI on a remote machine, e.g. your notebook, follow these additional steps:<br />
<br />
* Install the [http://qt-project.org/ Qt-libraries and QtCreator] on your system. This is just an installation - you do not have to compile it. Get the source code for the GUI and open the ".pro" file with QtCreator. After importing the project and selecting a built directory, click the green arrow on the left side. Compilation is started and as soon as it is finished the GUI will start. <br />
* In order to be able to connect to the Raspberry Pi you need to be connected to the same network. Therefore, make sure the Raspberry Pi USB wifi stick is working properly (see [https://2014.igem.org/Team:Aachen/Notebook/Engineering/WatsOn#pitrouble Troubleshooting & Useful Links]), and that you reside in the same network. Start the backend server on the Raspberry Pi. It will print the IP address on start up which you must enter in the GUI on your device running the GUI. Now you should be able to use all the features as if running the GUI on the Raspberry Pi.<br />
<br />
=== Troubleshooting & Useful Links ===<br />
<span class="anchor" id="pitrouble"></span><br />
<br />
* Display resolution: If your connected display is not working properly you may refer to<br />
** http://elinux.org/RPiconfig#Video<br />
** http://www.raspberrypi.org/forums/viewtopic.php?f=29&t=24679<br />
<br />
* Raspberry Pi Camera Module<br />
** http://elinux.org/Rpi_Camera_Module<br />
<br />
* Network configuration:<br />
** http://www.raspberrypi.org/documentation/configuration/wireless/README.md<br />
<br />
* General<br />
** [http://elinux.org/R-Pi_Troubleshooting Raspberry Pi Troubleshooting]<br />
** [http://raspberrywebserver.com/linux-basics/ Linux basics]<br />
** [http://www.raspberrypi.org/ Raspberry Pi Foundation]<br />
<br />
<br />
{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/Notebook/Engineering/WatsOnTeam:Aachen/Notebook/Engineering/WatsOn2014-10-18T03:43:09Z<p>Nbailly: /* Measurarty */</p>
<hr />
<div>__NOTOC__<br />
{{CSS/Main}}<br />
{{Team:Aachen/Stylesheet}}<br />
{{Team:Aachen/Header}}<br />
<br />
= ''WatsOn'' =<br />
<br />
This page contains technical details and construction manuals for our measurement device ''WatsOn'' as well as information on the software controlling the hardware. For more details, please click on the respective tile. For the image analysis software, please visit our [https://2014.igem.org/Team:Aachen/Notebook/Software/Measurarty ''Measurarty''] page.<br />
<br />
<html><br />
<center><br />
<ul class="team-grid" style="width:inherit;"><br />
<!-- Overview --><br />
<br />
<li style="margin-right:20px;margin-left:20px;"><br />
<a href="https://2014.igem.org/Team:Aachen/Notebook/Engineering/WatsOn#watsonhardware" style="color:black"><br />
<div class="team-item team-info" ><br />
<div class="menukachel">Hardware</div><br />
<!-- <br />
<br/><br/><br />
<b>Hardware</b><br />
<br/><br/><br />
click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/5/59/Aachen_14-10-16_Hardware_button_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%"> </div></a><br />
</li><br />
<br />
<br />
<li style="margin-right:20px;margin-left:20px;"><br />
<a href="https://2014.igem.org/Team:Aachen/Notebook/Engineering/WatsOn#watsonsoftware" style="color:black"><br />
<div class="team-item team-info" ><br />
<div class="menukachel">Software</div><br />
<!-- <br/><br><br />
<b>Software</b><br />
<br/><br/><br />
click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/1/13/Aachen_14-10-16_Software_button_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%"> </div></a><br />
</li><br />
<br />
<li style="margin-right:20px;margin-left:20px;"><br />
<a href="https://2014.igem.org/Team:Aachen/Notebook/Engineering/WatsOn#watsondiy" style="color:black"><br />
<div class="team-item team-info" ><br />
<div class="menukachel">DIY</div><br />
<!-- <br/><br/><br />
<b>DIY</b><br />
<br/><br/><br />
click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/9/9e/Aachen_14-10-15_DIY_Cellocks_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%"> </div></a><br />
</li><br />
<br />
</ul><br />
</center><br />
</html><br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
= Hardware =<br />
<span class="anchor" id="watsonhardware"></span><br />
<br />
{{Team:Aachen/FigureFloat|Aachen_Device_11.jpg|title=''WatsOn''|subtitle= |width=200px}}<br />
<br />
Our hardware consists of the casing and the electronical components. The casing which can be seen on the left was built from laser cut acrylic glass. A detailed description of the assembly is described in the section [https://2014.igem.org/Team:Aachen/Notebook/Engineering/WatsOn#watsondiy Build your own ''WatsOn].<br />
<br />
The connection between the different electronical elements is visualized below.<br />
<br />
{{Team:Aachen/Figure|Aachen_Device_Hardware_Graphics.png|title=Interaction of electronical components||width=750px}}<br />
<br />
* '''Raspberry Pi''' : The [http://www.raspberrypi.org/ Raspberry Pi] is a small single-board computer which runs a Linux operating system from an inserted SD card. The steps which are required to set up a fully working system are described in the [https://2014.igem.org/Team:Aachen/Notebook/Engineering/WatsOn#pisetup DIY section] of this page. The main purpose of the Raspberry Pi is to run the software described above, to control the attached camera and to show the GUI on the display. The big advantage of this board is that it is very powerful, cheap and therefore perfectly fit for our needs.<br />
<br />
* '''Raspberry Pi camera''': The camera is directly connected to the Raspberry Pi board and takes the images of the chips.<br />
<br />
* '''Arduino''': The [http://www.arduino.cc/ Arduino] board is also a single-board computer with less computing power than the Raspberry Pi but with a greater focus on controlling electronical components. Therefore, it is used to control the LEDs and the Peltier heater.<br />
<br />
* '''Relay''': The 2-channel relay works like two light switches which are either turned on or off. They control the 450&nbsp;nm and 480&nbsp;nm LEDs. The channels are connected and turned on and off by the Arduino board.<br />
<br />
* '''Peltier element''': A Peltier component transforms an applied power into a temperature gradient which leads to a hot surface on one side of the element and a cooler one on the other side. The Peltier element connected to the aluminum block heats up the interior of the device to incubate the sensing cells at 37°C.<br />
<br />
* '''USB WiFi stick''': The USB WiFi stick connects the Raspberry Pi to a local network.<br />
<br />
* '''Display''': A 8-digit display is connected to the Arduino board and shows the current interior temperature<br />
<br />
{{Team:Aachen/FigureFloatRight|Aachen_Filter_010.png|title=010|subtitle=|width=70px}}<br />
{{Team:Aachen/FigureFloatRight|Aachen_Filter_505.png|title=505|subtitle=|width=70px}}<br />
<br />
* '''Filter slides''': To block the undesired wavelenghts emitted from the LEDs a filter slide is placed in front of the camera. This step is taken to get a clear fluorescence signal from the chips. The characteristic of the filter slide is selected depending on the frequency of the LEDs which are either 450&nbsp;nm or 480&nbsp;nm ones. We used '505 Sally Green' for the 450&nbsp;nm and '010 Medium Yellow' for the 480&nbsp;nm LEDs. The filters are shown on the right.<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
= Software =<br />
<span class="anchor" id="watsonsoftware"></span><br />
<br />
The software consists of several parts which provide an user interface and manage the connection to the hardware.<br />
<br />
===GUI (Graphical User Interface)===<br />
On the graphical interface, the user can take images and time lapses of the chips inside the device. The software is written in C++. It makes use of the [http://qt-project.org/ Qt-Library] to provide a clear interface and a comfortable way to manage various software aspects such as handling images and establishing network connections. An advantage resulting from the utilization of Qt-Library is the multi-platform support for Windows, MacOS and Linux. Additionally, Qt is available with an Open Source license which can be used for free. The software can be used locally on the Raspberry Pi or remotely from a device in the same network.<br />
<br />
The scheme below shows the different components of the software:<br />
<br />
[[File:Aachen_Device_GUI.png|center|800px]]<br />
<br />
Features of the GUI include:<br />
* Change settings [1]:<br />
** The user can specify the iso-value and the shutter speed of the camera.<br />
** Custom settings can be labeled and saved for future reference.<br />
** Existing settings can be updated or deleted unless they are default configurations.<br />
* Take image/s [2]: <br />
** The excitation wavelength of GFP (480&nbsp;nm) and iLOV (450&nbsp;nm) can be selected.<br />
** The GUI offers two possibilities to take images:<br />
*** Take a single image with the active camera settings.<br />
*** Take time lapse shootings with the active camera settings and the specified interval. When activated, the images are saved automatically to a user defined directory with ascending filenames.<br />
** The last image which was taken by the camera is shown in the GUI, information containing the time stamp and used camera settings are displayed next to the image [3]. Previous images can be selected with the arrow buttons.<br />
* Analyze image [4]:<br />
** The image is analyzed by an image segmentation algorithm and shows whether the pathogen ''Pseudomonas&nbsp;aeruginosa'' is present on the chip or not<br />
<br />
''Download the GUI sourcecode:'' [https://static.igem.org/mediawiki/2014/9/90/Aachen_WatsOn_GUI.zip Download]<br />
<br />
===Backend===<br />
The backend is a software that runs on the Raspberry Pi and is responsible for the connection between the GUI and the hardware. If the user interface is executed on another device, e.g. a notebook, it has to be in the same network as the Raspberry Pi. The backend works like a web server that receives commands and acts according to the submitted parameters. It can take images and returns them to the GUI.<br />
<br />
Before an image is taken, the backend turns on the specified LEDs by sending a command to the connected Arduino board. Subsequently, the LEDs are turned off using the same mechanism. These steps are repeated in the given interval for a time lapse shooting.<br />
<br />
''Download the backend sourcecode:'' [https://static.igem.org/mediawiki/2014/7/77/Aachen_Device_Backend.zip Download]<br />
<html><br></html><br />
<br />
{{Team:Aachen/Figure|Aachen_Device_SoftwareBackend.png|title=Sample connection between GUI and backend for taking an image|subtitle= |width=900px}}<br />
<br />
===Arduino===<br />
The software on the Arduino board sets the power and thus controls the temperature of the Peltier heater. The power is set by evaluating the received values from the temperature sensors for the interior of the device and the aluminum block. Additionally, the Arduino receives commands from the Raspberry Pi to turn the LEDs on and off.<br />
<br />
''Download the Arduino sourcecode:'' [https://static.igem.org/mediawiki/2014/c/cd/Aachen_WatsOn_arduino.zip Download] <br />
<br />
===Measurarty===<br />
We have developed our own image analysis pipeline ''Measurarty''. Please go to the [https://2014.igem.org/Team:Aachen/Notebook/Software/Measurarty ''Measurarty''] project page for further information.<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_14-10-15_DIY_Cellocks_iNB.png|right|150px]]<br />
<br />
= DIY: How To Build Your Own ''WatsOn'' =<br />
<span class="anchor" id="watsondiy"></span><br />
<br />
==Technical Components==<br />
If you want to create your own ''WatsOn'' first take a look at the following list of necessary components. All parts except the laser cut acrylic glass can be readily purchased and do not require further adjustments.<br />
<br />
''Download the laser cutting plan here: [https://static.igem.org/mediawiki/2014/f/fd/Aachen_WatsOn_laser_cut.svg.zip Download] (for acrylic glass with a height of 6&nbsp;mm)<br />
<br />
'''All needed components, their quantities and prices for creating your own ''WatsOn'''''<br />
{| class="wikitable sortable"<br />
! align="center" |'''''WatsOn'''''<br />
!! align="center" | <br />
!! align="center" |''' 1€='''<br />
!! align="center" |''' $1.27'''<br />
!! align="center" |''' on 14/10/2014'''<br />
!! align="center" | <br />
|- class="unsortable"<br />
!Quantity !! Component !! Costs [€]!! Costs [$]!! Final [€]!! Final [$]<br />
|-<br />
| 1|| [http://www.prolighting.de/Zubehoer/Farbfilter/Lee-Filter_HT/Lee-Filters_Musterheft_Designer_Edition_i174_3965_0.htm filter slides] (medium yellow 010, sally green 505)||1.57||2.00||1.57||2.00<br />
|-<br />
| 1|| [http://www.dx.com/p/uno-r3-development-board-microcontroller-mega328p-atmega16u2-compat-for-arduino-blue-black-215600 Arduino UNO R3]||9.17||11.65||9.17||11.65<br />
|-<br />
| 1|| [http://www.dx.com/p/arduino-2-channel-relay-shield-module-red-144140 2-channel relay shield]||2.72||3.46||2.72||3.46<br />
|-<br />
| 40||jumper-wire cable||2.35||2.99||2.35||2.99<br />
|-<br />
| 1|| [http://www.dx.com/p/2-54mm-1x40-pin-breakaway-straight-male-header-10-piece-pack-144191 40er male header (10-Piece Pack)]||2.14||2.72||2.14||2.72<br />
|-<br />
| 1|| [http://www.dx.com/p/jtron-2-54mm-40-pin-single-row-seat-single-row-female-header-black-10-pcs-278953 40er female header (10-Piece Pack)]||2.05||2.60||2.05||2.60<br />
|-<br />
| 1|| [http://www.dx.com/p/prototype-universal-printed-circuit-board-breadboard-brown-5-piece-pack-130926 circuit board]||2.35||2.99||2.35||2.99<br />
|-<br />
| 1|| [http://www.newark.com/pro-signal/rp006/audio-video-cable-hdmi-1m-black/dp/96T7446 HDMI cable]||1.47||1.87||1.47||1.87<br />
|-<br />
| 1|| [http://www.dx.com/p/hd-053-high-speed-usb-2-0-7-port-hub-black-174817 7 port USB hub]||5.28||6.71||5.28||6.71<br />
|-<br />
| 1||[http://www.dx.com/p/dx-original-ultra-mini-usb-2-0-802-11n-b-g-150mbps-wi-fi-wlan-wireless-network-adapter-black-252716 USB WiFi stick]||4.21||5.35||4.21||5.35<br />
|-<br />
| 1||USB mouse and keyboard||9.84||12.50||9.84||12.50<br />
|-<br />
| 1|| [http://corporate.evonik.com/en/products/pages/default.aspx case acrylic glass XT 6mm~0.5<sup>2</sup>]||39.88||50.65||39.88||50.65<br />
|-<br />
| 1|| black spray paint for acrylic glass||5.15||6.54||5.15||6.54<br />
|-<br />
| 1|| [http://www.newark.com/raspberry-pi/raspberry-modb-512m/raspberry-pi-model-b-board/dp/68X0155 Raspberry Pi model B board]||27.56||35.00||27.56||35.00<br />
|-<br />
| 1||[http://www.newark.com/raspberry-pi/rpi-camera-board/add-on-brd-camera-module-raspberry/dp/69W0689 Raspberry Pi camera module]||19.69||25.00||19.69||25.00<br />
|-<br />
| 1||[http://www.pollin.de/shop/dt/NzUwOTc4OTk-/ 7” display]||39.35||49.97||39.35||49.97<br />
|-<br />
| 1||[http://www.dx.com/p/diy8-x-seven-segment-displays-module-for-arduino-595-driver-250813 8-segment display]||3.04||3.86||3.04||3.86<br />
|-11.81<br />
| 2|| [http://www.dx.com/p/arduino-dht11-digital-temperature-humidity-sensor-138531 digital temperature sensor DHT-22]||5.91||7.50||11.82||15.00<br />
|-<br />
| 1 ||aluminum block 100x100x15 mm||11.20||14.23||11.20||14.23<br />
|-<br />
| 1|| [http://www.dx.com/p/tec1-12706-semiconductor-thermoelectric-cooler-peltier-white-157283 Peltier heater 12V 60W]||3.54||4.49||3.54||4.49<br />
|-<br />
| 1||power supply||25.90||32.89||25.90||32.89<br />
|-<br />
| 6|| [http://www.leds.de/Low-Mid-Power-LEDs/SuperFlux-LEDs/Nichia-Superflux-LED-blau-3lm-100-NSPBR70BSS.html superflux LED 480nm]||0.99||1.26||5.94||7.54<br />
|-<br />
| 16||LED 450nm||0.37||0.47||5.94||7.54<br />
|-<br />
| 2|| Resistor 40 Ohm||0.12||0.15||0.24||0.30<br />
|-<br />
| 4|| Resistor 100 Ohm||0.12||0.15||0.48||0.60<br />
|-<br />
| 1||cupboard button||0.98||1.24||0.98||1.24<br />
|- class="sortbottom" style="background:#cfe2f4; border-top:2px #808080 solid; font-weight:bold"<br />
| -||Total||-||-||243.88||309.70<br />
|}<br />
<br />
You can find more economical information about ''WatsOn'' and the project on our [https://2014.igem.org/Team:Aachen/PolicyPractices/Economics Economical View] page.<br />
<br />
<br />
For building our '''''WatsOn''''' we used some tools that are not included in the list of necessary components because we assume that they are already available. We used a soldering iron to solder the resistors to the LEDs and fix the headers on the mount of the LEDs. For building electrical circuits our multimeter was very helpful. Furthermore, we applied special glue for plastic to hold the acrylic glass in place. All other components were fixed with tape or hot glue which is versatile and can be removed quickly during alignment of components.<br />
<br />
==Breadboard==<br />
<br />
{{Team:Aachen/Figure|Aachen_Device_Fritzing.png|align=center|title=Wiring of our device||width=900px}}<br />
<br />
==Construction Manual==<br />
<br />
{| class="wikitable centered"<br />
|-<br />
| [[File:Aachen_Device_1.jpg|300px]] || Start building your own ''WatsOn'' by assembling the base plate, the sides and the interior wall.<br />
|-style="border-top: 2px #808080 solid;"<br />
| [[File:Aachen_Device_2_3.jpg|350px] [File:Aachen_Device_.3jpg|300px]] || Fix the Peltier heater on the back of the aluminum block and place it in the hole of the interior wall.<html><br/></html>Arrange the 4x4 450&nbsp;nm LEDs and the 2x3 480&nbsp;nm LEDs<br />
|-style="border-top: 2px #808080 solid;"<br />
| [[File:Aachen_Device_7.jpg|350px]] || Assemble the camera holder with the camera and the corresponding filter slide on the lower part. Above the camera, you can place the temperature sensor for measuring the indoor temperature. Finally, put the fan on the back of the camera holder. <br />
|-style="border-top: 2px #808080 solid;"<br />
| [[File:Aachen_Device_8.jpg|350px]] || Connect the electronic components on the outside and the inside according to the wiring diagramm.<br />
|-style="border-top: 2px #808080 solid;"<br />
| [[File:Aachen_Device_4.jpg|350px]] || Put together the drawer.<br />
|-style="border-top: 2px #808080 solid;"<br />
| [[File:Aachen_Device_9.jpg|350px]] || Position the front panel and insert the drawer.<br />
|-style="border-top: 2px #808080 solid;"<br />
| [[File:Aachen_Device_10.jpg|350px]] || Place the temperature sensor measuring the aluminum block temperature directly on the block and put the back panel in front of it.<br />
|-style="border-top: 2px #808080 solid;"<br />
| [[File:Aachen_Device_6.jpg|350px]] || Setup the power supply<sup>[https://2014.igem.org/Team:Aachen/Notebook/Engineering/WatsOn#fn1 1]</sup> and connect all devices to either 5&nbsp;V or 12&nbsp;V. For security reasons it has been placed into an aluminium casing. Plug the USB hub connector into the Raspberry. If you use the GUI locally on the device a mouse and a keyboard need to be attached to the USB hub to navigate on the user interface. Follow the steps described in the section [https://2014.igem.org/Team:Aachen/Notebook/Engineering/WatsOn#pisetup Raspberry Pi - Setup].<br />
|-style="border-top: 2px #808080 solid;"<br />
| [[File:Aachen_Device_11.jpg|350px]] || Mount the device on top of the power supply casing. Add the display and apply some stickers to enjoy your custom-made ''WatsOn''.<br />
|}<br />
<br />
<span class="anchor" id="fn1"></span><br />
<sup>1</sup>By German law only certified electricians may work on 230&nbsp;V electronics. Therefore, the electrical workshop at our institute created the power supply specifically for our design.<br />
<br />
== Raspberry Pi - Setup ==<br />
<span class="anchor" id="pisetup"></span><br />
<br />
In order to get a running linux system on the Raspberry Pi which includes all required components and configurations the following steps have to be considered:<br />
<br />
* The Raspberry Pi needs an SD card on which the operating system will be installed. Go to the [http://www.raspberrypi.org/downloads/ download page of the Raspberry Pi Foundation] and select an operating system of your choice - we used Raspbian - or just download the NOOBS package which offers all different operating systems during setup. <br />
* Follow the specific image installation guidelines to install the downloaded system onto your SD card.<br />
* Once finished, insert the SD card in the slot on the Raspberry Pi board, connect a monitor over HDMI, plug in a USB mouse and keyboard and start the Raspberry Pi by connecting it to the micro USB power supply. Follow the installation instructions; these should be straightforward. After the installation you will be shown the desktop of your new system.<br />
* To be able to use the Raspberry Pi camera you need activate it over a terminal. Search for a desktop icon labeled "LxTerminal", double click it and a terminal will appear where you can enter commands which will be executed after you press Return. Enter "raspi-config", press Return and activate the camera with the displayed corresponding option.<br />
* To check if the camera works, enter "raspistill -t 5000 -o camera_test.jpg" in the terminal. An overlay shows a 5 second preview from the camera on the screen, then an image is taken and saved as "camera_test.jpg" in the current directory.<br />
* An issue concerning the Raspberry Pi camera is that it supports just a fixed-focus which is per default set to infinity. This can be solved by removing the glue dots fixing the lense und unscrewing it until the required distance is focused.<br />
* Download the source files for the backend server and the graphical user interface (GUI). To be able to compile the GUI, you need to install the Qt5-libraries. Follow [http://qt-project.org/wiki/Native_Build_of_Qt5_on_a_Raspberry_Pi this guide] on how to get the Qt source code, compile it and setup your environment correctly. Make sure that your Raspberry Pi is constantly running, since this process takes some time and must not be interrupted.<br />
* With the Qt-libraries installed, open a terminal and change to the directory where you put the source for the GUI (command "cd [path to source]"). Call "qmake" followed by "make" and you will start compilation of the program. When finished, you can launch the GUI with the command "./igem_GUI".<br />
* The backend - that will establish the connection between hardware and the user interface - requires you to install additional packages for Python which is a high-level general-purpose programming language and an interpreter that will ship with your system. Open the README in the "Backend" directory and follow the instructions.<br />
* You now should be able to launch the backend by calling "python takeimageserver.py &" from the terminal.<br />
* Now start the GUI. An input dialog will show up asking you to provide the IP address of the backend server or the Raspberry Pi, respectively. Since you are running the GUI and the backend on the same device, just press Enter to select the default entry which is the IP of the local host. After a few seconds, when the connection to the backend server has been established, the user interface gets enabled and you can start to take images and time lapse shootings. If the image is not focused you need to adjust the lense in front of the camera by rotating it. For the full list of features refer to the [https://2014.igem.org/Team:Aachen/Notebook/Engineering/WatsOn#watsonsoftware Software section] of this page.<br />
<br />
In case you want to run the GUI on a remote machine, e.g. your notebook, follow these additional steps:<br />
<br />
* Install the [http://qt-project.org/ Qt-libraries and QtCreator] on your system. This is just an installation - you do not have to compile it. Get the source code for the GUI and open the ".pro" file with QtCreator. After importing the project and selecting a built directory, click the green arrow on the left side. Compilation is started and as soon as it is finished the GUI will start. <br />
* In order to be able to connect to the Raspberry Pi you need to be connected to the same network. Therefore, make sure the Raspberry Pi USB wifi stick is working properly (see [https://2014.igem.org/Team:Aachen/Notebook/Engineering/WatsOn#pitrouble Troubleshooting & Useful Links]), and that you reside in the same network. Start the backend server on the Raspberry Pi. It will print the IP address on start up which you must enter in the GUI on your device running the GUI. Now you should be able to use all the features as if running the GUI on the Raspberry Pi.<br />
<br />
=== Troubleshooting & Useful Links ===<br />
<span class="anchor" id="pitrouble"></span><br />
<br />
* Display resolution: If your connected display is not working properly you may refer to<br />
** http://elinux.org/RPiconfig#Video<br />
** http://www.raspberrypi.org/forums/viewtopic.php?f=29&t=24679<br />
<br />
* Raspberry Pi Camera Module<br />
** http://elinux.org/Rpi_Camera_Module<br />
<br />
* Network configuration:<br />
** http://www.raspberrypi.org/documentation/configuration/wireless/README.md<br />
<br />
* General<br />
** [http://elinux.org/R-Pi_Troubleshooting Raspberry Pi Troubleshooting]<br />
** [http://raspberrywebserver.com/linux-basics/ Linux basics]<br />
** [http://www.raspberrypi.org/ Raspberry Pi Foundation]<br />
<br />
{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/Notebook/Protocols/Analytical_methodsTeam:Aachen/Notebook/Protocols/Analytical methods2014-10-18T03:42:46Z<p>Nbailly: /* Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) */</p>
<hr />
<div>__NOTOC__<br />
{{CSS/Main}}<br />
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<ul class="menusmall-grid"><br />
<br />
<li style="width:106px;margin-left: 12px;margin-right: 12px;" ><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Protocols/detection" style="color:black"><br />
<div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel" style="top: 10%; font-size: 14px;">2D Detection of<br/>IPTG & HSL</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/2/22/Aachen_14-10-14_button_chip_manufacturing_ipo.png); norepeat scroll 0% 0% transparent; background-size:100%; height:100px; width: 100px;"><br />
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<li style="width:106px;margin-left: 12px;margin-right: 12px;" ><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Protocols/Culture_medium_and_conditions" style="color:black"><br />
<div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel" style="top: 10%; font-size: 14px;">Culture Media</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/1/10/Aachen_14-10-13_Yellow_Flask_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%; height:100px; width: 100px;"><br />
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<li style="width:106px;margin-left: 12px;margin-right: 12px;" ><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Protocols/Molecular_biological_methods" style="color:black"><br />
<div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel" style="top: 10%; font-size: 14px;">Molecular biological methods</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/7/75/Aachen_14-10-14_Eppi_with_green_cells_panel_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%; height:100px; width: 100px;"><br />
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<li style="width:106px;margin-left: 12px;margin-right: 12px;" ><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Protocols/Analytical_methods" style="color:black"><br />
<div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel" style="top: 10%; font-size: 14px;">Analytical methods</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/4/4d/Aachen_14-10-14_Lense_panel_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%; height:100px; width: 100px;"><br />
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<br />
= Analytical Methods =<br />
To determine certain properties of proteins or contructed DNA fragments such as BioBricks, we have used different analytical methods. All used methods are listed below. <br />
== Agarose Gel Electrophoresis==<br />
The Agarose Gel Electrophoresis is used for separation of DNA or RNA fragments (e.g. after a PCR).<br />
<br />
# take 5&nbsp;µl of the PCR product<br />
# mix with 1&nbsp;µl loading dye<br />
# apply onto agarose gel together with a marker<br />
# run at 100&nbsp;V for 35&nbsp;minutes for a full gel<br />
<br />
== Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) ==<br />
The SDS-PAGE is used to determine certain features of the cells' proteome such as the strength of expression of a desired protein.<br />
<br />
'''Cell Preparation'''<br />
* lysis of cell pellet in lysis buffer<br />
* centrifuge for 15&nbsp;min at 13.000 rpm<br />
* mix the supernatant with 2x lammli buffer with β-mercaptoethanol<br />
* denatured for 5&nbsp;min at 95°C<br />
* sample to the gel <br />
<br />
For some SDS-PAGEs, we used BioRad ready made gels.<br />
<br />
Self-made SDS gels were made as described below:<br />
<br />
'''1.5x Buffer'''<br />
* 1.5&nbsp;M Tris-Cl pH = 8.8<br />
* in 1&nbsp;L is 40&nbsp;ml 10% SDS<br />
<br />
'''Gels'''<br />
<center><br />
{| class="wikitable" style="text-align: right;"<br />
! <br />
!! style="border-left: 2px solid #404040;" colspan="3"|0.75&nbsp;mm 12% RUNNING Gel <br />
!! style="border-left: 2px solid #404040; background-color:#8ebae5;" colspan="3"|1&nbsp;mm 4% STACKING Gel<br />
|-<br />
| <br />
| style="border-left: 2px solid #404040;"| '''1x''' || '''2x''' || '''4x''' <br />
| style="border-left: 2px solid #404040;"| '''1x''' || '''2x''' || '''4x'''<br />
|-<br />
| '''H{{sub|2}}O''' <br />
| style="border-left: 2px solid #404040;"| 1.65&nbsp;ml || 3.3&nbsp;ml || 6.6&nbsp;ml <br />
| style="border-left: 2px solid #404040;"| 1.5&nbsp;ml || 3&nbsp;ml || 6&nbsp;ml<br />
|-<br />
| '''1.5x Gel Buffer''' <br />
| style="border-left: 2px solid #404040;"| 1.3&nbsp;ml || 2.6&nbsp;ml || 5.2&nbsp;ml <br />
| style="border-left: 2px solid #404040;"| 0.65&nbsp;ml || 1.3&nbsp;ml || 2.6&nbsp;ml<br />
|-<br />
| '''30% Acrylamide (37.5:1)''' <br />
| style="border-left: 2px solid #404040;"| 2&nbsp;ml || 4&nbsp;ml || 8&nbsp;ml<br />
| style="border-left: 2px solid #404040;"| 0.325&nbsp;ml || 0.65&nbsp;ml || 1.3&nbsp;ml<br />
|-<br />
| '''10% APS''' <br />
| style="border-left: 2px solid #404040;"| 50&nbsp;µl || 100&nbsp;µl || 200&nbsp;µl <br />
| style="border-left: 2px solid #404040;"| 25&nbsp;µl || 50&nbsp;µl || 100&nbsp;µl<br />
|-<br />
| '''TEMED''' <br />
| style="border-left: 2px solid #404040;"| 10&nbsp;µl || 20&nbsp;µl || 40&nbsp;µl <br />
| style="border-left: 2px solid #404040;"| 5&nbsp;µl || 10&nbsp;µl || 20&nbsp;µl<br />
|-<br />
|}<br />
</center><br />
<br />
'''Run Gel'''<br />
* apply the prepared samples together with a protein marker on the gel<br />
* run the gel for 10&nbsp;min at 60&nbsp;V and after that for ca. 60&nbsp;min at 120&nbsp;V<br />
<br />
== Bradford Assay ==<br />
This assay is used for the determination of the protein concentration in a sample. <br />
* mix the Bradford solution with ddH{{sub|2}}O in a ratio of 1:4<br />
* prepare about 10 solutions 1&nbsp;ml, each between 125–1,000&nbsp;μg/ml BSA for a standard curve<br />
* use pure Bradford solution as a blank<br />
* mix equal amounts of BSA and samples with unknown concentrations (1-3&nbsp;µl) with 1&nbsp;ml of 1x&nbsp;Bradford solution, vortex and incubate for 5&nbsp;min. at room temperature<br />
* measure the OD with a spectrophotometer at 595&nbsp;nm<br />
* build a standard curve within the linear range of the BSA data (concentration against OD) <br />
* derive the concentration of your samples from the calibration curve<br />
<br />
== Measurement of Fluorescence ==<br />
The measurement of fluorescence was performed using the Synergy Mx Microplate Reader (BioTek) and the corresponding Gen5&nbsp;2.01 software. Unless stated otherwise following parameters were used:<br />
* volume of sample in each well: 100&nbsp;µl<br />
* measurement of GFP/eGFP/sfGFP<br />
** excitation wavelength: 496&nbsp;±&nbsp;9&nbsp;nm<br />
** emission wavelength: 516&nbsp;±&nbsp;9&nbsp;nm<br />
* measurement of iLOV<br />
** excitation wavelength: 450&nbsp;±&nbsp;9&nbsp;nm<br />
** emission wavelength: 495&nbsp;±&nbsp;9&nbsp;nm<br />
The fluorescence is given out in arbitrary units (a.u.).<br />
<br />
== Measurement of Optical Density ==<br />
Depending on the number of samples, two different devices were used for measurement of OD, the Unico Spectrophotometer 1201 (Fisher Bioblock Scientific) and the Synergy Mx Microplate Reader (BioTek) with the corresponding Gen5&nbsp;2.01 software.<br />
<br />
Unless stated otherwise following parameters were used for the plate reader measurements:<br />
* volume of sample in each well: 100&nbsp;µl<br />
* OD<sub>600</sub> with ''pathlength correction'' (including the height of liquid into the calculation of the optical density)<br />
<br />
The respective culture medium was used as reference.<br />
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{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/Notebook/Protocols/Culture_medium_and_conditionsTeam:Aachen/Notebook/Protocols/Culture medium and conditions2014-10-18T03:42:13Z<p>Nbailly: /* References */</p>
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<br />
= Culture Media =<br />
In our project we used different kinds of media for cultivation, transformation and chip preparation. While complex media such as LB offered an easy way of cultivation, minimal media such as M9 or HM provide a low autofluorescence for fluorescence measurements. All used media are listed below.<br />
<br />
== Complex media ==<br />
=== Luria-Bertani Medium (LB)===<br />
# weight components<br />
#: '''5&nbsp;g/L NaCl'''<br />
#: '''10&nbsp;g/L tryptone'''<br />
#: '''5&nbsp;g/L yeast extract'''<br />
#: (15&nbsp;g/L agar for plates)<br />
# fill up to 1&nbsp;L with deionized water<br />
# '''mix well''' by shaking<br />
# autoclave<br />
## autoclaving tape, caps slightly unscrewed<br />
## base of the pot has to be covered with deionized water<br />
## close lid<br />
## heat '''level 3 until the pressure valve opens'''<br />
## reduce '''heat level to 1.5'''<br />
## set timer to '''20 minutes'''<br />
## turn heater off<br />
## '''wait until the pressure valve retracts''' (30-45 minutes)<br />
## open, close caps & shake<br />
# for plates, wait until you can touch the bottle ('''<60°C''', clean bench!)<br />
# add antibiotics (1&nbsp;µl/ml) and '''shake''' (gloves!)<br />
<br />
=== Terrific-Broth-Medium (TB) ===<br />
# components for 1&nbsp;:<br />
#: '''4&nbsp;ml/L glycerol'''<br />
#: '''12&nbsp;g/L tryptone'''<br />
#: '''24&nbsp;g/L yeast extract''' <br />
# fill up to 900&nbsp;ml with deionized water<br />
# '''mix well''' by shaking<br />
# autoclave<br />
# components 2:<br />
#: '''0.17&nbsp;M KH<sub>2</sub>PO<sub>4</sub>'''<br />
#: '''0.72&nbsp;M K<sub>2</sub>HPO<sub>4</sub>'''<br />
# dissolve in 100&nbsp;ml deionized water and sterilize it by passing it through a filter<br />
# after autoclaving and cooling down, add sterile phosphate solutions<br />
<br />
=== Nutrient Agar medium (NA) ===<br />
#Components for 1&nbsp;L<br />
#: '''Enzymatic Digest of Gelatin 5&nbsp;g'''<br />
#: '''Beef Extract 3&nbsp;g'''<br />
#: '''Agar 15&nbsp;g'''<br />
# Final pH: 6.8 ± 0.2 at 25°C<br />
# Suspend 23&nbsp;g of the medium in one liter of purified water.<br />
# Heat with frequent agitation and boil for one minute to completely dissolve the medium.<br />
# Autoclave at 121°C for 15&nbsp;min.<br />
<br />
== Minimal Media ==<br />
=== Hartmans Minimal Medium (HM) ===<br />
<br />
This mineral salts medium is based on (Hartmans et al., 1989).<br />
<br />
Three '''100x stock solutions''' are prepared according to the following recipe and stored at 4°C:<br />
<br />
* '''100x Buffer''',composed of 388&nbsp;g dipotassium phosphate, 212&nbsp;g monosodium phosphate dihydrate per Liter, adjusted to a pH of 7.0 and sterilized by autoclaving.<br />
* '''100x ammonium sulfate''' composed of 200&nbsp;g/l ammonium sulfate and sterilized by autoclaving.<br />
* '''100x MM salts'''<br />
# Add 1&nbsp;g EDTA to 25&nbsp;ml water.<br />
# Add drops of 10&nbsp;M sodium hydroxide until EDTA is completely dissolved.<br />
# Adjust pH back to 4.0 with concentrated hypochloric acid.<br />
# Fill up with water to 800&nbsp;ml and dissolve following components:<br />
## 10&nbsp;g magnesium chloride sexahydrate<br />
## 200&nbsp;mg zinc sulfate heptahydrate<br />
## 100&nbsp;mg calcium chloride dihydrate<br />
## 500&nbsp;mg iron(II) sulfate heptahydrate<br />
## 20&nbsp;mg sodium molybdate dihydrate<br />
## 20&nbsp;mg copper(II) sulfate quintahydrate<br />
## 40&nbsp;mg cobalt(II) chloride sexahydrate<br />
## 100&nbsp;mg manganese(II) chloride sexahydrate<br />
<br />
For 1x medium without carbon source, pool 10&nbsp;ml of each '''100x stock solution''' and fill up to 1000&nbsp;ml with sterile, deionized water and store at 4°C.<br />
<br />
=== M9 Minimal Medium (M9) ===<br />
<center><br />
{| class="wikitable centered"<br />
! '''Components for 1&nbsp;L''' !! '''Volume'''<br />
|-<br />
| bidest. water || style="text-align:right"| 778.667&nbsp;ml<br />
|-<br />
| 10x Salt solution || style="text-align:right"| 100&nbsp;ml <br />
|- <br />
| Magnesiumsulfatehaptahydrate (10&nbsp;mM) || style="text-align:right"| 100&nbsp;ml <br />
|- <br />
| Glucose 20% (w/v) || style="text-align:right"| 20&nbsp;ml<br />
|- <br />
| 1000x Trace elements || style="text-align:right"| 1&nbsp;ml <br />
|-<br />
| Thiamin (1&nbsp;mM) || style="text-align:right"| 0.333&nbsp;ml <br />
|}<br />
<br />
<br />
{| class="wikitable centered"<br />
| colspan="3"| '''10x Salt solution''' <br />
|-<br />
! '''Component''' !!''Final concentration''' !! '''Concentration in stock solution'''<br />
|-<br />
| BisTris || style="text-align:right"| 95&nbsp;mM || style="text-align:right"| 200,000&nbsp;mg/L<br />
|-<br />
| Ammmonium chloride || style="text-align:right"| 60&nbsp;mM || style="text-align:right"| 32,100&nbsp;mg/L <br />
|-<br />
| Sodium citrate || style="text-align:right"| 12.5&nbsp;mM || style="text-align:right"| 27,000&nbsp;mg/L<br />
|-<br />
| Monopotassium phosphate || style="text-align:right"| 3&nbsp;mM || style="text-align:right"| 4,170&nbsp;mg/L<br />
|-<br />
| Dipotassium phosphate || style="text-align:right"| 0.7&nbsp;mM || style="text-align:right"| 1,590&nbsp;mg/L <br />
|- <br />
|}<br />
<br />
{| class="wikitable centered"<br />
| colspan="3"| '''1000x Trace elements'''<br />
|-<br />
! '''Component'''!!'''Final concentration''' !!'''Concentration in stock solution'''<br />
|- <br />
| Iron(III) chloride || style="text-align:right"| 50&nbsp;mM || style="text-align:right"| 13,515&nbsp;mg/L <br />
|- <br />
| Calcium chloride || style="text-align:right"| 20&nbsp;mM || style="text-align:right"| 2,220&nbsp;mg/L <br />
|-<br />
| Manganese(II) chloride || style="text-align:right"| 10&nbsp;mM || style="text-align:right"| 1,258&nbsp;mg/L <br />
|- <br />
| Zinc sulfate || style="text-align:right"| 10&nbsp;mM || style="text-align:right"| 1,615&nbsp;mg/L <br />
|- <br />
| Cobalt(II) chloride || style="text-align:right"| 2&nbsp;mM || style="text-align:right"| 260&nbsp;mg/L <br />
|- <br />
| Copper(II) chloride || style="text-align:right"| 2&nbsp;mM || style="text-align:right"| 269&nbsp;mg/L <br />
|-<br />
| Nickel(II) chloride || style="text-align:right"| 2&nbsp;mM || style="text-align:right"| 259&nbsp;mg/L <br />
|- <br />
| Sodium molybdate || style="text-align:right"| 2&nbsp;mM || style="text-align:right"| 412&nbsp;mg/L <br />
|-<br />
| Sodium selenite || style="text-align:right"| 2&nbsp;mM || style="text-align:right"| 346&nbsp;mg/L <br />
|-<br />
| Boric acid || style="text-align:right"| 2&nbsp;mM || style="text-align:right"| 124&nbsp;mg/L <br />
|-<br />
| Hydrochloric acid || style="text-align:right"| 1&nbsp;mM || style="text-align:right"| 20&nbsp;ml <br />
|-<br />
|}<br />
</center><br />
<br />
== Transformation Medium ==<br />
=== Super Optimal broth with Catabolite repression medium (SOC) ===<br />
# components<br />
#: '''0,5% yeast extract'''<br />
#: '''2% tryptone'''<br />
#: '''10&nbsp;mM NaCl'''<br />
#: '''2.5&nbsp;mM KCl'''<br />
#: '''20&nbsp;mM MgSO<sub>4</sub> '''<br />
# fill up with deionized water<br />
# adjust to pH 7.5 with NaOH<br />
# after autoclaving, add 20&nbsp;mM sterile glucose solution (filter sterilization)<br />
<br />
==References==<br />
* Hartmans, S., Smiths J.P., Volkering F., and de Brondth, J.A. (1989). Metabolism of Styrene Oxide and 2-Phenylethanol in the Styrene-Degrading Xanthobacter Strain 124X. Applied and Environmental Microbiology, Nov. Available at: http://www.ncbi.nlm.nih.gov/pubmed/?term=PMC203180.<br />
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<div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel" style="top: 10%; font-size: 14px;">2D Detection of<br/>IPTG & HSL</div></div><br />
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{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/Notebook/Protocols/Culture_medium_and_conditionsTeam:Aachen/Notebook/Protocols/Culture medium and conditions2014-10-18T03:40:24Z<p>Nbailly: /* References */</p>
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<div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel" style="top: 10%; font-size: 14px;">2D Detection of<br/>IPTG & HSL</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/2/22/Aachen_14-10-14_button_chip_manufacturing_ipo.png); norepeat scroll 0% 0% transparent; background-size:100%; height:100px; width: 100px;"><br />
</div><br />
</a><br />
</li><br />
<br />
<li style="width:106px;margin-left: 12px;margin-right: 12px;" ><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Protocols/Culture_medium_and_conditions" style="color:black"><br />
<div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel" style="top: 10%; font-size: 14px;">Culture Media</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/1/10/Aachen_14-10-13_Yellow_Flask_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%; height:100px; width: 100px;"><br />
</div><br />
</a><br />
</li><br />
<br />
<li style="width:106px;margin-left: 12px;margin-right: 12px;" ><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Protocols/Molecular_biological_methods" style="color:black"><br />
<div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel" style="top: 10%; font-size: 14px;">Molecular biological methods</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/7/75/Aachen_14-10-14_Eppi_with_green_cells_panel_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%; height:100px; width: 100px;"><br />
</div><br />
</a><br />
</li><br />
<br />
<li style="width:106px;margin-left: 12px;margin-right: 12px;" ><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Protocols/Analytical_methods" style="color:black"><br />
<div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel" style="top: 10%; font-size: 14px;">Analytical methods</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/4/4d/Aachen_14-10-14_Lense_panel_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%; height:100px; width: 100px;"><br />
</div><br />
</a><br />
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<br />
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</center><br />
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<br />
= Culture Media =<br />
In our project we used different kinds of media for cultivation, transformation and chip preparation. While complex media such as LB offered an easy way of cultivation, minimal media such as M9 or HM provide a low autofluorescence for fluorescence measurements. All used media are listed below.<br />
<br />
== Complex media ==<br />
=== Luria-Bertani Medium (LB)===<br />
# weight components<br />
#: '''5&nbsp;g/L NaCl'''<br />
#: '''10&nbsp;g/L tryptone'''<br />
#: '''5&nbsp;g/L yeast extract'''<br />
#: (15&nbsp;g/L agar for plates)<br />
# fill up to 1&nbsp;L with deionized water<br />
# '''mix well''' by shaking<br />
# autoclave<br />
## autoclaving tape, caps slightly unscrewed<br />
## base of the pot has to be covered with deionized water<br />
## close lid<br />
## heat '''level 3 until the pressure valve opens'''<br />
## reduce '''heat level to 1.5'''<br />
## set timer to '''20 minutes'''<br />
## turn heater off<br />
## '''wait until the pressure valve retracts''' (30-45 minutes)<br />
## open, close caps & shake<br />
# for plates, wait until you can touch the bottle ('''<60°C''', clean bench!)<br />
# add antibiotics (1&nbsp;µl/ml) and '''shake''' (gloves!)<br />
<br />
=== Terrific-Broth-Medium (TB) ===<br />
# components for 1&nbsp;:<br />
#: '''4&nbsp;ml/L glycerol'''<br />
#: '''12&nbsp;g/L tryptone'''<br />
#: '''24&nbsp;g/L yeast extract''' <br />
# fill up to 900&nbsp;ml with deionized water<br />
# '''mix well''' by shaking<br />
# autoclave<br />
# components 2:<br />
#: '''0.17&nbsp;M KH<sub>2</sub>PO<sub>4</sub>'''<br />
#: '''0.72&nbsp;M K<sub>2</sub>HPO<sub>4</sub>'''<br />
# dissolve in 100&nbsp;ml deionized water and sterilize it by passing it through a filter<br />
# after autoclaving and cooling down, add sterile phosphate solutions<br />
<br />
=== Nutrient Agar medium (NA) ===<br />
#Components for 1&nbsp;L<br />
#: '''Enzymatic Digest of Gelatin 5&nbsp;g'''<br />
#: '''Beef Extract 3&nbsp;g'''<br />
#: '''Agar 15&nbsp;g'''<br />
# Final pH: 6.8 ± 0.2 at 25°C<br />
# Suspend 23&nbsp;g of the medium in one liter of purified water.<br />
# Heat with frequent agitation and boil for one minute to completely dissolve the medium.<br />
# Autoclave at 121°C for 15&nbsp;min.<br />
<br />
== Minimal Media ==<br />
=== Hartmans Minimal Medium (HM) ===<br />
<br />
This mineral salts medium is based on (Hartmans et al., 1989).<br />
<br />
Three '''100x stock solutions''' are prepared according to the following recipe and stored at 4°C:<br />
<br />
* '''100x Buffer''',composed of 388&nbsp;g dipotassium phosphate, 212&nbsp;g monosodium phosphate dihydrate per Liter, adjusted to a pH of 7.0 and sterilized by autoclaving.<br />
* '''100x ammonium sulfate''' composed of 200&nbsp;g/l ammonium sulfate and sterilized by autoclaving.<br />
* '''100x MM salts'''<br />
# Add 1&nbsp;g EDTA to 25&nbsp;ml water.<br />
# Add drops of 10&nbsp;M sodium hydroxide until EDTA is completely dissolved.<br />
# Adjust pH back to 4.0 with concentrated hypochloric acid.<br />
# Fill up with water to 800&nbsp;ml and dissolve following components:<br />
## 10&nbsp;g magnesium chloride sexahydrate<br />
## 200&nbsp;mg zinc sulfate heptahydrate<br />
## 100&nbsp;mg calcium chloride dihydrate<br />
## 500&nbsp;mg iron(II) sulfate heptahydrate<br />
## 20&nbsp;mg sodium molybdate dihydrate<br />
## 20&nbsp;mg copper(II) sulfate quintahydrate<br />
## 40&nbsp;mg cobalt(II) chloride sexahydrate<br />
## 100&nbsp;mg manganese(II) chloride sexahydrate<br />
<br />
For 1x medium without carbon source, pool 10&nbsp;ml of each '''100x stock solution''' and fill up to 1000&nbsp;ml with sterile, deionized water and store at 4°C.<br />
<br />
=== M9 Minimal Medium (M9) ===<br />
<center><br />
{| class="wikitable centered"<br />
! '''Components for 1&nbsp;L''' !! '''Volume'''<br />
|-<br />
| bidest. water || style="text-align:right"| 778.667&nbsp;ml<br />
|-<br />
| 10x Salt solution || style="text-align:right"| 100&nbsp;ml <br />
|- <br />
| Magnesiumsulfatehaptahydrate (10&nbsp;mM) || style="text-align:right"| 100&nbsp;ml <br />
|- <br />
| Glucose 20% (w/v) || style="text-align:right"| 20&nbsp;ml<br />
|- <br />
| 1000x Trace elements || style="text-align:right"| 1&nbsp;ml <br />
|-<br />
| Thiamin (1&nbsp;mM) || style="text-align:right"| 0.333&nbsp;ml <br />
|}<br />
<br />
<br />
{| class="wikitable centered"<br />
| colspan="3"| '''10x Salt solution''' <br />
|-<br />
! '''Component''' !!''Final concentration''' !! '''Concentration in stock solution'''<br />
|-<br />
| BisTris || style="text-align:right"| 95&nbsp;mM || style="text-align:right"| 200,000&nbsp;mg/L<br />
|-<br />
| Ammmonium chloride || style="text-align:right"| 60&nbsp;mM || style="text-align:right"| 32,100&nbsp;mg/L <br />
|-<br />
| Sodium citrate || style="text-align:right"| 12.5&nbsp;mM || style="text-align:right"| 27,000&nbsp;mg/L<br />
|-<br />
| Monopotassium phosphate || style="text-align:right"| 3&nbsp;mM || style="text-align:right"| 4,170&nbsp;mg/L<br />
|-<br />
| Dipotassium phosphate || style="text-align:right"| 0.7&nbsp;mM || style="text-align:right"| 1,590&nbsp;mg/L <br />
|- <br />
|}<br />
<br />
{| class="wikitable centered"<br />
| colspan="3"| '''1000x Trace elements'''<br />
|-<br />
! '''Component'''!!'''Final concentration''' !!'''Concentration in stock solution'''<br />
|- <br />
| Iron(III) chloride || style="text-align:right"| 50&nbsp;mM || style="text-align:right"| 13,515&nbsp;mg/L <br />
|- <br />
| Calcium chloride || style="text-align:right"| 20&nbsp;mM || style="text-align:right"| 2,220&nbsp;mg/L <br />
|-<br />
| Manganese(II) chloride || style="text-align:right"| 10&nbsp;mM || style="text-align:right"| 1,258&nbsp;mg/L <br />
|- <br />
| Zinc sulfate || style="text-align:right"| 10&nbsp;mM || style="text-align:right"| 1,615&nbsp;mg/L <br />
|- <br />
| Cobalt(II) chloride || style="text-align:right"| 2&nbsp;mM || style="text-align:right"| 260&nbsp;mg/L <br />
|- <br />
| Copper(II) chloride || style="text-align:right"| 2&nbsp;mM || style="text-align:right"| 269&nbsp;mg/L <br />
|-<br />
| Nickel(II) chloride || style="text-align:right"| 2&nbsp;mM || style="text-align:right"| 259&nbsp;mg/L <br />
|- <br />
| Sodium molybdate || style="text-align:right"| 2&nbsp;mM || style="text-align:right"| 412&nbsp;mg/L <br />
|-<br />
| Sodium selenite || style="text-align:right"| 2&nbsp;mM || style="text-align:right"| 346&nbsp;mg/L <br />
|-<br />
| Boric acid || style="text-align:right"| 2&nbsp;mM || style="text-align:right"| 124&nbsp;mg/L <br />
|-<br />
| Hydrochloric acid || style="text-align:right"| 1&nbsp;mM || style="text-align:right"| 20&nbsp;ml <br />
|-<br />
|}<br />
</center><br />
<br />
== Transformation Medium ==<br />
=== Super Optimal broth with Catabolite repression medium (SOC) ===<br />
# components<br />
#: '''0,5% yeast extract'''<br />
#: '''2% tryptone'''<br />
#: '''10&nbsp;mM NaCl'''<br />
#: '''2.5&nbsp;mM KCl'''<br />
#: '''20&nbsp;mM MgSO<sub>4</sub> '''<br />
# fill up with deionized water<br />
# adjust to pH 7.5 with NaOH<br />
# after autoclaving, add 20&nbsp;mM sterile glucose solution (filter sterilization)<br />
<br />
==References==<br />
* Hartmans, S. et al., 1989. Metabolism of Styrene Oxide and 2-Phenylethanol in the Styrene-Degrading Xanthobacter Strain 124X. Applied and Environmental Microbiology, Nov. Available at: http://www.ncbi.nlm.nih.gov/pubmed/?term=PMC203180.<br />
<br />
<html><br />
<center><br />
<ul class="menusmall-grid"><br />
<br />
<li style="width:106px;margin-left: 12px;margin-right: 12px;" ><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Protocols/detection" style="color:black"><br />
<div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel" style="top: 10%; font-size: 14px;">2D Detection of<br/>IPTG & HSL</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/2/22/Aachen_14-10-14_button_chip_manufacturing_ipo.png); norepeat scroll 0% 0% transparent; background-size:100%; height:100px; width: 100px;"><br />
</div><br />
</a><br />
</li><br />
<br />
<li style="width:106px;margin-left: 12px;margin-right: 12px;" ><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Protocols/Culture_medium_and_conditions" style="color:black"><br />
<div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel" style="top: 10%; font-size: 14px;">Culture Media</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/1/10/Aachen_14-10-13_Yellow_Flask_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%; height:100px; width: 100px;"><br />
</div><br />
</a><br />
</li><br />
<br />
<li style="width:106px;margin-left: 12px;margin-right: 12px;" ><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Protocols/Molecular_biological_methods" style="color:black"><br />
<div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel" style="top: 10%; font-size: 14px;">Molecular biological methods</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/7/75/Aachen_14-10-14_Eppi_with_green_cells_panel_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%; height:100px; width: 100px;"><br />
</div><br />
</a><br />
</li><br />
<br />
<li style="width:106px;margin-left: 12px;margin-right: 12px;" ><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Protocols/Analytical_methods" style="color:black"><br />
<div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel" style="top: 10%; font-size: 14px;">Analytical methods</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/4/4d/Aachen_14-10-14_Lense_panel_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%; height:100px; width: 100px;"><br />
</div><br />
</a><br />
</li><br />
<br />
</ul><br />
</center><br />
</html><br />
<br />
{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/Notebook/IndexTeam:Aachen/Notebook/Index2014-10-18T03:39:36Z<p>Nbailly: /* List of abbreviations */</p>
<hr />
<div>{{Team:Aachen/Header}}<br />
<br />
= List of abbreviations =<br />
<center><br />
List of commonly used abbreviations:<br />
{| class="wikitable sortable"<br />
! align="center" |'''Abbreviation'''<br />
!! align="center" |'''Full term'''<br />
|-<br />
| ATP || Adenosinetriphosphate<br />
|-<br />
| BFP || Blue fluorescent protein<br />
|-<br />
| BSA || Bovine serum albumin<br />
|-<br />
| CAM|| Chloramphenicol<br />
|-<br />
| CFP|| Cyan fluorescent protein<br />
|-<br />
| CFU|| Colony forming units<br />
|-<br />
| CDS || Coding sequence<br />
|-<br />
| dNTP || Desoxynucleotidtriphosphate<br />
|-<br />
| EDTA || Ethylenediaminetetraacetic acid<br />
|-<br />
| DSMZ || German collection of microorganisms and cell cultures <br />
|-<br />
| DIY || Do it yourself<br />
|-<br />
| eYFP || Enhanced yellow flourescenct protein<br />
|-<br />
| F || Fluorescence <br />
|-<br />
| FRET || Förster resonance energy transfer<br />
|-<br />
| Gal-3 || Galactin-3 <br />
|-<br />
| GFP || Green fluorescent protein <br />
|-<br />
| GUI || Graphical user interface <br />
|-<br />
| His || Histidin<br />
|-<br />
| HM || Hartman Medium<br />
|-<br />
| HSL || Homoserine lactone<br />
|-<br />
| HSV || hue-saturation-value<br />
|-<br />
| HF || High frequency<br />
|-<br />
| IPTG || Isopropyl β-D-1-thiogalactopyranoside <br />
|-<br />
| LPS || Lipopolysaccharide <br />
|-<br />
| MRSA || Multi resistant ''Staphylococcus aureus'' <br />
|-<br />
| NIAID || National Institute of Allergy and Infectious Diseases<br />
|-<br />
| NTA || Nitrilotriacetic acid<br />
|-<br />
| OD || Optical density <br />
|-<br />
| 3-oxo-C{{sub|12}} HSL || 3-Oxo-dodecanoyl homoserine lactone<br />
|-<br />
| PBS || Phosphate buffered saline<br />
|-<br />
| PCR || Polymerase chain reaction<br />
|-<br />
| RBS || Ribosome binding site <br />
|-<br />
| REACh || Resonance Energy-Accepting Chromoprotein<br />
|-<br />
| RFP || Red fluorescent protein <br />
|-<br />
| SDS-PAGE || Sodium dodecyl sulfate polyacrylamide gel electrophoresis<br />
|-<br />
| SRM || Statistical region merging <br />
|-<br />
| TEV || Tobacco etch virus <br />
|-<br />
| WHO || World health organisation<br />
|-<br />
|}<br />
</center><br />
<br />
<br />
{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/Notebook/LabEquipmentTeam:Aachen/Notebook/LabEquipment2014-10-18T03:39:23Z<p>Nbailly: /* Lab Equipment */</p>
<hr />
<div>{{Team:Aachen/Header}}<br />
<br />
= Lab Equipment =<br />
<br />
<center><br />
Equipment used in our project:<br />
{| class="wikitable sortable"<br />
! align="center" |'''Equipment'''<br />
!! align="center" |'''Company'''<br />
|-<br />
| Avanti 30 Centrifuge || Beckman<br />
|-<br />
| Äkta Prime Plus || GE Healthcare<br />
|-<br />
| Biofuge "pico" Heraues || Thermo Scientific<br />
|-<br />
| Cleanbench || Scanlaf<br />
|-<br />
| Clark Electrode || Hamilton<br />
|-<br />
| GenePulser XCell || BioRad <br />
|-<br />
| Fermenter 1L || Sartorius<br />
|-<br />
| Fermenter control unit Biostat A-Plus || Sartorius<br />
|-<br />
| Freezer || Thermo Scientific <br />
|-<br />
| Geldoc || Herolab<br />
|-<br />
| Geldoc XR+ || Biorad<br />
|-<br />
| Heatplate || Yellow line <br />
|-<br />
| Megafuge 16R Heraeus || Thermo Scientific <br />
|-<br />
| Incubator || VWR<br />
|-<br />
| Mastercycler gradient || Eppendorf<br />
|-<br />
| Micro Centrifuge || Roth<br />
|-<br />
| Microscope ICG-50|| Leica<br />
|-<br />
| Microscope AF6000 LX Videomicroscope || Leica<br />
|-<br />
| Microscope Software LAS AF 3.2.09652 || Leica<br />
|-<br />
| Microscope Orthoplan || Leica<br />
|-<br />
| NanoVue Plus || GE Healthcare<br />
|-<br />
| Pico 17 Centrifuge || Thermo Scientific<br />
|-<br />
| PowerPac 200 + 300 || BioRad<br />
|-<br />
| pH Electrode || Mettler Toledo <br />
|-<br />
| pH/ORP Meter || Hanna Instruments<br />
|-<br />
| Programmable Thermal Controller || MJ Research<br />
|-<br />
| Pipet boy || Sartorius <br />
|-<br />
| Stirrer || Unkermotoren germany<br />
|-<br />
| Shaker || New Brunswick<br />
|-<br />
| Spectrophotometer 1200 || Fisher Bioblock Scientific<br />
|-<br />
| Platereader Synergy MX || BioTek<br />
|-<br />
| Platereader Software GenX || BioTek<br />
|-<br />
| Thermomixer Compact || Eppendorf<br />
|-<br />
| Thermocylcler Primus 25|| MWG Biotech<br />
|-<br />
| Waterbath || Lauda<br />
|-<br />
| Waterbath Frigomix 1000 || Sartorius<br />
|-<br />
| Varifuge 3.0R || Heraues <br />
|-<br />
| Vortexer || Fisher Scientific<br />
|-<br />
<br />
<br />
|}<br />
<br />
<br />
Kits:<br />
{| class="wikitable sortable"<br />
! align="center" |'''Kit'''<br />
!! align="center" |'''Company'''<br />
|-<br />
| GoTaq Green Mastermix || Promega <br />
|-<br />
| Gibson assembly cloning kit || NEB <br />
|-<br />
| Illustra Plasmid Prep Mini Spin Kit || GE Healthcare <br />
|-<br />
| PCR DNA and Gel Band Purification Kit || GE Healthcare <br />
|-<br />
| iGEM Biobrick Assembly Kit || NEB <br />
|-<br />
| Transformation efficiency kit || iGEM Headquarters<br />
|-<br />
| KAPA2G Fast ReadyMix || Kapa Biosystems<br />
|-<br />
|}<br />
</center><br />
<br />
<br />
{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/Collaborations/FreiburgTeam:Aachen/Collaborations/Freiburg2014-10-18T03:38:27Z<p>Nbailly: /* References */</p>
<hr />
<div>__NOTOC__<br />
{{Team:Aachen/Header}}<br />
<br />
= AcCELLoMatrix for [[Team:Freiburg/Team/Collaboration|Team Freiburg]]=<br />
<br />
At the iGEM meetup in Munich in May, members of both of our teams realized that our projects share a common objective: Analyzing 2-dimensional, visual signals. For the following months, we stayed in contact and developed a concept to encode information in 384-well plates in the form of data matrix codes.<br />
<br />
At the beginning of September, Michael spontaneously took a train to Freiburg and we met face-to-face for several hours to exchange details on our cooperation and general iGEM experiences.<br />
<br />
<br />
{{Team:Aachen/Figure|Aachen_FR-collaboration_group_photo.jpg|align=center|title=Spontaneous group photo|subtitle=Freíburg iGEMers and Michael (right) meeting to discuss our cooperation|width=350px}}<br />
<br />
<br />
== Data Matrix Masks ==<br />
<br />
We decided to use masks to locally induce the cells in 384-well plates in a data matrix pattern. To enable us to quickly design masks for different data to be encoded, we wrote a software to easily generate SVG-files for a laser cutter.<br />
<br />
{{Team:Aachen/Figure|Aachen_DataMatrixMaskDesigner.png|align=center|title=Making a datamatrix mask|subtitle=Upon entering a string, the software predicts if the datamatrix will fit into a 384-well plate.|width=350px}}<br />
<br />
After a string has been entered in the designer, an SVG-file can be saved and used for laser cutting the mask:<br />
<br />
{{Team:Aachen/Figure|Aachen_MammoMatrixMasks2.png|align=center|title=Cut Lines|subtitle=This AcCELLoMatrix mask can be lasercut and placed below a 384-well plate.|width=350px}}<br />
<br />
[https://static.igem.org/mediawiki/2014/a/a8/Aachen_DatamatrixMaskDesigner.zip Get the DatamatrixMaskDesigner] to make you own AcCELLoMatrixes! (requires Windows 7/8)<br />
<br />
You can also [https://static.igem.org/mediawiki/2014/c/ce/Aachen_DatamatrixMaskDesigner_Source.zip download the full source code] and compile or modify it with Visual Studio (Express) 2013.<br />
<br />
For the first experiments, we cut two different data matrix patterns from polypropylen foil. The masks can be used to cover the plates from the top or from the bottom.<br />
<br />
{{Team:Aachen/Figure|Aachen_MammoMatrixMasks1.jpg|align=center|title=AcCELLoMatrix masks|subtitle=These masks can be placed on top or beneath 384-well plates to facilitate localized illumination of wells.|width=350px}}<br />
<br />
== AcCELLoMatrix Reader App ==<br />
After mask have been designed and the information was encoded into the well plate by selectively illuminating the cells, how do you get it out again? We made an app for that: the AcCELLoMatrix&nbsp;Reader for Windows&nbsp;Phone&nbsp;8.<br />
<br />
We designed the app to be easy to use: A user can simply open the app<br />
<br />
{{Team:Aachen/Figure|Aachen_Collaboration-FRScreenshots.png|title=Usage of AcCELLoMatrix Reader|width=1018px|subtitle=(1) Start the app and read through some brief instructions (2). Then start scanning and aim for a datamatrix in the wild (3). As soon as a datamatrix is detected in the preprocessed frames (4, small rectangle) by the ZXing algorithm, the decoded text is displayed to the user (5).}}<br />
<br />
While the apps frontend is designed to be very minimalistic, there's actually going on a lot in the backend. Visual data coming from the camera feed has to be processed and fed into the ZXing decoding algorithm.<br />
<br />
{{Team:Aachen/Figure|Aachen_Collaboration-FRAMRflow.png|title=Activity diagram of the app|width=350px|subtitle=After the app was started and the scanning initiated by the user, a loop is initiated. Filters are applied to camera preview frames and the result is fed into the decoding algorithm. As soon as a datamatrix is detected, the loop is interrupted and the result presented to the user.}}<br />
<br />
== Outlook ==<br />
Due to the circumstance that this collaboration was started relatively late, we have not been able to test the decoding of real AcCELLoMatrixes. But we are confident, that with some threshold adjustments to the AcCELLoMatrixes&nbsp;Reader app and maybe the hole-size of the polypropylen masks, the coding and decoding of short text information in the genetic state of mammalian cells in a 384-well plate is perfectly possible.<br />
<br />
== References ==<br />
[http://www.nuget.org/packages/ZXing.Net/ ZXing.NET] by Michael Jahn is a port of [https://github.com/zxing/zxing ZXing] ("zebra crossing"), both licensed under [http://www.apache.org/licenses/LICENSE-2.0 Apache 2.0]<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
= Characterization of our OD Device by Team Freiburg =<br />
As we were helping the Freiburg team out with the encoding/decoding of text as datamatrixes, they also wanted to help us in a useful way. Our team was committed to characterize the OD measurement device, so we sent them one of prototypes and asked them to try a measurement of their mammalian cells.<br />
<br />
They prepared a dilution series with biological triplicates of their NIH3T3 mouse fibroblasts in the respective growth medium. The absolute cell number was determined at 10{{sup|6}}&nbsp;cells/ml. The transmission of light that was measured by our prototype, is plotted against the fraction of cell culture in the sample in the left chart: <br />
<br />
<br />
{{Team:Aachen/FigureDual|Aachen_Collaboration-FR_NIH3T3_1.png|Aachen_Collaboration-FR_NIH3T3_2.png|title1=Transmission at different concentrations|title2=Transmission versus true OD of the sample|subtitle1=A linear dependency was observed between the calculated concentration of cells transmission&nbsp;[%].|subtitle2=Calculation of the true ODs revealed, that the optical density of the samples was very low.|width=425px}} <br />
<br />
From the transmittance data of the dilution series, we calculated the [[Team:Aachen/Notebook/Engineering/ODF#Evaluation|true OD]] of the sample (right chart above).<br />
<br />
Finally, we incorporated the measurements by team Freiburg into our characterization of the OD-measurement device that now covers multiple strains over three orders of magnitude:<br />
<br />
{{Team:Aachen/Figure|Aachen_ODallstrains1.png|title=Transmission of different cell types at OD-values from 0.001-1|subtitle=The transmittance data of NIH&nbsp;3T3 cells align with the transmittance of ''P.&nbsp;putida'' and ''S.&nbsp;cerevisiae'' strains, even though the measured optical densities are lower by 1-2 orders of magnitude.|width=800px}}<br />
<br />
Our friends in Freiburg also tested a Nanodrop, but reported that this commercial device was unable to quantify the diluted samples. In the end we were very satisfied with the results of our cooperation!<br />
<br />
<br />
{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/MeetupTeam:Aachen/Meetup2014-10-18T03:37:40Z<p>Nbailly: </p>
<hr />
<div>__NOTOC__<br />
{{Team:Aachen/Header}}<br />
<html><br />
<script src="https://maps.googleapis.com/maps/api/js?v=3.exp"></script><br />
<script type="text/javascript" src="https://2014.igem.org/Template:Team:Aachen/MeetupMap1.js?action=raw&ctype=text/javascript"></script><br />
</html><br />
<br />
[[File:Aachen_Meetup_Logo.png|200px|right]]<br />
<br />
= Welcome =<br />
Welcome to our Meetup page! All teams are invited to attend the Aachen iGEM Meetup 2014. Please let us know beforehand if your team would like to take part in this unique opportunity to meet other iGEM teams, present and get feedback on our project, and have a lot of fun! Of course, non-iGEM participants are also highly welcome to attend our public event. You can find all the important dates on September 13th and 14th here!<br />
<br />
Für eine deutsche Version dieser Seite bitte [[Team:Aachen/Meetup/de|hier klicken]].<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
= News: iGEM Meetup Last Weekend - a great success! =<br />
<br />
Last weekend, six iGEM teams from all over Germany – namely Braunschweig, Darmstadt, Bielefeld, Tübingen, Freiburg and Munich – met with our team in Aachen.<br />
<br />
{{Team:Aachen/Figure|Aachen DSC 0523.JPG|align=center|title=Welcome teams!|subtitle=All teams were welcomed by our three beautiful receptionists.|width=500px}}<br />
<br />
The weekend started with a welcome ceremony for all iGEM teams in the Couven hall on our RWTH campus. This a game of “Synthetic Biology Jeopardy”, developed and programmed by our team member Michael. The game was divided into three rounds: two preliminary rounds and one final. In the first round, Munich alias “Bier”, Tübingen alias “Most” and Bielefeld alias “Bielefeld” challenged each other in the categories Abbreviations, Amino Acids, Bad Science Movies, Part Categories and Sponsor Logos. With 2400 and 800 points teams Most and Bier, respectively, advanced to the final round. Though the team Bielefeld came in last, they got another chance to proceed to the final round by substituting for Freiburg’s team which didn’t make it on time due to traffic problems.<br />
<br />
{{Team:Aachen/Figure|Aachen DSC 0549.JPG|align=center|title=First round|subtitle=Teams battled each other in the five categories Abbreviations, Amino Acids, Bad Science Movies, Part Categories and Sponsor Logos.|width=500px}}<br />
<br />
<br />
<br />
Team Darmstadt alias “Darmstadt” lead the second round by finding most questions to the answers in the categories Deadlines, DNA, Molecular Masses, Picture Puzzles and Sounds of Science. However, in the second last question, they stumbled across the last “double jeopardy”, set all their points – and lost, enabling team Braunschweig alias “Carola” and team Bielefeld to proceed to the final round. After coming up with questions to the answer belonging to the categories BioBricks, Chemical Structures, Inheritance, Plots and Propaganda, team Most came out as the winner, while Bier, Bielefeld and Carola came in second, third and forth, respectively.<br />
<br />
{{Team:Aachen/Figure|Aachen DSC 0565.JPG|align=center|title=Picture Puzzles|subtitle=What could these three pictures stand for?|width=500px}}<br />
<br />
After a break, all teams came together again at 7 pm to welcome guests to the public part of the event. In 15-minute presentations, the teams from Bielefeld, Darmstand, Braunschweig, Tübingen, Munich and Aachen presented their project ideas to the public audience and answered some questions in the subsequent discussion round. Then the teams walked over to the residence village where we finished the evening with some pizza and drinks.<br />
<br />
{{Team:Aachen/Figure|Aachen DSC 0603.JPG|align=center|title=Take a break!|subtitle=After the jeopardy meetup participants could enjoy a hearty snack before preparing for the public presentations.|width=500px}}<br />
{{Team:Aachen/Figure|Aachen DSC 0644.JPG|align=center|title=Public presentations|subtitle=Anna introduces iGEM team Braunschweig at the public presentations.|width=500px}}<br />
<br />
Although the night was quite short for some people, almost all teams made it to the Couven hall at 10 am on Sunday. We started the day with a presentation about SynBio research by Alexander Grünberg from the Jülich Research Centre, followed by Brian Claudill from IDT Biologica who introduced the concept of the company’s GBlocks® to the audience.<br />
<br />
Subsequently, the teams from Munich, Freiburg, Braunschweig, Bielefeld and Aachen gave more detailed presentations than the day before about their this year’s iGEM projects. Each presentation was vividly discussed and some valuable improvement suggestions were brainstormed. <br />
<br />
{{Team:Aachen/Figure|Aachen DSC 0699.JPG|align=center|title=BaKillus|subtitle=Members of iGEM team Munich present their project to the other teams.|width=500px}}<br />
<br />
Last but not least, the three winning teams of Saturday’s jeopardy game were rewarded with prizes, and we took this beautiful group photo:<br />
<br />
{{Team:Aachen/Figure|Aachen_IGEM_Aachen_Meetup_Gruppenbild_3.jpg|align=center|title=Group Picture|subtitle=It should be noted that Darmstadt also attended our meetup, but was indisposed when the picture was taken. Thanks to Carsten&nbsp;Ludwig from team Braunschweig for lending his camera!|width=500px}} <br />
<br />
The iGEM team Aachen hopes that everybody had a great time at our meetup, and that you could collect some valuable feedback about your projects! See you in Boston!<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
= Schedule =<br />
<br />
<br />
== Saturday ==<br />
<br />
<center style="font-size: 12pt;"><br />
{| border="1" cellpadding="10" cellspacing="0"<br />
! Time !! Session !! Location<br />
|-<br />
| noon || Arrival of the teams || Hotels and Hostels in Aachen<br />
|-<br />
| 2:30pm || Welcome reception || Couvenhalle<br />
|-<br />
| 3:00pm || Get-together || Couvenhalle<br />
|-<br />
| 3:30pm || Jeopardy! || Couvenhalle<br />
|-<br />
| 7:00pm || Public presentations of team projects || Couvenhalle<br />
|-<br />
| 10:00pm || Dinner and more || Student village & Pontstraße<br />
|}<br />
</center><br />
<br />
== Sunday ==<br />
<center style="font-size: 12pt;"><br />
{| border="1" cellpadding="10" cellspacing="0"<br />
! Time !! Session !! Location<br />
|-<br />
| 10am || Presentations by institutes and companies || Couvenhalle<br />
|-<br />
| 12am || Presentations of team projects || Couvenhalle<br />
|-<br />
| 4pm || Award ceremony of the Jeopardy! || Couvenhalle<br />
|-<br />
| 5pm || End || Couvenhalle<br />
|}<br />
</center><br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
= Location: Couvenhalle =<br />
{{Team:Aachen/Figure|DSC_0530.JPG|title=Couvenhalle|subtitle=Location|width=500px}}<br />
<html><br />
<div id="map-couvenhalle" style="width: 100%; height: 500px"></div><br />
</html><br />
<br />
== Address ==<br />
Couvenhalle<br />
<br />
Kármánstr. 17-19<br />
<br />
52062 Aachen<br />
<br />
== Getting there by bus ==<br />
=== Bus 13A/B ===<br />
If the bus timetable allows, take bus 13A/B to stop 'Technische Hochschule'.<br />
Take the way on the right hand side around the Kármán building and you will find the Couvenhalle on the right.<br />
<br />
=== Bus 3A/B ===<br />
Most of the times bus 13A/B will not operate. Then either take bus 3A/B to Audimax and walk in direction of RWTH Hauptgebäude/SuperC. You will find the bus stop 'Technische Hochschule' on your way.<br />
<br />
=== More busses ===<br />
Many buses stop at Driescher Gässchen.<br />
Exit the bus there and walk towards the Kármán Auditorium, RWTH Hauptgebäude/SuperC (in direction of a newly built street).<br />
<br />
To plan your bus trip visit the [http://aseag.de/ ASEAG] homepage.<br />
<br />
== Getting around in Aachen ==<br />
<br />
Aachen is, compared to other cities, quite compact. Basically, every route is well doable by foot. This way it is even possible to cross Aachen in 30 minutes! For instance, walking from Couvenhalle to Schanz is roughly 20 minutes by foot.<br />
<br />
Regarding bus tickets, and depending on whether you wish to get one yourself or not, we highly recommend to stick together in groups of 5 and get a 'Minigruppenticket'.<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
= Things to do in Aachen =<br />
<br />
If you are arriving early enough (or are departing late enough), there are a few things to do in Aachen.<br />
<br />
== Aachener Dom / Cathedral ==<br />
<br />
The [http://www.aachendom.de/ cathedral] is the major landmark of Aachen. It was the palatinate of Charlemagne, and several other ancient kings and emperors were crowned under the cathedral's dome. A relict from these days is the marble throne on the upper level of the cathedral. Since 1978, it is an UNESCO World Heritage, the first one in Germany.<br />
<br />
If you are a history geek or just want to learn more, the cathedral is a great place to visit. Taking a guided tour is highly recommended!<br />
<br />
== Lindt, Bahlsen & Lambertz Werksverkauf / Lindt, Bahlsen & Lambertz factory outlet ==<br />
<br />
Who does not like chocolate?!<br />
If you ever wanted to get Lindt chocolate for cheap, we highly recommend this opportunity! 1kg of parlines for less than 5€ seems tempting, doesn't it?<br />
<br />
'''Hours:''' Monday till Friday, 9am - 6pm and Saturday, 9am - 4pm <br /><br />
'''Address:''' Süsterfeldstraße 130, 52072 Aachen <br />
<br />
<br />
If you make it to Lindt, stopping by at the Bahlsen factory outlet is worth the time as well. It is just across the street from Lindt. Cookie monsters will love this place!<br />
<br />
'''Hours:''' Monday till Friday, 9am - 6pm and Saturday, 9am - 1pm <br /><br />
'''Address:''' Süsterfeldstr. 27, 52072 Aachen <br />
<br />
<br />
Just down the street, there is another factory outlet by Lambertz. Their specialties are gingerbread and the famous Aachener Printen which you can get all year round. Christmas in September!<br />
<br />
'''Hours:''' Monday till Friday, 9am - 6pm and Saturday, 9am - 1pm <br /><br />
'''Address:''' Ritterstraße 9, 52072 Aachen <br />
<br />
{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/MeetupTeam:Aachen/Meetup2014-10-18T03:37:15Z<p>Nbailly: /* Welcome */</p>
<hr />
<div>__NOTOC__<br />
{{Team:Aachen/Header}}<br />
<html><br />
<script src="https://maps.googleapis.com/maps/api/js?v=3.exp"></script><br />
<script type="text/javascript" src="https://2014.igem.org/Template:Team:Aachen/MeetupMap1.js?action=raw&ctype=text/javascript"></script><br />
</html><br />
<br />
[[File:Aachen_Meetup_Logo.png|200px|right]]<br />
<br />
= Welcome =<br />
Welcome to our Meetup page! All teams are invited to attend the Aachen iGEM Meetup 2014. Please let us know beforehand if your team would like to take part in this unique opportunity to meet other iGEM teams, present and get feedback on our project, and have a lot of fun! Of course, non-iGEM participants are also highly welcome to attend our public event. You can find all the important dates on September 13th and 14th here!<br />
<br />
Für eine deutsche Version dieser Seite bitte [[Team:Aachen/Meetup/de|hier klicken]].<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
= News: iGEM Meetup Last Weekend - a great success! =<br />
<br />
Last weekend, six iGEM teams from all over Germany – namely Braunschweig, Darmstadt, Bielefeld, Tübingen, Freiburg and Munich – met with our team in Aachen.<br />
<br />
{{Team:Aachen/Figure|Aachen DSC 0523.JPG|align=center|title=Welcome teams!|subtitle=All teams were welcomed by our three beautiful receptionists.|width=500px}}<br />
<br />
The weekend started with a welcome ceremony for all iGEM teams in the Couven hall on our RWTH campus. This a game of “Synthetic Biology Jeopardy”, developed and programmed by our team member Michael. The game was divided into three rounds: two preliminary rounds and one final. In the first round, Munich alias “Bier”, Tübingen alias “Most” and Bielefeld alias “Bielefeld” challenged each other in the categories Abbreviations, Amino Acids, Bad Science Movies, Part Categories and Sponsor Logos. With 2400 and 800 points teams Most and Bier, respectively, advanced to the final round. Though the team Bielefeld came in last, they got another chance to proceed to the final round by substituting for Freiburg’s team which didn’t make it on time due to traffic problems.<br />
<br />
{{Team:Aachen/Figure|Aachen DSC 0549.JPG|align=center|title=First round|subtitle=Teams battled each other in the five categories Abbreviations, Amino Acids, Bad Science Movies, Part Categories and Sponsor Logos.|width=500px}}<br />
<br />
<br />
<br />
Team Darmstadt alias “Darmstadt” lead the second round by finding most questions to the answers in the categories Deadlines, DNA, Molecular Masses, Picture Puzzles and Sounds of Science. However, in the second last question, they stumbled across the last “double jeopardy”, set all their points – and lost, enabling team Braunschweig alias “Carola” and team Bielefeld to proceed to the final round. After coming up with questions to the answer belonging to the categories BioBricks, Chemical Structures, Inheritance, Plots and Propaganda, team Most came out as the winner, while Bier, Bielefeld and Carola came in second, third and forth, respectively.<br />
<br />
{{Team:Aachen/Figure|Aachen DSC 0565.JPG|align=center|title=Picture Puzzles|subtitle=What could these three pictures stand for?|width=500px}}<br />
<br />
After a break, all teams came together again at 7 pm to welcome guests to the public part of the event. In 15-minute presentations, the teams from Bielefeld, Darmstand, Braunschweig, Tübingen, Munich and Aachen presented their project ideas to the public audience and answered some questions in the subsequent discussion round. Then the teams walked over to the residence village where we finished the evening with some pizza and drinks.<br />
<br />
{{Team:Aachen/Figure|Aachen DSC 0603.JPG|align=center|title=Take a break!|subtitle=After the jeopardy meetup participants could enjoy a hearty snack before preparing for the public presentations.|width=500px}}<br />
{{Team:Aachen/Figure|Aachen DSC 0644.JPG|align=center|title=Public presentations|subtitle=Anna introduces iGEM team Braunschweig at the public presentations.|width=500px}}<br />
<br />
Although the night was quite short for some people, almost all teams made it to the Couven hall at 10 am on Sunday. We started the day with a presentation about SynBio research by Alexander Grünberg from the Jülich Research Centre, followed by Brian Claudill from IDT Biologica who introduced the concept of the company’s GBlocks® to the audience.<br />
<br />
Subsequently, the teams from Munich, Freiburg, Braunschweig, Bielefeld and Aachen gave more detailed presentations than the day before about their this year’s iGEM projects. Each presentation was vividly discussed and some valuable improvement suggestions were brainstormed. <br />
<br />
{{Team:Aachen/Figure|Aachen DSC 0699.JPG|align=center|title=BaKillus|subtitle=Members of iGEM team Munich present their project to the other teams.|width=500px}}<br />
<br />
Last but not least, the three winning teams of Saturday’s jeopardy game were rewarded with prizes, and we took this beautiful group photo:<br />
<br />
{{Team:Aachen/Figure|Aachen_IGEM_Aachen_Meetup_Gruppenbild_3.jpg|align=center|title=Group Picture|subtitle=It should be noted that Darmstadt also attended our meetup, but was indisposed when the picture was taken. Thanks to Carsten&nbsp;Ludwig from team Braunschweig for lending his camera!|width=500px}} <br />
<br />
The iGEM team Aachen hopes that everybody had a great time at our meetup, and that you could collect some valuable feedback about your projects! See you in Boston!<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
= Schedule =<br />
<br />
<br />
== Saturday ==<br />
<br />
<center style="font-size: 12pt;"><br />
{| border="1" cellpadding="10" cellspacing="0"<br />
! Time !! Session !! Location<br />
|-<br />
| noon || Arrival of the teams || Hotels and Hostels in Aachen<br />
|-<br />
| 2:30pm || Welcome reception || Couvenhalle<br />
|-<br />
| 3:00pm || Get-together || Couvenhalle<br />
|-<br />
| 3:30pm || Jeopardy! || Couvenhalle<br />
|-<br />
| 7:00pm || Public presentations of team projects || Couvenhalle<br />
|-<br />
| 10:00pm || Dinner and more || Student village & Pontstraße<br />
|}<br />
</center><br />
<br />
== Sunday ==<br />
<center style="font-size: 12pt;"><br />
{| border="1" cellpadding="10" cellspacing="0"<br />
! Time !! Session !! Location<br />
|-<br />
| 10am || Presentations by institutes and companies || Couvenhalle<br />
|-<br />
| 12am || Presentations of team projects || Couvenhalle<br />
|-<br />
| 4pm || Award ceremony of the Jeopardy! || Couvenhalle<br />
|-<br />
| 5pm || End || Couvenhalle<br />
|}<br />
</center><br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
= Location: Couvenhalle =<br />
{{Team:Aachen/Figure|DSC_0530.JPG|title=Couvenhalle|subtitle=Location|width=500px}}<br />
<html><br />
<div id="map-couvenhalle" style="width: 100%; height: 500px"></div><br />
</html><br />
<br />
== Address ==<br />
Couvenhalle<br />
<br />
Kármánstr. 17-19<br />
<br />
52062 Aachen<br />
<br />
== Getting there by bus ==<br />
=== Bus 13A/B ===<br />
If the bus timetable allows, take bus 13A/B to stop 'Technische Hochschule'.<br />
Take the way on the right hand side around the Kármán building and you will find the Couvenhalle on the right.<br />
<br />
=== Bus 3A/B ===<br />
Most of the times bus 13A/B will not operate. Then either take bus 3A/B to Audimax and walk in direction of RWTH Hauptgebäude/SuperC. You will find the bus stop 'Technische Hochschule' on your way.<br />
<br />
=== More busses ===<br />
Many buses stop at Driescher Gässchen.<br />
Exit the bus there and walk towards the Kármán Auditorium, RWTH Hauptgebäude/SuperC (in direction of a newly built street).<br />
<br />
To plan your bus trip visit the [http://aseag.de/ ASEAG] homepage.<br />
<br />
== Getting around in Aachen ==<br />
<br />
Aachen is, compared to other cities, quite compact. Basically, every route is well doable by foot. This way it is even possible to cross Aachen in 30 minutes! For instance, walking from Couvenhalle to Schanz is roughly 20 minutes by foot.<br />
<br />
Regarding bus tickets, and depending on whether you wish to get one yourself or not, we highly recommend to stick together in groups of 5 and get a 'Minigruppenticket'.<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
= Things to do in Aachen =<br />
<br />
If you are arriving early enough (or are departing late enough), there are a few things to do in Aachen.<br />
<br />
== Aachener Dom / Cathedral ==<br />
<br />
The [http://www.aachendom.de/ cathedral] is the major landmark of Aachen. It was the palatinate of Charlemagne, and several other ancient kings and emperors were crowned under the cathedral's dome. A relict from these days is the marble throne on the upper level of the cathedral. Since 1978, it is an UNESCO World Heritage, the first one in Germany.<br />
<br />
If you are a history geek or just want to learn more, the cathedral is a great place to visit. Taking a guided tour is highly recommended!<br />
<br />
== Lindt, Bahlsen & Lambertz Werksverkauf / Lindt, Bahlsen & Lambertz factory outlet ==<br />
<br />
Who does not like chocolate?!<br />
If you ever wanted to get Lindt chocolate for cheap, we highly recommend this opportunity! 1kg of parlines for less than 5€ seems tempting, doesn't it?<br />
<br />
'''Hours:''' Monday till Friday, 9am - 6pm and Saturday, 9am - 4pm <br /><br />
'''Address:''' Süsterfeldstraße 130, 52072 Aachen <br />
<br />
<br />
If you make it to Lindt, stopping by at the Bahlsen factory outlet is worth the time as well. It is just across the street from Lindt. Cookie monsters will love this place!<br />
<br />
'''Hours:''' Monday till Friday, 9am - 6pm and Saturday, 9am - 1pm <br /><br />
'''Address:''' Süsterfeldstr. 27, 52072 Aachen <br />
<br />
<br />
Just down the street, there is another factory outlet by Lambertz. Their specialties are gingerbread and the famous Aachener Printen which you can get all year round. Christmas in September!<br />
<br />
'''Hours:''' Monday till Friday, 9am - 6pm and Saturday, 9am - 1pm <br /><br />
'''Address:''' Ritterstraße 9, 52072 Aachen <br />
<br />
{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/BlogTeam:Aachen/Blog2014-10-18T03:36:55Z<p>Nbailly: </p>
<hr />
<div>__NOTOC__<br />
{{Team:Aachen/Header}}<br />
{{Template:Team:Aachen/Stylesheet}}<br />
= Blog #21 - iGEM Meetup Last Weekend - A Great Success! =<br />
by [[User:Nbailly|Nina]] 09:27, 16 September 2014 (CDT)<br />
<br />
{{Team:Aachen/FigureFloat|Aachen_IGEM_Aachen_Meetup_Gruppenbild_3.jpg|title=Group Picture|subtitle=A final group picture taken by Carsten Ludwig from team Braunschweig.|left|width=500px}}<br />
<br />
Last weekend, six iGEM teams from all over Germany – namely Braunschweig, Darmstadt, Bielefeld, Tübingen, Freiburg and Munich – met with our team in Aachen.<br />
<br />
The weekend started with a welcome ceremony for all iGEM teams in the Couven hall on our RWTH campus. After our Jeopardy battle, the teams from Bielefeld, Darmstand, Braunschweig, Tübingen, Munich and Aachen presented their project ideas in 15-minute talks to the public audience. [https://2014.igem.org/Team:Aachen/Blog/14-09-16-01 Read more...]<br />
<br />
For a German version of this blog entry, please click [https://2014.igem.org/Team:Aachen/Blog/14-09-16-02 here].<br />
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= Blog #20 - Won a gene synthesis from Eurofins =<br />
by [[User:Mosthege|Michael]] 03:25, 18 July 2014 (CDT)<br />
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Two weeks ago we had an email in our inbox that made us very happy: We had won a gene synthesis from [http://eurofinsgenomics.eu/en/eurofins-genomics/corporate-information/deals-promotions/igem-sponsorship.aspx Eurofins Genomics]!<br />
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On Wednesday, we were then visited by Ms. Blumstein who handed us the voucher. Then we took this beautiful photo:<br />
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[[Image:Aachen_14-07-16_GruppenbildEurofins.jpg|center|300px]]<br />
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= Blog #19 - Noneffective Antibiotics (Part III): Have the Pharma Giants Lost The Race?=<br />
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By [[User:NBailly|NBailly]] 15:39, July 15 2014 (CDT)<br />
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{{Team:Aachen/FigureFloat|Aachen_David-Goliath-S1-300x261.jpg|left|width=300px|title=David and Goliath|subtitle=Small Companies have taken leading position in antibiotics research. Picture by LegalIT Lawyers.}}<br />
<html>It is not the big pharma companies that will develop the desperately needed new generation of antibiotics that can even cope with multi-resistant bacteria such as MRSA. Instead, some smaller companies who never gave up researching this field are now future market leaders. As the German business news magazine <a href="http://www.wiwo.de/technologie/forschung/wirkungslose-antibiotika-krankenhaeuser-als-sammelbecken-von-multiresistenten-keimen/10076108-2.html">Wirtschaftswoche</a> reported this June, it is companies like the British-Swedish Astra Zeneca or Hoffmann-La Roche spin off Basilea that are now in a leading position.</html><br />
[https://2014.igem.org/Team:Aachen/Blog/14-07-15-01 Read more...]<br />
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= Blog #18 - Noneffective Antibiotics (Part II): Hospitals as Receptacles for Multi-Resistant Pathogens =<br />
By [[User:NBailly|NBailly]] 17:21, July 14 2014 (CDT)<br />
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{{Team:Aachen/FigureFloat|Aachen_mrsa.jpg|width=200px|left|title=SEM of a human immune cell ingesting MRSA|subtitle=Picture by NIAID.}}<br />
<html>In their article <a href="http://www.wiwo.de/technologie/forschung/wirkungslose-antibiotika-krankenhaeuser-als-sammelbecken-von-multiresistenten-keimen/10076108-2.html">Noneffective Antibiotics</a> published in June, the German business news magazine Wirtschaftswoche explains it as follows: Antibiotics are the natural weapon of molds or soil microbes against competing bacterial growth. These bacteria under siege in turn counterattack with resistances: attack and defense – the natural course of evolution. Thus it was only natural, too, that since the first human use of penicillin and co., pathogens have developed strategies in order to escape the antibiotics’ effect albeit these drugs are quite insidious weapons.</html> [https://2014.igem.org/Team:Aachen/Blog/14-07-14-01 Read more...]<br />
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= Blog #17 - Open Hardware at the Maker Faire Hannover =<br />
[[User:Mosthege|Michael]] 03:55, 6 July 2014 (CDT)<br />
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This weekend [[User:fgohr|Florian]], [[User:R.hanke|René]] and [[User:mosthege|Michael]] joined [[User:Ansgar|Ansgar]] on a trip to the [http://makerfairehannover.com/ Maker Faire Hannover 2014].<br />
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The Maker Faire is an exhibition with things that people built on their own using readily available tools. Dozens of 3D printers and microcontrollers can be examined and there are a lot of devices visitors can interact with.<br />
Apart from the fire-spitting dragon, our OD and fluorescence measurement devices attracted a lot of attention. We spoke with hundreds of visitors and had a lot of interesting conversations. [https://2014.igem.org/Team:Aachen/Blog/14-07-06-01 Read more...]<br />
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= Blog #16 - iGEM team at RWTH Open House =<br />
By [[User:NBailly|NBailly]] 15:22, June 29 2014 (CDT)<br />
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{{Team:Aachen/FigureFloat|Aachen_14-06-28_Erstinfotag_(2).JPG|left|width=300px|title=Our iGEM stand|subtitle=[[User:NBailly|Nina]] and [[User:fgohr|Florian]] showed students how they can get involved in research outside of the programs..}}<br />
<html>Yesterday, RWTH organized an <a href="http://www.rwth-aachen.de/cms/root/Studium/Vor_dem_Studium/Liste/~tfg/ErstInfoTag-Entdecke-die-Welt-des-Studi/">open house</a> for students from ages 14 to 16. Different departments, institutes and student councils presented exhibits, latest research projects, and the many programs they offer. Students could actively try out science and research in prepared experiments at each stand. Various groups of student representatives also offered first hand advice to students with respect to what to study and when and where to collect information. In presentations about the different fields of studies, students gained first insights into the world of academic professions and other career perspectives.</html> [https://2014.igem.org/Team:Aachen/Blog/14-06-29-01 Read more...]<br />
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= Blog #15 - Noneffective Antibiotics (Part I): Dangerous Battle against Killer Germs =<br />
By [[User:NBailly|NBailly]] 17:06, June 23 2014 (CDT)<br />
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{{Team:Aachen/FigureFloat|Aachen_SEM_of_Pseudomonas_aeruginosa.jpg|width=400px|title= SEM of ''Pseudomonas aeruginosa''|subtitle=Picture by Janice Haney Carr.}}<br />
<html>Are we soon going to be dying of pneumonia again? It is almost unimaginable, but the danger that our antibiotics will soon fail against plagues thought to be conquered a long time ago, is eminent. In fact, the situation has grown very acute. The all-purpose weapon antibiotic is on the verge of losing its vigor since many pathogens, such as the pneumonia causing bacterium <i>Pseudomonas aeruginosa</i> , have become resistant. Experts of the World Health Organization (WHO) have already raised a loud alarm: In their recently published first <a href="http://www.who.int/drugresistance/documents/surveillancereport/en/" title="WHO Antimicrobial resistance: global report on surveillance 2014" target="_blank">global resistance report</a>, they are drawing a rather apocalyptic picture. If nothing is done, doctors soon might not be able to do anything but stand helplessly next to their patients while they are dying from nowadays easily curable diseases or even smallest wound infections.</html> [https://2014.igem.org/Team:Aachen/Blog/14-06-23-01 Read more...]<br />
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= Blog #14 - Students Explore Careers in Synthetic Biology =<br />
By [[User:NBailly|NBailly]] 21:11, June 12 2014 (CDT)<br />
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{{Team:Aachen/FigureFloat|Cellock_liegend.png|left|width=300px}}<br />
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Our "Synthetic Biology" teaching module has come to an end. Today was our last day at Kaiser-Karl-Gymnasium in Aachen, where we have worked together with a grade 9 biology-chemistry class for past 1 1/2 months. <!--more-->The students have learned about the iGEM and synthetic biology in general. We presented our project to the class, and explained various aspects for our endeavour in more detail.<br />
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To give interested students some overview of how they can get involved in synthetic biology, Vera, Florian, Ansgar and Björn gave short presentations about each university program at RWTH represented in our team. [https://2014.igem.org/Team:Aachen/Blog/14-06-12-01 Read more...]<br />
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= Blog #13 - Teaching Module Wrap-Up =<br />
By [[User:NBailly|NBailly]] 16:32, June 05 2014 (CDT)<br />
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Today [[User:NBailly|Nina]] and [[User:AZimmermann|Arne]] discussed last class's experiment. Arne explained to the students the meaning behind the values measured, and how the students can use the value to determine the concentration of riboflavin in their vanilla pudding samples. [https://2014.igem.org/Team:Aachen/Blog/14-06-05-01 Read more...]<br />
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[[File:Cellock_stehend.png|center|300px|frameless]]<br />
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= Blog #12 - Glowing vanilla pudding =<br />
By [[User:NBailly|NBailly]] 16:16, June 02 2014 (CDT)<br />
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{{Team:Aachen/FigureFloat|Aachen KKG (15).jpg|left|width=300px|title=Our iGEM stand|subtitle=Things needed for a cool experiment.}}<br />
The topics of today's double lesson was quorum sensing as well as measurement of fluorescence. At the beginning of class, the students form 6 groups and start an experiment dealing with fluorescence: Each group weighs and dissolves 4g of vanilla pudding powder in 50mL of water. While conducting the experiment, each group is supervised by a member of our iGEM team. [https://2014.igem.org/Team:Aachen/Blog/14-06-02-01 Read more...]<br />
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= Blog #11 - No time for high politics =<br />
By [[User:NBailly|NBailly]] 15:42, May 31 2014 (CDT)<br />
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{{Team:Aachen/FigureFloat|Aachen HuClimateUN.jpg|width=300px|title=UN Climate Change Conference|subtitle=Summits like the UN Climate Change Conference are the wrong approach to problems such as antibiotic resistances, says Lars Fischer. Picture from the Intern Blog of American University.}}<br />
International collaborations against antibiotic resistances are all well and good, but the UN Climate Change conference of all collaborations shows why coordination at the government level is the wrong approach, says Lars Fischer, editorial journalist at "Spektrum Der Wissenschaft".<br />
Not even a century ago, the discovery of antibiotics caused a decisive turn for the better in the millennia-long battle against infectious diseases. But while many already saw pathogenic agents eradicated and a golden age of medicine seemed within reach, we have now however reached an abyss: a dramatic relapse into a world looms ahead, where microorganisms are again rulers over life and death. <br />
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The threat emanating from the increasing antibiotic resistances is existential—for every one of us, as well as the countries and society itself. That the world community unites in a supranational board in order to face the latter, suggests itself. However, as alluring as the vision of an intergovernmental panel on antimicrobial resistance may be, as evoked by Mark Woolhouse and Jeremy Farrar (authors of the Nature article "Policy: An intergovernmental panel on antimicrobial resistance", published May 2014), this thought is gravely mistaken. [https://2014.igem.org/Team:Aachen/Blog/14-05-31-01 Read more...]<br />
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= Blog #10 - Microorganisms on the rise =<br />
By [[User:NBailly|NBailly]] 16:05, May 26 2014 (CDT)<br />
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{{Team:Aachen/FigureFloat|10404745_641111075966352_965198584_o.jpg|right|width=200px}}<br />
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Last lesson students at Kaiser-Karl-Gymnasium took environmental samples using a variety of different contact agar plates. On the weekend, René examined the plates under the microscope and took photos. Today [[User:R.hanke|René]] and [[User:NBailly|Nina]] present the students the results of the experiment.<!--more--> But first, we showed the students an excerpt from the TV show "Planetopia" that broaches the issue "Hygiene in every-day life". [https://2014.igem.org/Team:Aachen/Blog/14-05-26-01 Read more...]<br />
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= Blog #9 - Going to Munich =<br />
By [[User:AZimmermann|AZimmermann]] 15:02, May 23 2014 (CDT)<br />
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Hello iGEM enthusiasts,<br />
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on the coming weekend 3 of our team ([[User:Mjoppich|Markus]], [[User:Mosthege|Michael]] and [[User:AZimmermann|Arne]]) are going to travel to Munich for the iGEM meetup iGEM meets CAS. We are very excited for this and can’t wait to meet all the other teams who will be attending. Stay tuned to this blog to see the first pictures when we come back!<br />
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So stay tuned!<br />
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= Blog #8 - iGEM team members back 2 school =<br />
By [[User:NBailly|NBailly]] 16:00, May 22 2014 (CDT)<br />
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{{Team:Aachen/FigureFloat|Aachen_Kaiser-Karls-Gymnasium.jpg|right|width=200px}}<br />
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Hello iGEM enthusiasts,<br />
Since our last visit, the student of the biology-chemistry course at Kaiser-Karl-Gymnasium in Aachen have been busy studying the basics of protein biosynthesis and the "lock and key" model. Now the 9th graders are prepared to have a closer look at synthetic biology and our project.<br />
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[https://2014.igem.org/Team:Aachen/Blog/14-05-22-01 Read more...]<br />
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= Blog #7 - The Biobricks have arrived =<br />
By [[User:AZimmermann|AZimmermann]] 16:02, May 12 2014 (CDT)<br />
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{{Team:Aachen/FigureFloat|Aachen_Biobricks_arrival_2014.jpg|left|width=300px}}<br />
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Hello iGEM enthusiasts,<br />
we have finally gotten the iGEM 2014 biobricks!<br />
Now we can really start working on the molecular side of our project.<br />
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The reveal of our project, coupled with the launch of our new website will be done by the end of next week.<br />
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So stay tuned!<br />
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= Blog #6 - Preparing Cool Experiments =<br />
By [[User:AZimmermann|AZimmermann]] 12:12, May 7 2014 (CDT)<br />
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Hello iGEM enthusiasts,<br />
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We are currently working together with the Kaiser-Karl-Gymnasium in Aachen. Together we are teaching the students about synthetic biology, iGEM and our project in particular. As a part of this, we are designing cool experiments for the students to do themselves. Here a some nice pictures of us preparing them:<br />
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<li><a><img src="https://static.igem.org/mediawiki/2014/thumb/2/27/Aachen_prepping_school_project_2.jpg/450px-Aachen_prepping_school_project_2.jpg"/></a><div class="label_text"><p></p></div></li><br />
<li><a><img src="https://static.igem.org/mediawiki/2014/thumb/1/1b/Aachen_prepping_scholl_project_3.jpg/450px-Aachen_prepping_scholl_project_3.jpg"/></a><div class="label_text"><p></p></div></li><br />
<li><a><img src="https://static.igem.org/mediawiki/2014/thumb/6/65/Aachen_prepping_school_project_4.jpg/450px-Aachen_prepping_school_project_4.jpg"/></a><div class="label_text"><p>We got a fluorescence readout!</p></div></li><br />
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= Blog #5 - Assembly in progress =<br />
By [[User:AZimmermann|AZimmermann]] 12:56, May 5 2014 (CDT)<br />
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Hello iGEM enthusiasts,<br />
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if you are like us and can't wait for our wiki page to go fully online (see last post for more information) and the project to be fully revealed, here are some pictures to tide you over until then:<br />
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Can you guess what we are assembling?<br />
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<li><a><img src="https://static.igem.org/mediawiki/2014/thumb/e/e8/Aachen_device_building_1_2.JPG/780px-Aachen_device_building_1_2.JPG"></a><div class="label_text"><p>Hier Text möglich</p></div></li><br />
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= Blog #4 - We are approved! =<br />
By [[User:AZimmermann|AZimmermann]] 11:00, May 4 2014 (CDT)<br />
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{{Team:Aachen/Figure|Aachen_NEB_Kit_Arrival.jpg|width=400px|title= Also in this blog...|subtitle=NEB is awesome!}}<br />
Hello iGEM enthusiasts,<br />
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we are more than happy to finally be able to announce that our application has been finally approved by the iGEM headquarters. Now nothing will stand between us and our journey towards the giant Jamboree at the end of October! <br />
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[https://2014.igem.org/Team:Aachen/Blog/14-05-04-01 Read more...]<br />
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= Blog #3 - School Project Kick-Off =<br />
By [[User:NBailly|NBailly]] 20:48, April 28 2014 (CDT)<br />
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Today was the kick-off for the cooperation between our iGEM team and the Kaiser-Karl-Gymnasium, a secondary school in Aachen.<br />
In the upcoming weeks, exciting school lessons with members of our iGEM team await the students of the 9th grade biology-chemistry class. Different aspects of our project will be highlighted and explained to the students. Demonstrative experiments will explain the practical relevance.<br />
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In today's lesson, our members [[User:NBailly|Nina]] and [[User:R.hanke|René]] offered the students a short impression of what to expect in the course of this teaching module. We also explained synthetic biology and the goal of our project.<br />
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= Blog #2 - The box is opened =<br />
By [[User:AZimmermann|AZimmermann]] 9:46, April 28 2014 (CDT)<br />
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Hello iGEM enthusiasts,<br />
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interested to see what is inside the box from the previous post?<br />
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<li><a><img src="https://static.igem.org/mediawiki/2014/thumb/e/e1/Aachen_Peltier_Temperatursensor.jpg/780px-Aachen_Peltier_Temperatursensor.jpg"/></a><div class="label_text"><p>Hier Text möglich</p></div></li><br />
<li><a><img src="https://static.igem.org/mediawiki/2014/thumb/c/c4/Aachen_Widerstaende_LEDs.jpg/780px-Aachen_Widerstaende_LEDs.jpg"/></a><div class="label_text"><p>Hier Text möglich</p></div></li><br />
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Guess what we are building?<br />
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= Blog #1 - The opening =<br />
By [[User:AZimmermann|AZimmermann]] 10:31, April 24 2014 (CDT)<br />
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{{Team:Aachen/FigureFloat|Aachen_Opening_Arduino_Box.jpg|title=Group Picture|width=250px}}<br />
Hello everyone,<br />
the iGEM competition has officially started and we are taking part!<br />
If you are as excited as we are stay tuned as we will unveil everything about our project in the upcoming weeks. This blog will function as a behind the scenes look of the project and the team. So stay tuned for everything iGEM Aachen!<br />
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In the meantime you can check us out on [http://www.facebook.com/iGEMAachen Facebook] or [http://twitter.com/igemaachen Twitter]<br />
for more updates.<br />
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This year Aachen is rocking iGEM!<br />
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{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/SafetyTeam:Aachen/Safety2014-10-18T03:34:48Z<p>Nbailly: /* Safety */</p>
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[[File:Aachen_14-10-13_Pathogen_Cell_iNB.png|right|150px]]<br />
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= Safety =<br />
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Our iGEM team is committed to consider all aspects of the entire project, including biosafety. For this project, two biosafety aspects have to be considered. On the one hand, we are using ''E. coli'' as '''genetically modified organism''', and on the other hand, we are detecting ''Pseudomonas aeruginosa'', an '''opportunistic human pathogen'''. It infects immunodeficient people as well as severe burns and open wounds. When sampling ''P. aeruginosa'', we should prevent proliferation and spread of the bacterium. For ''E. coli'', we have to take care of biological containment of a genetically modified organism. <br />
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In general, we developed and designed the measurement device as '''closed system''' for a better safety handling. This way, neither the sampled pathogens nor the genetically modified sensor cells can escape our biosensor unit. For the detection, we are using one-time usage sampling and sensor chips which can be disposed of after '''autoclaving or irradation''' with strong UV light. Moreover, the electronic components are in a separate compartment and inaccessible for the user, preventing electric shock or other injuries.<br />
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{{Team:Aachen/Figure|Aachen_Security_WatsOn.png|title=Biosafety level for ''WatsOn''|subtitle=Don't forget to use ''WatsOn'' only in laboratoris with the biosafty standard 1|width=500px}}<br />
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To detect ''P. aeruginosa'', a sampling agar chip is slightly pressed against the solid surface to be tested. The sampling chip is placed on the sensor chip in a petri dish which will be closed and not open at all. The cells have no contact with the device during and after the measurement, but the sensor chips must be handled in '''S1 environments only''' since they contain genetically modified ''E. coli''. Afterwards, both chips can be autoclaved and disposed. The whole lining of the measurement device is built from plastic so that it can be disinfected easily.<br />
<!-- Afterwards, the sampling chip is immediately introduced into our measurement device and will not be removed until the detection is finished and the chips have been disinfected. The sensor chips must be handled in '''S1 environments only''' since they contain genetically modified ''E. coli''. However, once introduced into the measurement device, the sensor chips, too, will not be removed before disinfection. The living cells inside the measurement device are effectively killed after a measurement by '''using desinfectants''' such as Bacillol. For this procedure, the drawer of the measurement device is opened and Bacillol is poured over the sampling and sensor chips. --> <br />
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To '''simulate the worst case scenario''', we did replica plating of some exemplary sensor chips. In three experiments, we got an arithmetic mean of five colonies which were picked up. From that we concluded that the '''risk of infection is really low''' even if the measurement device and chips are not handled properly.<br />
<br />
For further analysis of our project from a safety perspective, please view our [https://igem.org/Safety/Safety_Form?team_id=1319 safety form].<br />
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{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/Collaborations/MakerFaireTeam:Aachen/Collaborations/MakerFaire2014-10-18T03:34:36Z<p>Nbailly: /* Representing iGEM and Synthetic Biology at the MakerFaire Hannover */</p>
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= Representing iGEM and Synthetic Biology at the MakerFaire Hannover =<br />
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On July 4th-7th, [[User:fgohr|Florian]], [[User:R.hanke|René]] and [[User:mosthege|Michael]] joined [[User:Ansgar|Ansgar]] on a trip to the [http://makerfairehannover.com/ MakerFaire Hannover 2014].<br />
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The MakerFaire is an exhibition with things that people built on their own using readily available tools. Dozens of 3D printers and microcontrollers can be examined, and there are a lot of devices visitors can interact with.<br />
Among the fire-spitting dragon, our OD and fluorescence measurement devices attracted a lot of attention. We spoke with hundreds of visitors and had a lot of interesting conversations.<br />
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Saturday evening, Carsten Ludwig from the [[Team:Braunschweig|iGEM Team Braunschweig]] visited us. We showed him the most fascinating stands and afterwards went to the city to walk the [http://www.roterfaden-hannover.de/ ''Red Thread of Hannover''], a track going past 36 very central tourist attractions as e.g. the roofless church.<br />
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On this quick sightseeing trip we took a nice [[Media:Aachen_MakerFairePanorama.jpg|panorama of the ''Aegidienkirche'']].<br />
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When the #NEDCRC soccer game started, we settled for some hamburgers, fermentation broth and exchanged funny stories about our lab experience.<br />
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On Saturday, the run on our stand had been so enormous that we had to improvise and print new flyers at our hostel for Sunday, the second and last day of the fair. Just like the day before, a lot of people showed interest in our stand, and especially René passionately demonstrated our devices and informed about synthetic biology as well as our project ''Cellock Holmes''.<br />
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At about midday, Manuel, advisor of [[Team:Bielefeld-CeBiTec|Team Bielefeld]], visited us and got to experience our measurement device, too.<br />
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When we left Hannover Monday morning, we decided to stop by in Bielefeld to meet the rest of their team. This way they, too, got to test our OD and fluorescence measurement device and we got to eat some ''delicious'' ribes cake :)<br />
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Five hours later, we left the Bielefeld team to eat some incredibly spicy pizza and continued our journey back home to Aachen. On the autobahn, we took the time to write down all experiences and things we learned to share them with our team in the coming days.<br />
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'''tl;dr:''' We had an awesome trip to the MakerFaire Hannover, presented our devices to hundreds of people and met the teams Braunschweig and Bielefeld.<br />
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'''If you like to see more about our weekend have a look at the following gallery =)'''<br />
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{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/Collaborations/Kaiser-Karls-GymnasiumTeam:Aachen/Collaborations/Kaiser-Karls-Gymnasium2014-10-18T03:34:09Z<p>Nbailly: </p>
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= Teaching Module "Synthetic Biology" for Highschools =<br />
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In the course of our cooperation with the grade 9 biology-chemistry class at Kaiser-Karl-Gymnasium, a secondary school in Aachen, we developed a teaching module about “Synthetic Biology”. The scope of the module is 8 school classes 45 min. each. Topics include<br />
*sources of and exposure to microorganisms in our environment<br />
*antibiotic resistances<br />
*quorum sensing<br />
*fluorescence and measurement thereof<br />
*the bio-molecular aspect of our iGEM project. <br />
To wrap up the module we did some career-exploration in synthetic biology and related fields.<br />
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The teaching module is suitable for the subjects biology, biology-chemistry and sciences. The students should have completed grade 8, and know about the central dogma of biology as well as protein biosynthesis. Because our work is going to be evaluated based on this wiki, we would like to take photos during the lessons. In order to publish the photos on our homepage and our wiki, we need a consent form signed by the students’ parents. If you, too, are interested in collaborating with our team write us an email at igem@rwth-aachen.de<br />
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We have carried out this teaching module in cooperation with Kaiser-Karls-Gymnasium. Here are some impressions of this collaboration:<br />
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For each of the lessons we also recorded our experience. Read about the outcome of our work below.<br />
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== Lesson 1 - School Project Kick-Off ==<br />
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By [[Nina:NBailly|Nina]] 20:48, April 28 2014 (CDT) <br />
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Today was the kick-off for the cooperation between our iGEM team and the Kaiser-Karl-Gymnasium, a secondary school in Aachen. In the upcoming weeks, exciting school lessons with members of our iGEM team await the students of the 9th grade biology-chemistry class. Different aspects of our project will be highlighted and explained to the students. Demonstrative experiments will explain the practical relevance. <br />
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In today's lesson, our members Nina and René offered the students a short impression of what to expect in the course of this teaching module. We also explained synthetic biology and the goal of our project. <br />
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== Lesson 2 - iGEM team members back 2 school ==<br />
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By [[User:NBailly|Nina]] 16:00, May 22 2014 (CDT) <br />
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Since our last visit, the student of the biology-chemistry course at Kaiser-Karl-Gymnasium in Aachen have been busy studying the basics of protein biosynthesis and the "lock and key" model. Now the 9th graders are prepared to have a closer look at synthetic biology and our project.<br />
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[[File:Aachen_Kaiser-Karls-Gymnasium.jpg|800px]]<br />
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Each year, many people die of nosocominal infections - infections that the patients acquired in a hospital. However, these infections would be preventable in large part through better hygiene programs. Diseases caused by multi-resistant pathogens are especially critical because therapy options are very limited in these cases.<br />
<br />
But actually, what are pathogens exactly and where do they come from? In order to answer these questions, we start the teaching module with the topic "Microorganisms in our Environment". In this part, we explain which germs we encounter every day, and how be can best protect ourselves from them.<br />
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In order to demonstrate how many and which microorganisms are our steady companions, we conduct an experiment with the students. A pair of students received 3 agar plates: one with regular LB agar, one plate with agar supplemented by an antibiotic, and one selective agar plate for yeasts and fungi. Equipped with the plates, the students wander through their school and take contact samples from places they think many microorganisms grow there. We will incubate the plates over the weekend so that we can show the students in the next lesson what has grown on their samples.<br />
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== Lessons 3 & 4 - Microorganisms on the rise ==<br />
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By [[User:NBailly|Nina]] 16:05, May 26 2014 (CDT) <br />
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Last lesson students at Kaiser-Karl-Gymnasium took environmental samples using a variety of different contact agar plates. On the weekend, René examined the plates under the microscope and took photos. Today René and Nina presented the results of the experiment. Most of the students have been really astonished about the diverse microbial fauna found on themselves and their classroom. But first, we showed the students an excerpt from the TV show "Planetopia" that broaches the issue "Hygiene in every-day life".<br />
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<em>Staphylo-</em> and <em>micrococci</em> as well as <em>Enterobacteria</em>, <em>Candida</em> yeasts and miscellaneous fungi are found on the majority of plates. But why is there no growth on the plates containing the antibiotic? Actually, what is an antibiotic, how does it work and why are there multi-resistant pathogens? Nina explained the answer to all these questions to the students using descriptive graphics, and concluded the topic "Microorganisms in our Environment" with a short summmary.<br />
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As a starter for our next topic that is relevant to our project, we distribute a worksheet about quorum sensing among the students. For the remainder of the lesson, the students read the text and begin to answer the attached questions.<br />
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== Lessons 5 & 6 - Glowing Vanilla Pudding ==<br />
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By [[User:NBailly|Nina]] 16:16, June 02 2014 (CDT)<br />
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The topics of today's double lesson was quorum sensing as well as measurement of fluorescence. At the beginning of class, the students form 6 groups and start an experiment dealing with fluorescence: Each group weighs and dissolves 4g of vanilla pudding powder in 50mL of water. While conducting the experiment, each group is supervised by a member of our iGEM team.<br />
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{{Team:Aachen/Figure|Aachen School pudding.png|width=590px}}<br />
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While we give the powder some time to dissolve, René discusses the worksheet about quorum sensing that we gave out to the students last week. In doing so, the students learn what quorum sensing actually is, how it is used in different ways by a variety of bacteria, and how we want to use this function for our project.<br />
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{{Team:Aachen/Figure|Aachen School Vfischeri.png|width=590px}}<br />
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Meanwhile the powder had dissolved in the water. Excess powder accumulated at the bottom of the beaker. Using a syringe, the students suck 2mL of supernatant out of the beaker, and press it through a filter into a cuvette. A part of our project involves the development of a fluorescence measurement device named ''Cellock Holmes''. Each group of students at a time places its cuvette in the ''Cellock Holmes'' prototype, and notes down the values displayed on the cellphone display. Of course, each group also measures the fluorescence of a positive (pure riboflavin in water) and a negative (chalk dust in water) control.<br />
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{{Team:Aachen/Figure|Aachen School experiment.png|width=590px}}<br />
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While the other groups wait for their turn, their supervisors explain the background of this experiment: The electrons in some molecules change into energy states when irradiated with electromagnetic waves. When returning to the normal state, the electrons dissipate excess energy, also in the form of electromagnetic radiation. This process is called fluorescence. Vanilla pudding contains the molecule riboflavin (vitamin B2). When riboflavin is irradiated with blue light, the molecule fluoresces green. The intensity of the green light is visually recorded by our device. Special software processes the data and the measured value is displayed on the cellphone connected to the device via Bluetooth. We will discuss the results of the experiment next class.<br />
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== Lesson 7 - Teaching Module Wrap-Up ==<br />
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By [[User:NBailly|Nina]] 16:32, June 05 2014 (CDT)<br />
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Today Nina and Arne discussed last class's experiment. Arne explained to the students the meaning behind the values measured, and how the students can use the value to determine the concentration of riboflavin in their vanilla pudding samples.<br />
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{{Team:Aachen/Figure|Aachen Cellock.png|width=268px}}<br />
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Subsequently, Nina explained the students how we are going to manipulate regular E. coli cells, using synthetic biology methods, to carry out the desired functions. The students also learned, what role promoters play in gene regulation, and how our iGEM team wants to regulate the expression of the genes artificially inserted into the E. coli cells.<br />
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Now we have individually discussed all aspects of our project. Therefore, Nina summarized all the topic together with the students, and outlined the link between all these different topics and our iGEM project idea. In doing so, we almost arrived at the end of our teaching module "Synthetic Biology". Next class, we will visit the biology-chemistry course of Kaiser-Karl-Gymnasium for the last time, and do some career-exploration in synthetic biology and related fields.<br />
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== Lesson 8 - Students Explore Careers in Synthetic Biology ==<br />
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By [[User:NBailly|Nina]] 21:11, June 12 2014 (CDT) <br />
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Our "Synthetic Biology" teaching module has come to an end. Today was our last day at Kaiser-Karl-Gymnasium in Aachen, where we have worked together with a grade 9 biology-chemistry class for past 1 1/2 months. <!--more-->The students have learned about the iGEM and synthetic biology in general. We presented our project to the class, and explained various aspects for our endeavour in more detail.<br />
<br />
To give interested students some overview of how they can get involved in synthetic biology, Vera, Florian, Ansgar and Björn gave short presentations about each university program at RWTH represented in our team: Biology, Biotechnology, Computational Engineering Science and Informatics, respectively. We gave them examples for typical classes and specialization options as well as career perspectives in each program.<br />
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<html><a style="text-align: center; display: block;" href="https://2014.igem.org/File:Cellock_liegend.png"><img class="size-medium wp-image-218" src="https://static.igem.org/mediawiki/2014/b/ba/Cellock_liegend.png" alt="cellock_liegend" width="300" height="194" /></a></html><br />
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While discussing some more career perspectives, we handed an evaluation sheet out to each student. This way the class could give us some feedback about our work, and leave some comments on what they liked best and what we can still improve on. On average, the students gave us an A (1,9 or 87%) for the teaching module. We are really happy about this great mark, and will try to improve on all the things the students suggested.<br />
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Unfortunately, the bell already rang when the students finished handing in their evaluation sheets. However, some students still followed our invitation to stay after class to ask more questions about paths into synthetic biology, and for some cookies.<br />
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Overall, all parties seemed really satisfied with our collaboration and we are looking forward to repeating the teaching module with other high school classes!<br />
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{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/PolicyPracticesTeam:Aachen/PolicyPractices2014-10-18T03:33:22Z<p>Nbailly: /* Intellectual Property on BioBricks */</p>
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= Policy & Practices =<br />
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During the past summer, we not only refined the technical and biological sides of ''Cellock Holmes'' but also considered other aspects of our iGEM project such as '''social acceptance''', '''biosafety''' and '''economical relevance'''. Will society accept the technology we develop? How can we convince skeptics that synthetic biology is safe? Does our product have economical relevance and how can we best market what we built? What is the target group that might benefit from our devices, and can we make our developments available to not only the privileged population but to everybody in the world? At the meetup of the German iGEM teams in Munich earlier this summer, we also prepared a suggestion on how to handle '''intellectual proporty rights on BioBricks'''.<br />
<br />
These are only a few of the questions we discussed within our team. To read more about the different aspects of our Policy & Practices work, please click on a panel below: <br />
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<center><br />
<html><ul class="team-grid" style="width:1040px"><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/PolicyPractices#ppsocialacceptance" style="color:black"><br />
<div class="menusmall-item menusmall-info" style="height: 180px; width: 180px;" > <div class="menukachel" style="top: 32%;line-height: 1.5em;">Social Acceptance</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/3/35/Aachen_14-10-13_Love_Cell_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/PolicyPractices#ppbiosafety" style="color:black"><br />
<div class="menusmall-item menusmall-info" style="height: 180px; width: 180px;" > <div class="menukachel" style="top: 40%;line-height: 1.5em;">Biosafety</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/1/11/Aachen_14-10-13_Pathogen_Cell_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"><br />
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<a class="menulink" href="https://2014.igem.org/Team:Aachen/PolicyPractices#ppeconomics" style="color:black"><br />
<div class="menusmall-item menusmall-info" style="height: 180px; width: 180px;" > <div class="menukachel" style="top: 32%;line-height: 1.5em;">Economical View</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/b/bd/Aachen_14-10-13_Money_Cell_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/PolicyPractices#ppbbaip" style="color:black"><br />
<div class="menusmall-item menusmall-info" style="height: 180px; width: 180px;"> <div class="menukachel" style="top: 23%;line-height: 1.5em;">Intellectual Property on BioBricks</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/9/9b/Aachen_14-10-14_IP_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"><br />
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<a class="menulink" href="https://2014.igem.org/Team:Aachen/PolicyPractices#ppblog" style="color:black"><br />
<div class="menusmall-item menusmall-info" style="height: 180px; width: 180px;"> <div class="menukachel" style="top: 40%;line-height: 1.5em;">Blog</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/b/b8/Aachen_14-10-13_Blogger_Cell_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"><br />
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[[File:Aachen_14-10-13_Love_Cell_iNB.png|right|150px]]<br />
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== Spreading the Idea of Synthetic Biology ==<br />
<span class="anchor" id="ppsocialacceptance"></span><br />
<br />
How can we convince people that the technology we develop is safe to use and that the problems we tackle with our project concern everybody? Unfortunately, lots of people around the world are scared of genetically modified organisms and any application related to them. Though we believe that '''natural skepticism''' towards new and unproved technologies is not just good but especially desirable, the current fear some people encounter gene technology with is a bit disproportionate and might be counterproductive to technological and scientific advance in related fields.<br />
<br />
However, as reported, for example, in an [http://www.rundschau-online.de/magazin/gentechnik--risiko-oder-chance-,15184902,15929266.html article] published in a major local newspaper's magazine, Kölner Stadtanzeiger, the social acceptance of biotechnological products could be higher if people felt informed better and understood the underlying science. Following up on this, we thought about how we can inform people '''factually but in a comprehensible way''' about gene technology and synthetic biology. Before we talk about fancy devices in synthetic biology, how can we '''get down to the underlying issue''' of social rejection of gene technology in general? <br />
<br />
At the same time, '''young students''' interested in science and engineering are the most valuable future source of innovation. One day, they might be the researchers who develop the solutions to the most pressing issues of our world. For that reason, informing this group of people is of utmost importance and was therefore prioritized in our Policy & Practices work.<br />
<br />
Combining these two thoughts, we visited '''two schools''', the [https://2014.igem.org/Team:Aachen/Collaborations/Kaiser-Karls-Gymnasium Kaiser-Karls-Gymnasium] in Aachen and the [https://2014.igem.org/Team:Aachen/Collaborations/Neanderlab NEAnderLab] in Hilden, where we talked to students about synthetic biology and the iGEM competition, but also explained the scientific background and social aspects of our project. A delegation of our team also visited the [https://2014.igem.org/Team:Aachen/Collaborations/MakerFaire MakerFaire] in Hannover, a family-friendly '''exhibition for tinkerers''' of all kinds, to spread the idea of synthetic biology and to discuss our project with the public. When we organized the [https://2014.igem.org/Team:Aachen/Meetup Aachen iGEM Meetup 2014], we also made sure to include a '''public part''' where all teams who participated in our meetup had the opportunity to present their project to a general audience.<br />
<br />
In general, we received '''positive feeback throughout'''. The students we worked with in the NEAnderLab also filled out evaluation sheets, giving us [https://2014.igem.org/Team:Aachen/Collaborations/Neanderlab#neanderlabevaluation very good marks] for our collaboration. Through our work with the different people we encountered during our Policy & Practices work, we were able to inform people in a comprehensible way about synthetic biology and gene technology in general. We see this as a ''' successful first step towards a society that embraces the possibilities provided by Synthetic Biology''', and recommend approaches like the school collaborations to other iGEM teams who want the spread the idea of synthetic biology.<br />
<br />
To read more about our different public projects, please click on the respective logo below.<br />
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<html><ul class="team-grid" style="width:1064px;"><br />
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<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;"><br />
<a href="https://2014.igem.org/Team:Aachen/Collaborations/Kaiser-Karls-Gymnasium" style="color:black"><br />
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<b> Kaiser-Karls-Gymnasium</b><br />
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Teaching Module "Synthetic Biology" for High Schools<br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/f/f7/Aachen_14-10-10_Logo_Kaiser-Karls-Gymnasium.png); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a><br />
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<a href="https://2014.igem.org/Team:Aachen/Collaborations/Neanderlab" style="color:black"><br />
<div class="team-item team-info" style="width:214px;height:214px;"><br />
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<b> NEAnderLab </b><br />
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In Cooperation with the Gymnasium an Neandertal<br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/d/d8/Aachen_14-10-10_Logo_NEAnderLab.jpg); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a><br />
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<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;"><br />
<a href="https://2014.igem.org/Team:Aachen/Collaborations/MakerFaire" style="color:black"><br />
<div class="team-item team-info" style="width:214px;height:214px;"><br />
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<b> MakerFaire </b><br />
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Visit of a DIY Exhibition<br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/5/53/Aachen_14-10-10_Logo_MakerFaire.png); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a><br />
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<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;"><br />
<a href="https://2014.igem.org/Team:Aachen/Meetup" style="color:black"><br />
<div class="team-item team-info" style="width:214px;height:214px;"><br />
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<b> Aachen iGEM Meetup 2014 </b><br />
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Including Public Presentations<br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/5/5b/Aachen_14-10-10_Meetup_Logo_white_background_iVA.png); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a><br />
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[[File:Aachen_14-10-13_Pathogen_Cell_iNB.png|right|150px]]<br />
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== Biosafety ==<br />
<span class="anchor" id="ppbiosafety"></span><br />
<br />
Our iGEM team is committed to reflect all aspects of the entire project, including biosafety. From the beginning on, the team thoroughly discussed safety issues that could potentially arise with the implementation of ''Cellock Holmes''. The results of these discussions fundamentally influenced the design of [https://2014.igem.org/Team:Aachen/Project/Measurement_Device ''Watson''] and the choice of potential application fields. Read more about our safety considerations on our [https://2014.igem.org/Team:Aachen/Safety Safety] page.<br />
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[[File:Aachen_14-10-13_Money_Cell_iNB.png|right|150px]]<br />
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== Economical View ==<br />
<span class="anchor" id="ppeconomics"></span><br />
<br />
The economical considerations regarding our project were carried out according to the motto: <br />
<br />
'''Make the world a better place - Open access for scientific advance'''<br />
<br />
For both our [https://2014.igem.org/Team:Aachen/Project/Measurement_Device ''WatsOn''] and our [https://2014.igem.org/Team:Aachen/OD/F_device OD/F Device], we are following an economical strategy focused on the open source principle. Low cost and the use of easily available parts have '''heavily influenced the design choices''' made when developing our devices. You can find more information on our page [https://2014.igem.org/Team:Aachen/PolicyPractices/Economics Economical View].<br />
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[[File:Aachen_14-10-14_IP_iNB.png|right|150px]]<br />
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== Intellectual Property on BioBricks ==<br />
<span class="anchor" id="ppbbaip"></span><br />
<br />
During the meetup of the German iGEM teams from 23rd to 25th May also workshops 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 />
<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 where only upon a closer look it becomes clear that the rights are company–owned. Because the issue of '''uncertain legal status of parts''' in the registry persists, the German iGEM teams '''wrote a proposal''' on how to deal with intellectual property rights in the Registry of Standard Biological Parts.<br />
<br />
For for information on intellectual property on BioBricks, read the [https://2014.igem.org/Team:Aachen/PolicyPractices/BioBrickIntellectualProperty full proposal] the German iGEM teams composed.<br />
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[[File:Aachen_14-10-13_Blogger_Cell_iNB.png|right|150px]]<br />
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== Blog ==<br />
<span class="anchor" id="ppblog"></span><br />
<br />
On our [https://2014.igem.org/Team:Aachen/Blog Blog] we post entries about recent news concerning our team's work and activities. We also write about general news from the field of synthetic biology, biotechnology and medicine. <br />
<br />
{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/PolicyPracticesTeam:Aachen/PolicyPractices2014-10-18T03:33:13Z<p>Nbailly: /* Economical View */</p>
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<div>__NOTOC__<br />
{{CSS/Main}}<br />
{{Team:Aachen/Stylesheet}}<br />
{{Team:Aachen/Header}}<br />
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= Policy & Practices =<br />
<br />
During the past summer, we not only refined the technical and biological sides of ''Cellock Holmes'' but also considered other aspects of our iGEM project such as '''social acceptance''', '''biosafety''' and '''economical relevance'''. Will society accept the technology we develop? How can we convince skeptics that synthetic biology is safe? Does our product have economical relevance and how can we best market what we built? What is the target group that might benefit from our devices, and can we make our developments available to not only the privileged population but to everybody in the world? At the meetup of the German iGEM teams in Munich earlier this summer, we also prepared a suggestion on how to handle '''intellectual proporty rights on BioBricks'''.<br />
<br />
These are only a few of the questions we discussed within our team. To read more about the different aspects of our Policy & Practices work, please click on a panel below: <br />
<br />
<center><br />
<html><ul class="team-grid" style="width:1040px"><br />
<br />
<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/PolicyPractices#ppsocialacceptance" style="color:black"><br />
<div class="menusmall-item menusmall-info" style="height: 180px; width: 180px;" > <div class="menukachel" style="top: 32%;line-height: 1.5em;">Social Acceptance</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/3/35/Aachen_14-10-13_Love_Cell_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/PolicyPractices#ppbiosafety" style="color:black"><br />
<div class="menusmall-item menusmall-info" style="height: 180px; width: 180px;" > <div class="menukachel" style="top: 40%;line-height: 1.5em;">Biosafety</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/1/11/Aachen_14-10-13_Pathogen_Cell_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"><br />
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</a><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/PolicyPractices#ppeconomics" style="color:black"><br />
<div class="menusmall-item menusmall-info" style="height: 180px; width: 180px;" > <div class="menukachel" style="top: 32%;line-height: 1.5em;">Economical View</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/b/bd/Aachen_14-10-13_Money_Cell_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/PolicyPractices#ppbbaip" style="color:black"><br />
<div class="menusmall-item menusmall-info" style="height: 180px; width: 180px;"> <div class="menukachel" style="top: 23%;line-height: 1.5em;">Intellectual Property on BioBricks</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/9/9b/Aachen_14-10-14_IP_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"><br />
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<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/PolicyPractices#ppblog" style="color:black"><br />
<div class="menusmall-item menusmall-info" style="height: 180px; width: 180px;"> <div class="menukachel" style="top: 40%;line-height: 1.5em;">Blog</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/b/b8/Aachen_14-10-13_Blogger_Cell_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"><br />
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{{Team:Aachen/BlockSeparator}}<br />
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[[File:Aachen_14-10-13_Love_Cell_iNB.png|right|150px]]<br />
<br />
== Spreading the Idea of Synthetic Biology ==<br />
<span class="anchor" id="ppsocialacceptance"></span><br />
<br />
How can we convince people that the technology we develop is safe to use and that the problems we tackle with our project concern everybody? Unfortunately, lots of people around the world are scared of genetically modified organisms and any application related to them. Though we believe that '''natural skepticism''' towards new and unproved technologies is not just good but especially desirable, the current fear some people encounter gene technology with is a bit disproportionate and might be counterproductive to technological and scientific advance in related fields.<br />
<br />
However, as reported, for example, in an [http://www.rundschau-online.de/magazin/gentechnik--risiko-oder-chance-,15184902,15929266.html article] published in a major local newspaper's magazine, Kölner Stadtanzeiger, the social acceptance of biotechnological products could be higher if people felt informed better and understood the underlying science. Following up on this, we thought about how we can inform people '''factually but in a comprehensible way''' about gene technology and synthetic biology. Before we talk about fancy devices in synthetic biology, how can we '''get down to the underlying issue''' of social rejection of gene technology in general? <br />
<br />
At the same time, '''young students''' interested in science and engineering are the most valuable future source of innovation. One day, they might be the researchers who develop the solutions to the most pressing issues of our world. For that reason, informing this group of people is of utmost importance and was therefore prioritized in our Policy & Practices work.<br />
<br />
Combining these two thoughts, we visited '''two schools''', the [https://2014.igem.org/Team:Aachen/Collaborations/Kaiser-Karls-Gymnasium Kaiser-Karls-Gymnasium] in Aachen and the [https://2014.igem.org/Team:Aachen/Collaborations/Neanderlab NEAnderLab] in Hilden, where we talked to students about synthetic biology and the iGEM competition, but also explained the scientific background and social aspects of our project. A delegation of our team also visited the [https://2014.igem.org/Team:Aachen/Collaborations/MakerFaire MakerFaire] in Hannover, a family-friendly '''exhibition for tinkerers''' of all kinds, to spread the idea of synthetic biology and to discuss our project with the public. When we organized the [https://2014.igem.org/Team:Aachen/Meetup Aachen iGEM Meetup 2014], we also made sure to include a '''public part''' where all teams who participated in our meetup had the opportunity to present their project to a general audience.<br />
<br />
In general, we received '''positive feeback throughout'''. The students we worked with in the NEAnderLab also filled out evaluation sheets, giving us [https://2014.igem.org/Team:Aachen/Collaborations/Neanderlab#neanderlabevaluation very good marks] for our collaboration. Through our work with the different people we encountered during our Policy & Practices work, we were able to inform people in a comprehensible way about synthetic biology and gene technology in general. We see this as a ''' successful first step towards a society that embraces the possibilities provided by Synthetic Biology''', and recommend approaches like the school collaborations to other iGEM teams who want the spread the idea of synthetic biology.<br />
<br />
To read more about our different public projects, please click on the respective logo below.<br />
<br />
<html><ul class="team-grid" style="width:1064px;"><br />
<br />
<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;"><br />
<a href="https://2014.igem.org/Team:Aachen/Collaborations/Kaiser-Karls-Gymnasium" style="color:black"><br />
<div class="team-item team-info" style="width:214px;height:214px;" ><br />
<br/><br/><br />
<b> Kaiser-Karls-Gymnasium</b><br />
<br/><br/><br />
Teaching Module "Synthetic Biology" for High Schools<br />
<br/><br/><br />
<!-- click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/f/f7/Aachen_14-10-10_Logo_Kaiser-Karls-Gymnasium.png); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a><br />
</li><br />
<br />
<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;"><br />
<a href="https://2014.igem.org/Team:Aachen/Collaborations/Neanderlab" style="color:black"><br />
<div class="team-item team-info" style="width:214px;height:214px;"><br />
<br/><br/><br />
<b> NEAnderLab </b><br />
<br/><br/><br />
In Cooperation with the Gymnasium an Neandertal<br />
<br/><br/><br />
<!-- click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/d/d8/Aachen_14-10-10_Logo_NEAnderLab.jpg); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a><br />
</li><br />
<br />
<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;"><br />
<a href="https://2014.igem.org/Team:Aachen/Collaborations/MakerFaire" style="color:black"><br />
<div class="team-item team-info" style="width:214px;height:214px;"><br />
<br/><br/><br />
<b> MakerFaire </b><br />
<br/><br/><br />
Visit of a DIY Exhibition<br />
<br/><br/><br />
<!-- click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/5/53/Aachen_14-10-10_Logo_MakerFaire.png); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a><br />
</li><br />
<br />
<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;"><br />
<a href="https://2014.igem.org/Team:Aachen/Meetup" style="color:black"><br />
<div class="team-item team-info" style="width:214px;height:214px;"><br />
<br/><br/><br />
<b> Aachen iGEM Meetup 2014 </b><br />
<br/><br/><br />
Including Public Presentations<br />
<br/><br/><br />
<!-- click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/5/5b/Aachen_14-10-10_Meetup_Logo_white_background_iVA.png); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a><br />
</li><br />
<br />
</ul></html><br />
</center><br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_14-10-13_Pathogen_Cell_iNB.png|right|150px]]<br />
<br />
== Biosafety ==<br />
<span class="anchor" id="ppbiosafety"></span><br />
<br />
Our iGEM team is committed to reflect all aspects of the entire project, including biosafety. From the beginning on, the team thoroughly discussed safety issues that could potentially arise with the implementation of ''Cellock Holmes''. The results of these discussions fundamentally influenced the design of [https://2014.igem.org/Team:Aachen/Project/Measurement_Device ''Watson''] and the choice of potential application fields. Read more about our safety considerations on our [https://2014.igem.org/Team:Aachen/Safety Safety] page.<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_14-10-13_Money_Cell_iNB.png|right|150px]]<br />
<br />
== Economical View ==<br />
<span class="anchor" id="ppeconomics"></span><br />
<br />
The economical considerations regarding our project were carried out according to the motto: <br />
<br />
'''Make the world a better place - Open access for scientific advance'''<br />
<br />
For both our [https://2014.igem.org/Team:Aachen/Project/Measurement_Device ''WatsOn''] and our [https://2014.igem.org/Team:Aachen/OD/F_device OD/F Device], we are following an economical strategy focused on the open source principle. Low cost and the use of easily available parts have '''heavily influenced the design choices''' made when developing our devices. You can find more information on our page [https://2014.igem.org/Team:Aachen/PolicyPractices/Economics Economical View].<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_14-10-14_IP_iNB.png|right|150px]]<br />
<br />
== Intellectual Property on BioBricks ==<br />
<span class="anchor" id="ppbbaip"></span><br />
<br />
During the meetup of the German iGEM teams from 23rd to 25th May also workshops 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 />
<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 where only upon a closer look it becomes clear that the rights are company–owned. Because the issue of '''uncertain legal status of parts''' in the registry persists, the German iGEM teams '''wrote a proposal''' on how to deal with intellectual property rights in the Registry of Standard Biological Parts.<br />
<br />
For for information on intellectual property on BioBricks, read the [https://2014.igem.org/Team:Aachen/PolicyPractices/BioBrickIntellectualProperty full proposal] the German iGEM teams composed.<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_14-10-13_Blogger_Cell_iNB.png|right|150px]]<br />
<br />
== Blog ==<br />
<span class="anchor" id="ppblog"></span><br />
<br />
On our [https://2014.igem.org/Team:Aachen/Blog Blog] we post entries about recent news concerning our team's work and activities. We also write about general news from the field of synthetic biology, biotechnology and medicine. <br />
<br />
{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/PolicyPracticesTeam:Aachen/PolicyPractices2014-10-18T03:33:06Z<p>Nbailly: /* Biosafety */</p>
<hr />
<div>__NOTOC__<br />
{{CSS/Main}}<br />
{{Team:Aachen/Stylesheet}}<br />
{{Team:Aachen/Header}}<br />
<br />
= Policy & Practices =<br />
<br />
During the past summer, we not only refined the technical and biological sides of ''Cellock Holmes'' but also considered other aspects of our iGEM project such as '''social acceptance''', '''biosafety''' and '''economical relevance'''. Will society accept the technology we develop? How can we convince skeptics that synthetic biology is safe? Does our product have economical relevance and how can we best market what we built? What is the target group that might benefit from our devices, and can we make our developments available to not only the privileged population but to everybody in the world? At the meetup of the German iGEM teams in Munich earlier this summer, we also prepared a suggestion on how to handle '''intellectual proporty rights on BioBricks'''.<br />
<br />
These are only a few of the questions we discussed within our team. To read more about the different aspects of our Policy & Practices work, please click on a panel below: <br />
<br />
<center><br />
<html><ul class="team-grid" style="width:1040px"><br />
<br />
<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/PolicyPractices#ppsocialacceptance" style="color:black"><br />
<div class="menusmall-item menusmall-info" style="height: 180px; width: 180px;" > <div class="menukachel" style="top: 32%;line-height: 1.5em;">Social Acceptance</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/3/35/Aachen_14-10-13_Love_Cell_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"><br />
</div><br />
</a><br />
</li><br />
<br />
<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/PolicyPractices#ppbiosafety" style="color:black"><br />
<div class="menusmall-item menusmall-info" style="height: 180px; width: 180px;" > <div class="menukachel" style="top: 40%;line-height: 1.5em;">Biosafety</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/1/11/Aachen_14-10-13_Pathogen_Cell_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"><br />
</div><br />
</a><br />
</li><br />
<br />
<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/PolicyPractices#ppeconomics" style="color:black"><br />
<div class="menusmall-item menusmall-info" style="height: 180px; width: 180px;" > <div class="menukachel" style="top: 32%;line-height: 1.5em;">Economical View</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/b/bd/Aachen_14-10-13_Money_Cell_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"><br />
</div><br />
</a><br />
</li><br />
<br />
<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/PolicyPractices#ppbbaip" style="color:black"><br />
<div class="menusmall-item menusmall-info" style="height: 180px; width: 180px;"> <div class="menukachel" style="top: 23%;line-height: 1.5em;">Intellectual Property on BioBricks</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/9/9b/Aachen_14-10-14_IP_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"><br />
</div><br />
</a><br />
</li><br />
<br />
<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/PolicyPractices#ppblog" style="color:black"><br />
<div class="menusmall-item menusmall-info" style="height: 180px; width: 180px;"> <div class="menukachel" style="top: 40%;line-height: 1.5em;">Blog</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/b/b8/Aachen_14-10-13_Blogger_Cell_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%; height: 180px; width: 180px;"><br />
</div><br />
</a><br />
</li><br />
<br />
</ul><br />
</center><br />
</html><br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_14-10-13_Love_Cell_iNB.png|right|150px]]<br />
<br />
== Spreading the Idea of Synthetic Biology ==<br />
<span class="anchor" id="ppsocialacceptance"></span><br />
<br />
How can we convince people that the technology we develop is safe to use and that the problems we tackle with our project concern everybody? Unfortunately, lots of people around the world are scared of genetically modified organisms and any application related to them. Though we believe that '''natural skepticism''' towards new and unproved technologies is not just good but especially desirable, the current fear some people encounter gene technology with is a bit disproportionate and might be counterproductive to technological and scientific advance in related fields.<br />
<br />
However, as reported, for example, in an [http://www.rundschau-online.de/magazin/gentechnik--risiko-oder-chance-,15184902,15929266.html article] published in a major local newspaper's magazine, Kölner Stadtanzeiger, the social acceptance of biotechnological products could be higher if people felt informed better and understood the underlying science. Following up on this, we thought about how we can inform people '''factually but in a comprehensible way''' about gene technology and synthetic biology. Before we talk about fancy devices in synthetic biology, how can we '''get down to the underlying issue''' of social rejection of gene technology in general? <br />
<br />
At the same time, '''young students''' interested in science and engineering are the most valuable future source of innovation. One day, they might be the researchers who develop the solutions to the most pressing issues of our world. For that reason, informing this group of people is of utmost importance and was therefore prioritized in our Policy & Practices work.<br />
<br />
Combining these two thoughts, we visited '''two schools''', the [https://2014.igem.org/Team:Aachen/Collaborations/Kaiser-Karls-Gymnasium Kaiser-Karls-Gymnasium] in Aachen and the [https://2014.igem.org/Team:Aachen/Collaborations/Neanderlab NEAnderLab] in Hilden, where we talked to students about synthetic biology and the iGEM competition, but also explained the scientific background and social aspects of our project. A delegation of our team also visited the [https://2014.igem.org/Team:Aachen/Collaborations/MakerFaire MakerFaire] in Hannover, a family-friendly '''exhibition for tinkerers''' of all kinds, to spread the idea of synthetic biology and to discuss our project with the public. When we organized the [https://2014.igem.org/Team:Aachen/Meetup Aachen iGEM Meetup 2014], we also made sure to include a '''public part''' where all teams who participated in our meetup had the opportunity to present their project to a general audience.<br />
<br />
In general, we received '''positive feeback throughout'''. The students we worked with in the NEAnderLab also filled out evaluation sheets, giving us [https://2014.igem.org/Team:Aachen/Collaborations/Neanderlab#neanderlabevaluation very good marks] for our collaboration. Through our work with the different people we encountered during our Policy & Practices work, we were able to inform people in a comprehensible way about synthetic biology and gene technology in general. We see this as a ''' successful first step towards a society that embraces the possibilities provided by Synthetic Biology''', and recommend approaches like the school collaborations to other iGEM teams who want the spread the idea of synthetic biology.<br />
<br />
To read more about our different public projects, please click on the respective logo below.<br />
<br />
<html><ul class="team-grid" style="width:1064px;"><br />
<br />
<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;"><br />
<a href="https://2014.igem.org/Team:Aachen/Collaborations/Kaiser-Karls-Gymnasium" style="color:black"><br />
<div class="team-item team-info" style="width:214px;height:214px;" ><br />
<br/><br/><br />
<b> Kaiser-Karls-Gymnasium</b><br />
<br/><br/><br />
Teaching Module "Synthetic Biology" for High Schools<br />
<br/><br/><br />
<!-- click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/f/f7/Aachen_14-10-10_Logo_Kaiser-Karls-Gymnasium.png); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a><br />
</li><br />
<br />
<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;"><br />
<a href="https://2014.igem.org/Team:Aachen/Collaborations/Neanderlab" style="color:black"><br />
<div class="team-item team-info" style="width:214px;height:214px;"><br />
<br/><br/><br />
<b> NEAnderLab </b><br />
<br/><br/><br />
In Cooperation with the Gymnasium an Neandertal<br />
<br/><br/><br />
<!-- click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/d/d8/Aachen_14-10-10_Logo_NEAnderLab.jpg); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a><br />
</li><br />
<br />
<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;"><br />
<a href="https://2014.igem.org/Team:Aachen/Collaborations/MakerFaire" style="color:black"><br />
<div class="team-item team-info" style="width:214px;height:214px;"><br />
<br/><br/><br />
<b> MakerFaire </b><br />
<br/><br/><br />
Visit of a DIY Exhibition<br />
<br/><br/><br />
<!-- click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/5/53/Aachen_14-10-10_Logo_MakerFaire.png); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a><br />
</li><br />
<br />
<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;"><br />
<a href="https://2014.igem.org/Team:Aachen/Meetup" style="color:black"><br />
<div class="team-item team-info" style="width:214px;height:214px;"><br />
<br/><br/><br />
<b> Aachen iGEM Meetup 2014 </b><br />
<br/><br/><br />
Including Public Presentations<br />
<br/><br/><br />
<!-- click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/5/5b/Aachen_14-10-10_Meetup_Logo_white_background_iVA.png); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a><br />
</li><br />
<br />
</ul></html><br />
</center><br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_14-10-13_Pathogen_Cell_iNB.png|right|150px]]<br />
<br />
== Biosafety ==<br />
<span class="anchor" id="ppbiosafety"></span><br />
<br />
Our iGEM team is committed to reflect all aspects of the entire project, including biosafety. From the beginning on, the team thoroughly discussed safety issues that could potentially arise with the implementation of ''Cellock Holmes''. The results of these discussions fundamentally influenced the design of [https://2014.igem.org/Team:Aachen/Project/Measurement_Device ''Watson''] and the choice of potential application fields. Read more about our safety considerations on our [https://2014.igem.org/Team:Aachen/Safety Safety] page.<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_14-10-13_Money_Cell_iNB.png|right|150px]]<br />
<br />
== Economical View ==<br />
<span class="anchor" id="ppeconomics"></span><br />
<br />
The economical considerations regarding our project were carried out according to the motto: <br />
<br />
'''Make the world a better place - Open access for scientific advance'''<br />
<br />
For both our [https://2014.igem.org/Team:Aachen/Project/Measurement_Device ''WatsOn''] and our [https://2014.igem.org/Team:Aachen/OD/F_device OD/F Device], we are following an economical strategy focused on the open source principle. Low cost and the use of easily available parts have '''heavily influenced the design choices''' made when developing our devices. You can find more information on our page [https://2014.igem.org/Team:Aachen/PolicyPractices/Economics Economical View].<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_14-10-14_IP_iNB.png|right|150px]]<br />
<br />
== Intellectual Property on BioBricks ==<br />
<span class="anchor" id="ppbbaip"></span><br />
<br />
During the meetup of the German iGEM teams from 23rd to 25th May also workshops 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 />
<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 where only upon a closer look it becomes clear that the rights are company–owned. Because the issue of '''uncertain legal status of parts''' in the registry persists, the German iGEM teams '''wrote a proposal''' on how to deal with intellectual property rights in the Registry of Standard Biological Parts.<br />
<br />
For for information on intellectual property on BioBricks, read the [https://2014.igem.org/Team:Aachen/PolicyPractices/BioBrickIntellectualProperty full proposal] the German iGEM teams composed.<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_14-10-13_Blogger_Cell_iNB.png|right|150px]]<br />
<br />
== Blog ==<br />
<span class="anchor" id="ppblog"></span><br />
<br />
On our [https://2014.igem.org/Team:Aachen/Blog Blog] we post entries about recent news concerning our team's work and activities. We also write about general news from the field of synthetic biology, biotechnology and medicine. <br />
<br />
{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/AchievementsTeam:Aachen/Achievements2014-10-18T03:32:34Z<p>Nbailly: /* Medal Fulfillments */</p>
<hr />
<div>__NOTOC__<br />
{{CSS/Main}}<br />
{{Team:Aachen/Stylesheet}}<br />
{{Team:Aachen/Header}}<br />
<br />
=Achievements=<br />
<br />
On this page, we summarized all things we accomplished throughout our project "''Cellock Holmes'' - A Case of Identity", and present the medal criteria we have fulfilled for the iGEM competition 2014:<br />
<br />
<html><br />
<center><br />
<ul class="menusmall-grid" style="width:1040px"><br />
<!-- Overview --><br />
<br />
<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/Achievements#achmedals" style="color:black"><br />
<div class="menusmall-item menusmall-info" style="height: 180px; width: 180px;"><div class="menukachel" style="top:32%; line-height:1.5em;">Medal Fulfillments</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/2/2d/Aachen_14-10-14_Medals_iFG.png); norepeat scroll 0% 0% transparent; background-size:100%;height: 180px; width: 180px;"><br />
</div><br />
</a><br />
</li><br />
<br />
<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/Achievements#achbiosensor" style="color:black"><br />
<div class="menusmall-item menusmall-info" style="height: 180px; width: 180px;"><div class="menukachel" style="top:40%;">Biosensor</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/5/5a/Aachen_14-10-14_cellock_liegend_panel_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%;height: 180px; width: 180px;"><br />
</div><br />
</a><br />
</li><br />
<br />
<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/Achievements#achwatson" style="color:black"><br />
<div class="menusmall-item menusmall-info" style="height: 180px; width: 180px;"><div class="menukachel" style="top:40%;"><i>WatsOn</i></div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/c/c7/Aachen_WatsOn_easy.png); norepeat scroll 0% 0% transparent; background-size:100%;height: 180px; width: 180px;"><br />
</div><br />
</a><br />
</li><br />
<br />
<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/Achievements#achdevice" style="color:black"><br />
<div class="menusmall-item menusmall-info" style="height: 180px; width: 180px;"><div class="menukachel" style="top:40%;">OD/F Device</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/0/0f/Aachen_14-10-10_ODF_Button_ipo.png); norepeat scroll 0% 0% transparent; background-size:100%;height: 180px; width: 180px;"><br />
</div><br />
</a><br />
</li><br />
<br />
<li style="width:186px;margin-left: 11px;margin-right: 11px;margin-bottom: 11px;margin-top: 11px;"><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/Achievements#achinterlab" style="color:black"><br />
<div class="menusmall-item menusmall-info" style="height: 180px; width: 180px;"><div class="menukachel" style="top:32%; line-height:1.5em;">Interlab<br>Study</br></div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/3/39/Aachen_Interlab_Cellocks.png); norepeat scroll 0% 0% transparent; background-size:100%;height: 180px; width: 180px;"><br />
</div><br />
</a><br />
</li><br />
<br />
</ul><br />
</center><br />
</html><br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==Medal Fulfillments==<br />
<span class="anchor" id="achmedals"></span><br />
<br />
{| style="float:left; margin-left:30px; margin-bottom:20px"<br />
| https://static.igem.org/mediawiki/2014/thumb/b/b8/Aachen_14-10-14_Bronze_iFG.png/230px-Aachen_14-10-14_Bronze_iFG.png<br />
|<span style="font-size:150%">'''Bronze Requirements (6/6)'''</span><br />
# Our team was registered successfully.<br />
# The [https://igem.org/2014_Judging_Form?id=1319 Judging form] was completed.<br />
# Team [https://2014.igem.org/Team:Aachen/ Wiki] was created.<br />
# We are looking forward to present a poster and a talk at the iGEM Jamboree.<br />
# We successfully participated in the [https://2014.igem.org/Team:Aachen/Interlab_Study Measurement Interlab Study].<br />
# Several [https://2014.igem.org/Team:Aachen/Parts parts] were submitted to the [http://parts.igem.org/Main_Page Registry of Standard Biological Parts].<br />
|-<br />
|colspan="2" | &nbsp;<br />
|-<br />
|https://static.igem.org/mediawiki/2014/thumb/4/42/Aachen_14-10-14_Silver_iFG.png/230px-Aachen_14-10-14_Silver_iFG.png<br />
|<span style="font-size:150%;">'''Silver Requirements (4/4)</span><br />
# It was experimentally validated that [http://parts.igem.org/Part:BBa_K1319001 K1319001], [http://parts.igem.org/Part:BBa_K1319004 K1319004] and other [https://2014.igem.org/Team:Aachen/Parts BioBrick Parts] and Devices of our own design work as expected.<br />
# The characterization of [http://parts.igem.org/Part:BBa_K1319001 K1319001], [http://parts.igem.org/Part:BBa_K1319004 K1319004] and other [https://2014.igem.org/Team:Aachen/Parts BioBrick Parts] is documented in the 'Main Page' section of that Registry entry and in the parts section of our Wiki.<br />
# [http://parts.igem.org/Part:BBa_K1319001 K1319001], [http://parts.igem.org/Part:BBa_K1319004 K1319004] and other [https://2014.igem.org/Team:Aachen/Parts BioBrick Parts] were submitted to the iGEM Parts Registry.<br />
# During the project we addressed several [https://2014.igem.org/Team:Aachen/PolicyPractices important questions beyond the bench]. Together with students of the [https://2014.igem.org/Team:Aachen/Collaborations/Kaiser-Karls-Gymnasium Kaiser-Karls-Gymnasium] and the [https://2014.igem.org/Team:Aachen/Collaborations/Neanderlab Gymnasiun am Neandertal] as well as people at the [https://2014.igem.org/Team:Aachen/Collaborations/MakerFaire MakerFaire] and at the [https://2014.igem.org/Team:Aachen/Meetup iGEM Meetup] we discussed several issues regarding our project, microorganisms in our environment and synthetic biology. Since we use genetically engineered bacteria to detect human pathogens, [https://2014.igem.org/Team:Aachen/Safety safety] was an important aspect for us throughout the whole project. Together with other German iGEM Teams we encountered an issue regarding [https://2014.igem.org/Team:Aachen/PolicyPractices/BioBrickIntellectualProperty intellectual property rights] in context of the legal status of Biobricks.<br />
|-<br />
|colspan="2" | &nbsp;<br />
|-<br />
|https://static.igem.org/mediawiki/2014/thumb/5/5b/Aachen_14-10-14_Gold_iFG.png/230px-Aachen_14-10-14_Gold_iFG.png<br />
|<span style="font-size:150%;">'''Gold Requirements (4/1)'''</span><br />
# We demonstrated a substantial improvement in cost efficiency of our [https://2014.igem.org/Team:Aachen/OD/F_device OD/F Device] in comparison to commercially available spectrophotometers. For more information see [https://2014.igem.org/Team:Aachen/PolicyPractices/Economics Economical View].<br />
# We increased the accessability of our new measurement techniques by sharing instructions to build our DIY devices [https://2014.igem.org/Team:Aachen/Notebook/Engineering/WatsOn#watsondiy ''WatsOn''] and the [https://2014.igem.org/Team:Aachen/Notebook/Engineering/ODF#diy OD/F Device].<br />
# Helped three other iGEM Teams:<br />
#:* We built a [https://2014.igem.org/Team:Aachen/Collaborations/Braunschweig low-cost DIY methane sensor] for [https://2014.igem.org/Team:Braunschweig Team Braunschweig].<br />
#:* We built [https://2014.igem.org/Team:Aachen/Collaborations/Freiburg AcCELLoMatrix Masks] for [https://2014.igem.org/Team:Freiburg Team Freiburg].<br />
#:* We [https://2014.igem.org/Team:Aachen/Collaborations/Heidelberg tested different expression vectors] constructed by [https://2014.igem.org/Team:Heidelberg Team Heidelberg].<br />
# On top of the Policy & Practice aspects already mentioned in the Silver Medal Requirements, we also critically [https://2014.igem.org/Team:Aachen/Collaborations/Neanderlab#neanderlabevaluation evaluated] the effectiveness of our approach to increase the social acceptance of gene technology in general, and of our initiative to spark interest for synthetic biology and DIY hardware.<br />
|}<br />
<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_14-10-14_cellock_liegend_panel_iNB.png|right|150px]]<br />
<br />
==Biosensor==<br />
<span class="anchor" id="achbiosensor"></span><br />
<br />
We developed and optimized a novel [https://2014.igem.org/Team:Aachen/Project/2D_Biosensor method] for detecting pathogens using a two dimensional biosensor. In our approach, the sensor cells are immobilized in '''[https://2014.igem.org/Team:Aachen/Project/2D_Biosensor#biosensordevelopment optimized agar chips]'''. It has been '''experimentally [https://2014.igem.org/Team:Aachen/Project/2D_Biosensor#biosensorachievements validated]''' that this method works for different kinds of reporter cells, induction substances and readout methods including our newly designed system [https://2014.igem.org/Team:Aachen/Project/Measurement_Device ''WatsOn''].<br />
<br />
In order to improve the response of our sensor cells to induction we '''designed and engineered a novel [https://2014.igem.org/Team:Aachen/Project/FRET_Reporter reporter system]''' using a TEV protease to activate a reporter protein by cleaving off a quencher. A faster response in comparison to regular expression of the reporter protein was shown by '''[https://2014.igem.org/Team:Aachen/Project/Model computational modeling] as well as in our [https://2014.igem.org/Team:Aachen/Project/FRET_Reporter#reachachievements experiments]'''. All parts needed to build this kind of reporter system have been '''submitted to the parts registry'''.<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen WatsOn easy.png|right|150px]]<br />
<br />
==''WatsOn''==<br />
<span class="anchor" id="achwatson"></span><br />
<br />
[https://2014.igem.org/Team:Aachen/Project/Measurement_Device ''WatsOn''] is a measuring device designed to read out novel two dimensional biosensor. Using ''WatsOn'', we could demonstrate the '''successful detection''' of ''Pseudomonas&nbsp;aeruginosa'' with our chip technology. This measurement device is built from inexpensive and easily available parts to make it ideal for use in low-budget institutions. Following our economic strategy embracing the Open Access principle, we published all technical details and constructional manuals for ''WatsOn'' on our wiki.<br />
<br />
In addition, we have shown that our '''image analysis software [https://2014.igem.org/Team:Aachen/Project/Measurement_Device#watsonmeasurarty ''Measurarty'']''' can analyze photos of the sensor chips taken by ''WatsOn'' and can '''effectively segment the image''' into pathogenic regions and non-pathogenic regions.<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_14-10-10_ODF_Button_ipo.png|right|150px]]<br />
<br />
==OD/F Device==<br />
<span class="anchor" id="achdevice"></span><br />
<br />
In addition to our main project, we built an [https://2014.igem.org/Team:Aachen/OD/F_device OD/F Device] capable of measuring optical density and flourescence. It '''beats commercially available devices in cost and portability'''. It can be built with common, inexpensive and easily available parts. A [https://2014.igem.org/Team:Aachen/Notebook/Engineering/ODF#diy construction manual] is available on our wiki.<br />
<br />
The device has been '''successfully tested''' with one of the target groups, high school students, in our collaboration with the [https://2014.igem.org/Team:Aachen/Collaborations/Neanderlab NEAnderLab].<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_Interlab_Cellocks.png|right|150px]]<br />
<br />
==Interlab Study==<br />
<span class="anchor" id="achinterlab"></span><br />
<br />
Since we compete in the Measurement Track of this year's iGEM competition, one of our bronze medal criteria was to participate in the [https://2014.igem.org/Team:Aachen/Interlab_Study Measurement Interlab Study]. Our team was able to measure optical density and fluorescence of ''E.&nbsp;coli'' cells containing the three specified genetic devices, and '''obtained high quality data''' supporting the hypotheses regarding different expression levels depending on plamid copy number and promoter strength.<br />
<br />
<br />
{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/PartsTeam:Aachen/Parts2014-10-18T03:32:12Z<p>Nbailly: /* TEV protease with anti-self cleavage mutation S219V, codon optimized for E. coli */</p>
<hr />
<div>__NOTOC__<br />
{{Team:Aachen/Header}}<br />
= iGEM Team Aachen BioBricks =<br />
<br />
This page lists the collection of BioBricks developed by our team for the project ''Cellock Holmes - A Case of Identity''.<br />
<br />
<center><br />
{| class="wikitable"<br />
! BioBrick !! Description<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319000 K1319000]<br />
||RFC[25] version of E0030<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319001 K1319001]<br />
||RFC[25]-compatible dark quencher based on K1319000 (E0030)<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319002 K1319002]<br />
||RFC[25]-compatible dark quencher based on K1319000 (E0030)<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319003 K1319003]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319004 K1319004]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319008 K1319008]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319009 K1319009]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319010 K1319010]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319011 K1319011]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319012 K1319012]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319013 K1319013]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319014 K1319014]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319015 K1319015]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319016 K1319016]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319017 K1319017]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319020 K1319020]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319042 K1319042]<br />
|} </center><br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319000 K1319000]==<br />
<span class="anchor" id="partsK1319000"></span><br />
<br />
===RFC &#91;25&#93; Version of E0020===<br />
<br />
This part is an RFC [25]-compatible version of BBa_E0030. The start and stop codons have been removed to make it RFC [25]-compatible and the part is flanked by the RFC [25] prefix- and suffix-sequences.<br />
<br />
The coding sequence encodes EYFP (enhanced yellow fluorescent protein) which is derived from ''A. victoria'' GFP. The excitation is 512&nbsp;nm and the emission is 534 nm. This part was used to create the parts K1319001 and K1319002. It can also be used in a fusion protein instead of E0030 due to its RFC[25] compability.<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319001 K1319001]==<br />
<span class="anchor" id="partsK1319001"></span><br />
<br />
===RFC [25] - compatible dark quencher based on K1319000 (E0030) called REACh1===<br />
<br />
This part is a RFC[25] dark quencher that is based upon K1319000 (the RFC[25] version of E0030/EYFP) called REACh1.<br />
<br />
Two mutations were introduced that eliminated fluorescence:<br />
* L90I<br />
* Y145W<br />
<br />
===References===<br />
* Ganesan S, Ameer-Beg SM, Ng TT, Vojnovic B, Wouters FS. A dark yellow fluorescent protein (YFP)-based Resonance Energy-Accepting Chromoprotein (REACh) for Förster resonance energy transfer with GFP. Proc Natl Acad Sci U S A. 2006 Mar 14;103(11):4089-94. Epub 2006 Mar 6. PubMed PMID: 16537489; PubMed Central PMCID: PMC1449651. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1449651/?report=classic PubMed Central]<br />
<br />
<br />
===Usage and Biology===<br />
This protein is designed to be a dark quencher for GFP ([http://parts.igem.org/Part:BBa_E0040 E0040]) in a FRET system. When used in a fusion protein with GFP it reduces the observed fluorescence of GFP drastically. In the biobrick [http://parts.igem.org/Part:BBa_K1319013 K1319013] this is realized and the proteins are fused together with the linker [http://parts.igem.org/Part:BBa_K1319016 K1319016] which includes a specific TEV protease (available as [http://parts.igem.org/Part:BBa_K1319004 K1319004]) cleavage site. The fusion of the proteins bring GFP and REACh1 in proximity to each other which allows GFP and REACh1 to act as donors and acceptors in a FRET (Förster Energy Transfer System) system. GFPs emission energy is thereby taken up by REACh1 and released as thermal energy instead of visible light. This eliminates the GFP fluorescence and allows the release of a strong fluorescence signal, if a TEV protease is expressed and the linker is cut. The cutting separates GFP and REACh1 cancelling the FRET interaction and providing a GFP fluorescence response.<br />
<br />
===Characterization===<br />
<br />
In order to characterize K1319001 it was expressed as a fusion protein together with GFP to show its quenching ability. This fusion protein, produced by K1319013, was located on the plasmid backbone pSB3K3. To show that the reduced fluorescence was due to the quenching ability of K1319001 and not based on a faulty expression, K1319008 was also introduced into the cells on a pSB1C3 backbone to create a double plasmid system. Both constructs were put into ''E. coli'' BL21 (DE3) and compared to [http://parts.igem.org/Part:BBa_I20260 I20260] and [http://parts.igem.org/Part:BBa_B0015 B0015]. B0015 was used as a negative control and I20260 was chosen as a positive control due to the expressed GFP being identical to the GFP being expressed in the fusion protein of K1319013 and having the same promoter, RBS, Terminator and plasmid backbone. <br />
<br />
The double plasmid system, B0015 and I20260 were measured in biological triplicates and after 2 h one set of triplicates of each of the three systems were induced with 50 µl 100mM IPTG in their 50 ml culture (500 ml shake flasks). Fluorescence measurement was performed with the [http://www.biotek.com/products/microplate_detection/synergymx_monochromator_based_multimode_microplate_reader.html Synergy Mx from BioTek] with an excitation wavelength of 496 ± 9 nm and emission wavelength of 516 ± 9 nm. The following graph shows the resulting fluorescence adjusted for the measured optical density to account for difference in growth of the cultures and to only show the fluorescence per cell.<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|800px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/5/56/Aachen_Graph2_13.PNG" width="800px"></html><br />
|-<br />
|'''{{{title|Comparison of the fluorescence adjusted for OD of I20260, B0015 and the double plasmid system K1319013 + K1319008}}}'''<br />{{{subtitle|After induction with IPTG after 2 h the double plasmid system produced a fast fluorescence response with an over 9-fold increase compared to the non induced state. I20260 served as positive control and B0015 as negative control.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
The strong fluorescence response (9-fold increase) after induction with IPTG shows the functionality of GFP inside the fusion protein. After the induction a TEV protease is produced which is specifically cutting the recognition sequence build inside the linker (K1319016) between GFP and K1319001. The cutting results in a separation of GFP and REACh1 collapsing the FRET system between the two. This results in a fluorescence signal of GFP due to the fact that the emission is no longer absorbed by REACh1 and emitted as heat but rather as fluorescence with a wavelength of 511 nm.<br />
<br />
The very low fluorescence in the non induced double plasmid system of K1319013 and K1319008 shows the functionality of K1319001. As established before, the GFP is being expressed correctly inside the fusion protein, therefore the reduction in fluorescence in the non induced double plasmid system is a direct result of the quenching ability of K1319001. The quenching, after subtraction of the background fluorescence, reduced the fluorescence of GFP by a factor of 12,5. This also includes the slight leakiness of the TEV protease.<br />
<br />
To eliminate the effect of the leakiness of the K1319008 construct in determining the quenching ability of K1319001, K1319013 was also compared against I20260 and B0015 on its own under the same condition as above (again in a biological triplicate).<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|800px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/b/bb/Aachen_K1319001_comparison_positive_negative.PNG" width="800px"></html><br />
|-<br />
|'''{{{title|Comparison of the fluorescence adjusted for OD of I20260, B0015 and K1319013}}}'''<br />{{{subtitle|The expressed fusion protein K1319013 exhibits a fluorescence more than 30 fold smaller as the positive control of I20260.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
<br />
This experiments shows that the fluorescence of the fusion protein GFP-REACh1 is more than 30-fold lower than normal GFP expression under identical circumstances (same backbone, promoter, RBS, terminator and cultivation circumstances). This demonstrates a quenching percentage of GFP of >96 %!<br />
<br />
To prove that the measured constructs were the same as assumed the plasmids were tested with specially designed Check PCRs with one primer binding upstream on the plasmid backbone and one primer binding specifically inside the insert. The following results were obtained for K1319013.<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|500px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/7/74/Aachen_K1319013_Gel_Check_PCR.png" width="500px"></html><br />
|-<br />
|'''{{{title|Check PCR for K1319013}}}'''<br />{{{subtitle|The length of the PCR product matches the length of the control plasmid.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
<br />
The PCR clearly shows that the used plasmid had the correct insert. After a positive identification of K1319013 the double plasmid system was also checked for the correct TEV protease plasmid K1319008.<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|500px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/0/0c/Aachen_K1319008_Gel_Check_PCR.png" width="500px"></html><br />
|-<br />
|'''{{{title|Check PCR for K1319008}}}'''<br />{{{subtitle|The length of the PCR product matches the length of the control plasmid.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
<br />
This PCR also positively identified K1319008. Additionally both parts were sequenced. The sequencing data for [http://parts.igem.org/Part:BBa_K1319008 K1319008] and [http://parts.igem.org/Part:BBa_K1319013 K1319013] can be found in the parts registry.<br />
<br />
===Characterization of K1319001 with the iGEM Team Aachen [https://2014.igem.org/Team:Aachen/Project/2D_Biosensor 2D Biosensor]===<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 40px; border:{{{border|0px solid #aaa}}};width:{{{width|500px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/6/6d/Aachen_K1319014%2B8_and_K1319013%2B8_pixelig_minus_bg.gif" width="500px"></html><br />
|-<br />
|'''{{{title|K1319013 + K1319008 and K1319014 + K1319008 non-induced (top) and induced (bottom) in sensor cells }}}'''<br />{{{subtitle|The induced double plasmid systems K1319013 + K1319008 and K1319014&nbsp;+&nbsp;K1319008 exhibit a clear fluorescence response in our sensor cells which in response to induction with 2 µl IPTG. }}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
To further characterize the REACh construct, they were introduced into the sensor cells which were then induced with 2&nbsp;µL IPTG with a concentration of 100&nbsp;mM. Subsequently, we took fluorescence measurement read-outs (GFP, excitation 496&nbsp;±&nbsp;9&nbsp;nm, emission&nbsp;516&nbsp;±&nbsp;9&nbsp;nm) roughly every 10&nbsp;min in the plate reader. The results were plotted in the heatmap shown on the left. <br />
<br />
The heatmap shows an increase of fluorescence from blue (no fluorescence) to red (high fluorescence). It is clearly visible that the induced chips are exhibiting a significantly higher fluorescence than the non-induced chips. This again shows that the constructs work as intended: The TEV protease cuts the linker so that the fusion protein is separated into GFP and a dark quencher, disabling the quenching. GFP has a clear fluorescence emission after the fusion protein has been successfully cut into two pieces by the TEV protease. <br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319002 K1319002]==<br />
<span class="anchor" id="partsK1319002"></span><br />
<br />
===RFC [25] - compatible dark quencher based on K1319000 (E0030) called REACh2===<br />
<br />
This part is a RFC[25] dark quencher that is based upon K1319000 (the RFC[25] version of E0030/EYFP) called REACh2. <br />
<br />
Three mutations were introduced that eliminated fluorescence: <br />
* L90I <br />
* Y145W <br />
* H148R<br />
<br />
===References===<br />
* Ganesan S, Ameer-Beg SM, Ng TT, Vojnovic B, Wouters FS. A dark yellow fluorescent protein (YFP)-based Resonance Energy-Accepting Chromoprotein (REACh) for Förster resonance energy transfer with GFP. Proc Natl Acad Sci U S A. 2006 Mar 14;103(11):4089-94. Epub 2006 Mar 6. PubMed PMID: 16537489; PubMed Central PMCID: PMC1449651. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1449651/?report=classic PubMed Central]<br />
<br />
===Usage and Biology===<br />
This protein is designed to be a dark quencher for GFP ([http://parts.igem.org/Part:BBa_E0040 E0040]) in a FRET system. When used in a fusion protein with GFP it reduces the observed fluorescence of GFP drastically. In the biobrick [http://parts.igem.org/Part:BBa_K1319014 K1319014] this is realized and the proteins are fused with the linker [http://parts.igem.org/Part:BBa_K1319016 K1319016] which includes a specific TEV protease (available as [http://parts.igem.org/Part:BBa_K1319004 K1319004]) cleavage site. The fusion of the proteins brings GFP and REACh2 in proximity to each other which allows GFP and REACh2 to act as donors and acceptors in a FRET (Förster Energy Transfer System) system. GFPs emission energy is thereby taken up by REACh2 and released as thermal energy instead of visible light. This eliminates the GFP fluorescence and allows a release of a strong fluorescence signal if a TEV protease is expressed and the linker is cut. The cutting separates GFP and REACh2 cancelling the FRET interaction and providing a GFP fluorescence response.<br />
<br />
===Characterization===<br />
<br />
In order to characterize K1319002 it was expressed as a fusion protein together with GFP to show its quenching ability. This fusion protein, produced by K1319014, was located on the plasmid backbone pSB3K3. To show that the reduced fluorescence was due to the quenching ability of K1319002 and not based on a faulty expression, K1319008 was also introduced into the cells on a pSB1C3 backbone to allow for a double plasmid system. Both constructs were put into ''E. coli'' BL21 (DE3) and compared to [http://parts.igem.org/Part:BBa_I20260 I20260] and [http://parts.igem.org/Part:BBa_B0015 B0015]. B0015 was used as a negative control and I20260 was chosen as a positive control due to the expressed GFP being identical to the GFP being expressed in the fusion protein of K1319014 and having the same promoter, RBS, Terminator and plasmid backbone. <br />
<br />
The double plasmid system, B0015 and I20260 were measured in biological triplicates and after 2 h one set of triplicates of each of the three systems were induced with 50 µl 100mM IPTG in their 50 ml culture (500 ml shake flasks). Fluorescence measurement was performed with the [http://www.biotek.com/products/microplate_detection/synergymx_monochromator_based_multimode_microplate_reader.html Synergy Mx from BioTek] with an excitation wavelength of 496 ± 9 nm and emission wavelength of 516 ± 9 nm. The following graph shows the resulting fluorescence adjusted for the measured optical density to account for difference in growth of the cultures and to only show the fluorescence per cell.<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|800px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/3/3e/Aachen_Graph2_14.PNG" width="800px"></html><br />
|-<br />
|'''{{{title|Comparison of the fluorescence adjusted for OD of I20260, B0015 and the double plasmid system K1319014 + K1319008}}}'''<br />{{{subtitle|After induction with IPTG after 2 h the double plasmid system produced a fast fluorescence response with an over 10-fold increase compared to the non induced state. I20260 served as positive control and B0015 as negative control.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
The strong fluorescence response (10-fold increase) after induction with IPTG shows the functionality of GFP inside the fusion protein. After the induction a TEV protease is produced which is specifically cutting the recognition sequence build inside the linker (K1319016) between GFP and K1319002. The cutting results in a separation of GFP and REACh2 resulting in a collapse of the FRET system between the two. This results in a fluorescence signal of GFP due to the fact that the emission is no longer absorbed by REACh2 and emitted as heat but rather as fluorescence with a wavelength of 511 nm. The overall fluorescence of the double plasmid system reaches the fluorescence level of the positive control indicating a total clavage of all fusion proteins by the produced TEV protease. <br />
<br />
The very low fluorescence in the non induced double plasmid system of K1319014 and K1319008 shows the functionality of K1319002. As established before, the GFP is being expressed correctly inside the fusion protein, therefore the reduction in fluorescence in the non induced double plasmid system is a direct result of the quenching ability of K1319002. The quenching, after subtraction of the background fluorescence, reduced the fluorescence of GFP by a factor of 10. This also includes the slight leakiness of the TEV protease.<br />
<br />
To eliminate the effect of the leakiness of the K1319008 construct in determining the quenching ability of K1319002, K1319014 was also compared against I20260 and B0015 on its own under the same condition as above (again in a biological triplicate).<br />
<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|800px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/6/61/Aachen_K1319002_characterization_positive_negative.PNG" width="800px"></html><br />
|-<br />
|'''{{{title|Comparison of the fluorescence adjusted for OD of I20260, B0015 and K1319014}}}'''<br />{{{subtitle|The expressed fusion protein K1319014 exhibits a fluorescence more than nearly 25-fold smaller as the positive control of I20260.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
<br />
This experiments shows that the fluorescence of the fusion protein GFP-REACh2 is nearly 25-fold lower than normal GFP expression under identical circumstances (same backbone, promoter, RBS, terminator and cultivation circumstances). This demonstrates a quenching percentage of GFP of ~96 %!<br />
<br />
To prove that the measured constructs were the same as assumed the plasmids were tested with specially designed Check PCRs with one primer binding upstream on the plasmid backbone and one primer binding specifically inside the insert. The following results were obtained for K1319014.<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|500px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/0/08/Aachen_K1319014_Gel_Check_PCR.png" width="500px"></html><br />
|-<br />
|'''{{{title|Check PCR for K1319014}}}'''<br />{{{subtitle|The length of the PCR product matches the length of the control plasmid.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
<br />
The PCR clearly shows that the used plasmid had the correct insert. After a positive identification of K1319014 the double plasmid system was also checked for the correct TEV protease plasmid K1319008.<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|500px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/0/0c/Aachen_K1319008_Gel_Check_PCR.png" width="500px"></html><br />
|-<br />
|'''{{{title|Check PCR for K1319008}}}'''<br />{{{subtitle|The length of the PCR product matches the length of the control plasmid.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
<br />
This PCR also positively identified K1319008. Additionally both parts were sequenced. The sequencing data for [http://parts.igem.org/Part:BBa_K1319008 K1319008] and [http://parts.igem.org/Part:BBa_K1319014 K1319014] can be found in the parts registry.<br />
<br />
===Characterization of K1319001 with the iGEM Team Aachen [https://2014.igem.org/Team:Aachen/Project/2D_Biosensor 2D Biosensor]===<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 40px; border:{{{border|0px solid #aaa}}};width:{{{width|500px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/6/6d/Aachen_K1319014%2B8_and_K1319013%2B8_pixelig_minus_bg.gif" width="500px"></html><br />
|-<br />
|'''{{{title|K1319013 + K1319008 and K1319014 + K1319008 non-induced (top) and induced (bottom) in sensor cells }}}'''<br />{{{subtitle|The induced double plasmid systems K1319013 + K1319008 and K1319014&nbsp;+&nbsp;K1319008 exhibit a clear fluorescence response in our sensor cells which in response to induction with 2 µl IPTG. }}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
To further characterize the REACh construct, they were introduced into the sensor cells which were then induced with 2&nbsp;µL IPTG with a concentration of 100&nbsp;mM. Subsequently, we took fluorescence measurement read-outs (GFP, excitation 496&nbsp;±&nbsp;9&nbsp;nm, emission&nbsp;516&nbsp;±&nbsp;9&nbsp;nm) roughly every 10&nbsp;min in the plate reader. The results were plotted in the heatmap shown on the left. <br />
<br />
The heatmap shows an increase of fluorescence from blue (no fluorescence) to red (high fluorescence). It is clearly visible that the induced chips are exhibiting a significantly higher fluorescence than the non-induced chips. This again shows that the constructs work as intended: The TEV protease cuts the linker so that the fusion protein is separated into GFP and a dark quencher, disabling the quenching. GFP has a clear fluorescence emission after the fusion protein has been successfully cut into two pieces by the TEV protease. <br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319003 K1319003]==<br />
<span class="anchor" id="partsK1319003"></span><br />
<br />
===Human galectin-3, codon-optimized for ''E.&nbsp;coli''===<br />
<br />
Galectin-3 is a 26 kDa protein that binds certain LPS patterns. It especially bind the O-section of the LPS.<br />
<br />
Galectins are proteins of the lectin family, which posess '''carbonhydrate recognition domains''' binding specifically to β-galactoside sugar residues. In humans, 10 different galectines have been identified, among which is galectin-3. <br />
<br />
Galectin-3 has a size of about 31&nbsp;kDA and is encoded by a single gene, LGALS3. It has many physiological functions, such as '''cell adhesion, cell growth and differentiation,''' and contributes to the development of '''cancer, inflammation, fibrosis and others'''.<br />
<br />
Human galectin-3 is a protein of the lectin-family that was shown to bind the LPS of multiple human pathogens.<br />
Some of them, including ''Pseudomonas&nbsp;aeruginosa'' protect themselves against the human immune system by mimicking the lipopolysaccharides (LPS) present on human erythrocytes.<br />
<br />
===Usage and Biology===<br />
<br />
K1319003 was used to create [http://parts.igem.org/Part:BBa_K1319020 K1319020], a Galectin-mRFP fusion protein with a C-terminal His tag in the [https://2014.igem.org/Team:Heidelberg/Team/Collaborations Heidelberger expression vector] pSBX1A3.<br />
<br />
We also cloned our K1319003 into the pET17 expression vector and expressed all combinations of fusion proteins in E.&nbsp;coli&nbsp;BL21(DE3). An SDS-PAGE showed that all fusion proteins were fully translated:<br />
<center><br />
<div class="figure" style="float:{{{align|center}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|800px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/thumb/5/52/Aachen_14-10-04_Expression_Pellets_iMO.png/425px-Aachen_14-10-04_Expression_Pellets_iMO.png" width="400px"></html><br />
|-<br />
|'''{{{title|Pellets of different fusion protein expressions}}}'''<br />{{{subtitle|Expression in the pET17 vector was much more leaky than the expression in the pSBX vectors.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
<center><br />
<div class="figure" style="float:{{{align|center}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|800px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/thumb/6/62/Aachen_Gal3_Expression.png/425px-Aachen_Gal3_Expression.png" width="400px"></html><br />
|-<br />
|'''{{{title|SDS-PAGE of K1319020 expression}}}'''<br />{{{subtitle|The fusion protein was fully translated to the correct molecular mass of 74&nbsp;kDa.}}}<br />
|}<br />
</div><br />
</center><br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319004 K1319004]==<br />
<span class="anchor" id="partsK1319004"></span><br />
<br />
===TEV protease with anti-self cleavage mutation S219V, codon-optimized for ''E. coli''===<br />
<br />
This part is a TEV protease in RFC25 that was optimized for expression in E. coli. The part contains the S219V anti-self cleavage mutation.<br />
<br />
The TEV Protease (also known as Tobaco Edge Virus nuclear inclusion a endopeptidase) is a highly sequence specific cysteine protease from the Tobacco Edge Virus (TEV). The protease is highly sequence specific. The consensus sequence for the cut is ENLYFQ\S with \ denoting the cleaved peptide bond. This sequence can be found in the part [http://parts.igem.org/Part:BBa_K1319016 K1319016]. <br />
<br />
ENLYFQ\S is the optimal cleavage site with the highest activity but the protease is also active to a greater or lesser extent on a range of substrates. The highest cleavage is of sequences closest to the consensus EXLYΦQ\φ where X is any residue, Φ is any large or medium hydrophobic amino acid and φ is any small hydrophobic amino acid.<br />
<br />
The TEV protease is commonly used as a biochemical tool to cleave affinity tags from purified proteins like [http://parts.igem.org/Part:BBa_K1319007 His-Tags]. The high specifity makes the protease relatively non-toxic ''in vitro'' and ''in vivo''. The molecular weight of the TEV protease is 27 kDa.<br />
<br />
===Usage and Biology===<br />
<br />
The TEV Protease was used and characterizes in the [http://parts.igem.org/Part:BBa_K1319008 K1319008] construct.<br />
<br />
To characterize the TEV protease we used the fusion protein [http://parts.igem.org/Part:BBa_K1319014 K1319014]. This fusion protein contains GFP ([http://parts.igem.org/Part:BBa_E0040 E0040]) bound to a dark quencher ([http://parts.igem.org/Part:BBa_K1319002 REACh2/K1319002]) over a [http://parts.igem.org/Part:BBa_K1319016 linker] which includes the TEV protease cleavage site. If the TEV protease successfully cuts the linker, GFP and its quencher would separate and the FRET (Förster Resonance Energy Transfer) system would be shut down. This would result in an increased GFP fluorescence.<br />
<br />
To demonstrate this behaviour a double plasmid system was designed using the biobrick K1319013 in a pSB3K3 backbone and K1319008 in a pSB1C3 backbone. Also [http://parts.igem.org/Part:BBa_I20260 I20260] was used as a positive control because it produces the same GFP as used in the fusion protein and is regulated by the same promoter, RBS and Terminator on the same plasmid backbone. [http://parts.igem.org/Part:BBa_B0015 B0015] was used as negative control. Induction of the double plasmid constructs occured after 2 h with 50 µl of 100mM IPTG in a 50 ml shake flask culture. <br />
<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|800px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/3/3e/Aachen_Graph2_14.PNG" width="800px"></html><br />
|-<br />
|'''{{{title|Comparison of the fluorescence adjusted for OD of I20260, B0015 and the double plasmid system K1319014 + K1319008}}}'''<br />{{{subtitle|After induction with IPTG after 2 h the double plasmid system produced a fast fluorescence response with an over 10-fold increase compared to the non induced state. I20260 served as positive control and B0015 as negative control.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
<br />
The increase in fluorescence after induction with IPTG is clear sign of funtional expression of the TEV protease. The difference between not induced and induced plasmid is proof that the increase in fluorescence is only attributed to the successful cleavage of the linker. Therefore this is proof of a functional expression of the TEV protease after induction with IPTG.<br />
<br />
Furthermore we validated the used plasmid with a Check PCR with one primer binding upstream on the plasmid backbone and one specifically in the insert.<br />
<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|500px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/0/0c/Aachen_K1319008_Gel_Check_PCR.png" width="500px"></html><br />
|-<br />
|'''{{{title|Check PCR for K1319008}}}'''<br />{{{subtitle|The length of the PCR product matches the length of the control plasmid.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
<br />
This validates that the construct is indeed the TEV protease and thereby the functionality of the TEV protease is established. The construct K1319008 was also sequenced. The sequencing data can be seen in the parts registry [http://parts.igem.org/Part:BBa_K1319008 here].<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319008 K1319008]==<br />
<span class="anchor" id="partsK1319008"></span><br />
<br />
=== IPTG-induced and T7-driven expression of TEV protease ===<br />
<br />
This protein generator produces TEV protease when induced with IPTG in a DE3 strain.<br />
<br />
===Usage and Biology===<br />
More information about the characterization of this part is available on the parts registry page for [http://parts.igem.org/Part:BBa_K1319004 K1319004]. This part was alos used in the validation and characterization of the parts [http://parts.igem.org/Part:BBa_K1319001 K1319001] and [http://parts.igem.org/Part:BBa_K1319001 K1319001].<br />
<br />
This biobrick is used in our [https://2014.igem.org/Team:Aachen/Project/FRET_Reporter REACh construct].<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319009 K1319009]==<br />
<br />
===mRFP-gal3-his fusion protein CDS===<br />
<span class="anchor" id="partsK1319009"></span><br />
<br />
This is a fusion protein created from E1010, K1319003 and K1319007.<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319010 K1319010]==<br />
<span class="anchor" id="partsK1319010"></span><br />
<br />
=== Constitutive expression of K1319000 ===<br />
<br />
This part expresses [http://parts.igem.org/Part:BBa_K1319000 K1319000] behind a [http://parts.igem.org/Part:BBa_J23101 J23101] constitutive promoter.<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319011 K1319011]==<br />
<span class="anchor" id="partsK1319011"></span><br />
<br />
=== Constitutive expression of K1319001 ===<br />
<br />
This part expresses [http://parts.igem.org/Part:BBa_K1319001 K1319001] behind a [http://parts.igem.org/Part:BBa_J23101 J23101] constitutive promoter.<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
== [http://parts.igem.org/Part:BBa_K1319012 K1319012] ==<br />
<span class="anchor" id="partsK1319012"></span><br />
<br />
=== Constitutive expression of K1319002 ===<br />
<br />
This part expresses [http://parts.igem.org/Part:BBa_K1319002 K1319002] behind a [http://parts.igem.org/Part:BBa_J23101 J23101] constitutive promoter.<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
== [http://parts.igem.org/Part:BBa_K1319013 K1319013] ==<br />
<span class="anchor" id="partsK1319013"></span><br />
<br />
=== Constitutive expression of GFP-REACh1 fusion protein ===<br />
<br />
This part expresses a [http://parts.igem.org/Part:BBa_E0040 E0040].[http://parts.igem.org/Part:BBa_K1319001 K1319001] fusion protein (GFP-REACh1) behind a [http://parts.igem.org/Part:BBa_J23101 J23101] promoter. <br />
<br />
===Usage and Biology===<br />
More information about the characterization of this part can be found on the part page of [http://parts.igem.org/Part:BBa_K1319001 K1319001].<br />
<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
== [http://parts.igem.org/Part:BBa_K1319014 K1319014] ==<br />
<span class="anchor" id="partsK1319014"></span><br />
<br />
===Constitutive expression of GFP-REACh2 fusion protein===<br />
<br />
This part expresses a [http://parts.igem.org/Part:BBa_E0040 E0040].[http://parts.igem.org/Part:BBa_K1319002 K1319002] fusion protein (GFP-REACh1) behind a [http://parts.igem.org/Part:BBa_J23101 J23101] promoter. <br />
[http://parts.igem.org/Part:BBa_K1319002 K1319002] is a '''dark quencher''' that eliminates the fluorescence of the GFP-domain by Förster Resonance Energy Transfer (FRET), but does not exhibit strong fluorescence itself.<br />
<br />
===Usage and Biology===<br />
The two domains can be separated from each other via a TEV protease cleavage site in the linker.<br />
<br />
More information about the characterization of this part can be found on the part page of [http://parts.igem.org/Part:BBa_K1319002 K1319002].<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
== [http://parts.igem.org/Part:BBa_K1319015 K1319015] ==<br />
<span class="anchor" id="partsK1319015"></span><br />
<br />
===Constitutive expression of GFP-EYFP fusion protein===<br />
<br />
This part expresses a [http://parts.igem.org/Part:BBa_E0040 E0040].[http://parts.igem.org/Part:BBa_K1319000 K1319000] fusion protein (GFP-EYFP) behind a [http://parts.igem.org/Part:BBa_J23101 J23101] promoter.<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
== [http://parts.igem.org/Part:BBa_K1319016 K1319016] ==<br />
<span class="anchor" id="partsK1319016"></span><br />
<br />
===TEV protease cleavage site===<br />
<br />
This sequence codes for a [http://parts.igem.org/Part:BBa_K1319004 TEV protease] cleavage site.<br />
<br />
ENLYFQ\S is the optimal cleavage site with the highest activity but the protease is also active to a greater or lesser extent on a range of substrates. The highest cleavage is of sequences closest to the consensus EXLYΦQ\φ where X is any residue, Φ is any large or medium hydrophobic amino acid and φ is any small hydrophobic amino acid.<br />
<br />
===Usage and Biology===<br />
<br />
A TEV protease is available codon optimised for ''E. coli'' with the part [http://parts.igem.org/Part:BBa_K1319004 K1319004].<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319017 K1319017]==<br />
<span class="anchor" id="partsK1319017"></span><br />
<br />
=== LasI induced iLOV ===<br />
<br />
This device produces iLOV (K660004) in response to a quorum sensing input.<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319020 K1319020]==<br />
<span class="anchor" id="partsK1319020"></span><br />
<br />
===Translational unit of mRFP-galectin3-His===<br />
<br />
This part is a translational unit of a mRFP-galectin-3-his (B0032.E1010.K1319003.K1319016.B0015)<br />
<br />
===Usage and Biology===<br />
<br />
For more information about the characterization of this part check out [http://parts.igem.org/Part:BBa_K1319003 K1319003].<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319042 K1319042]==<br />
<span class="anchor" id="partsK1319042"></span><br />
<br />
===IPTG inducible iLOV===<br />
<br />
This part can be used for IPTG-induced expression of K660004 (iLOV).<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
===iGEM Team Aachen Biobrick overview===<br />
<br />
<groupparts>iGEM14 Aachen</groupparts><br />
<br />
{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/PartsTeam:Aachen/Parts2014-10-18T03:31:35Z<p>Nbailly: /* human galectin-3, codon optimized for E. coli */</p>
<hr />
<div>__NOTOC__<br />
{{Team:Aachen/Header}}<br />
= iGEM Team Aachen BioBricks =<br />
<br />
This page lists the collection of BioBricks developed by our team for the project ''Cellock Holmes - A Case of Identity''.<br />
<br />
<center><br />
{| class="wikitable"<br />
! BioBrick !! Description<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319000 K1319000]<br />
||RFC[25] version of E0030<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319001 K1319001]<br />
||RFC[25]-compatible dark quencher based on K1319000 (E0030)<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319002 K1319002]<br />
||RFC[25]-compatible dark quencher based on K1319000 (E0030)<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319003 K1319003]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319004 K1319004]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319008 K1319008]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319009 K1319009]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319010 K1319010]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319011 K1319011]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319012 K1319012]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319013 K1319013]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319014 K1319014]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319015 K1319015]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319016 K1319016]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319017 K1319017]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319020 K1319020]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319042 K1319042]<br />
|} </center><br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319000 K1319000]==<br />
<span class="anchor" id="partsK1319000"></span><br />
<br />
===RFC &#91;25&#93; Version of E0020===<br />
<br />
This part is an RFC [25]-compatible version of BBa_E0030. The start and stop codons have been removed to make it RFC [25]-compatible and the part is flanked by the RFC [25] prefix- and suffix-sequences.<br />
<br />
The coding sequence encodes EYFP (enhanced yellow fluorescent protein) which is derived from ''A. victoria'' GFP. The excitation is 512&nbsp;nm and the emission is 534 nm. This part was used to create the parts K1319001 and K1319002. It can also be used in a fusion protein instead of E0030 due to its RFC[25] compability.<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319001 K1319001]==<br />
<span class="anchor" id="partsK1319001"></span><br />
<br />
===RFC [25] - compatible dark quencher based on K1319000 (E0030) called REACh1===<br />
<br />
This part is a RFC[25] dark quencher that is based upon K1319000 (the RFC[25] version of E0030/EYFP) called REACh1.<br />
<br />
Two mutations were introduced that eliminated fluorescence:<br />
* L90I<br />
* Y145W<br />
<br />
===References===<br />
* Ganesan S, Ameer-Beg SM, Ng TT, Vojnovic B, Wouters FS. A dark yellow fluorescent protein (YFP)-based Resonance Energy-Accepting Chromoprotein (REACh) for Förster resonance energy transfer with GFP. Proc Natl Acad Sci U S A. 2006 Mar 14;103(11):4089-94. Epub 2006 Mar 6. PubMed PMID: 16537489; PubMed Central PMCID: PMC1449651. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1449651/?report=classic PubMed Central]<br />
<br />
<br />
===Usage and Biology===<br />
This protein is designed to be a dark quencher for GFP ([http://parts.igem.org/Part:BBa_E0040 E0040]) in a FRET system. When used in a fusion protein with GFP it reduces the observed fluorescence of GFP drastically. In the biobrick [http://parts.igem.org/Part:BBa_K1319013 K1319013] this is realized and the proteins are fused together with the linker [http://parts.igem.org/Part:BBa_K1319016 K1319016] which includes a specific TEV protease (available as [http://parts.igem.org/Part:BBa_K1319004 K1319004]) cleavage site. The fusion of the proteins bring GFP and REACh1 in proximity to each other which allows GFP and REACh1 to act as donors and acceptors in a FRET (Förster Energy Transfer System) system. GFPs emission energy is thereby taken up by REACh1 and released as thermal energy instead of visible light. This eliminates the GFP fluorescence and allows the release of a strong fluorescence signal, if a TEV protease is expressed and the linker is cut. The cutting separates GFP and REACh1 cancelling the FRET interaction and providing a GFP fluorescence response.<br />
<br />
===Characterization===<br />
<br />
In order to characterize K1319001 it was expressed as a fusion protein together with GFP to show its quenching ability. This fusion protein, produced by K1319013, was located on the plasmid backbone pSB3K3. To show that the reduced fluorescence was due to the quenching ability of K1319001 and not based on a faulty expression, K1319008 was also introduced into the cells on a pSB1C3 backbone to create a double plasmid system. Both constructs were put into ''E. coli'' BL21 (DE3) and compared to [http://parts.igem.org/Part:BBa_I20260 I20260] and [http://parts.igem.org/Part:BBa_B0015 B0015]. B0015 was used as a negative control and I20260 was chosen as a positive control due to the expressed GFP being identical to the GFP being expressed in the fusion protein of K1319013 and having the same promoter, RBS, Terminator and plasmid backbone. <br />
<br />
The double plasmid system, B0015 and I20260 were measured in biological triplicates and after 2 h one set of triplicates of each of the three systems were induced with 50 µl 100mM IPTG in their 50 ml culture (500 ml shake flasks). Fluorescence measurement was performed with the [http://www.biotek.com/products/microplate_detection/synergymx_monochromator_based_multimode_microplate_reader.html Synergy Mx from BioTek] with an excitation wavelength of 496 ± 9 nm and emission wavelength of 516 ± 9 nm. The following graph shows the resulting fluorescence adjusted for the measured optical density to account for difference in growth of the cultures and to only show the fluorescence per cell.<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|800px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/5/56/Aachen_Graph2_13.PNG" width="800px"></html><br />
|-<br />
|'''{{{title|Comparison of the fluorescence adjusted for OD of I20260, B0015 and the double plasmid system K1319013 + K1319008}}}'''<br />{{{subtitle|After induction with IPTG after 2 h the double plasmid system produced a fast fluorescence response with an over 9-fold increase compared to the non induced state. I20260 served as positive control and B0015 as negative control.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
The strong fluorescence response (9-fold increase) after induction with IPTG shows the functionality of GFP inside the fusion protein. After the induction a TEV protease is produced which is specifically cutting the recognition sequence build inside the linker (K1319016) between GFP and K1319001. The cutting results in a separation of GFP and REACh1 collapsing the FRET system between the two. This results in a fluorescence signal of GFP due to the fact that the emission is no longer absorbed by REACh1 and emitted as heat but rather as fluorescence with a wavelength of 511 nm.<br />
<br />
The very low fluorescence in the non induced double plasmid system of K1319013 and K1319008 shows the functionality of K1319001. As established before, the GFP is being expressed correctly inside the fusion protein, therefore the reduction in fluorescence in the non induced double plasmid system is a direct result of the quenching ability of K1319001. The quenching, after subtraction of the background fluorescence, reduced the fluorescence of GFP by a factor of 12,5. This also includes the slight leakiness of the TEV protease.<br />
<br />
To eliminate the effect of the leakiness of the K1319008 construct in determining the quenching ability of K1319001, K1319013 was also compared against I20260 and B0015 on its own under the same condition as above (again in a biological triplicate).<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|800px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/b/bb/Aachen_K1319001_comparison_positive_negative.PNG" width="800px"></html><br />
|-<br />
|'''{{{title|Comparison of the fluorescence adjusted for OD of I20260, B0015 and K1319013}}}'''<br />{{{subtitle|The expressed fusion protein K1319013 exhibits a fluorescence more than 30 fold smaller as the positive control of I20260.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
<br />
This experiments shows that the fluorescence of the fusion protein GFP-REACh1 is more than 30-fold lower than normal GFP expression under identical circumstances (same backbone, promoter, RBS, terminator and cultivation circumstances). This demonstrates a quenching percentage of GFP of >96 %!<br />
<br />
To prove that the measured constructs were the same as assumed the plasmids were tested with specially designed Check PCRs with one primer binding upstream on the plasmid backbone and one primer binding specifically inside the insert. The following results were obtained for K1319013.<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|500px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/7/74/Aachen_K1319013_Gel_Check_PCR.png" width="500px"></html><br />
|-<br />
|'''{{{title|Check PCR for K1319013}}}'''<br />{{{subtitle|The length of the PCR product matches the length of the control plasmid.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
<br />
The PCR clearly shows that the used plasmid had the correct insert. After a positive identification of K1319013 the double plasmid system was also checked for the correct TEV protease plasmid K1319008.<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|500px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/0/0c/Aachen_K1319008_Gel_Check_PCR.png" width="500px"></html><br />
|-<br />
|'''{{{title|Check PCR for K1319008}}}'''<br />{{{subtitle|The length of the PCR product matches the length of the control plasmid.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
<br />
This PCR also positively identified K1319008. Additionally both parts were sequenced. The sequencing data for [http://parts.igem.org/Part:BBa_K1319008 K1319008] and [http://parts.igem.org/Part:BBa_K1319013 K1319013] can be found in the parts registry.<br />
<br />
===Characterization of K1319001 with the iGEM Team Aachen [https://2014.igem.org/Team:Aachen/Project/2D_Biosensor 2D Biosensor]===<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 40px; border:{{{border|0px solid #aaa}}};width:{{{width|500px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/6/6d/Aachen_K1319014%2B8_and_K1319013%2B8_pixelig_minus_bg.gif" width="500px"></html><br />
|-<br />
|'''{{{title|K1319013 + K1319008 and K1319014 + K1319008 non-induced (top) and induced (bottom) in sensor cells }}}'''<br />{{{subtitle|The induced double plasmid systems K1319013 + K1319008 and K1319014&nbsp;+&nbsp;K1319008 exhibit a clear fluorescence response in our sensor cells which in response to induction with 2 µl IPTG. }}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
To further characterize the REACh construct, they were introduced into the sensor cells which were then induced with 2&nbsp;µL IPTG with a concentration of 100&nbsp;mM. Subsequently, we took fluorescence measurement read-outs (GFP, excitation 496&nbsp;±&nbsp;9&nbsp;nm, emission&nbsp;516&nbsp;±&nbsp;9&nbsp;nm) roughly every 10&nbsp;min in the plate reader. The results were plotted in the heatmap shown on the left. <br />
<br />
The heatmap shows an increase of fluorescence from blue (no fluorescence) to red (high fluorescence). It is clearly visible that the induced chips are exhibiting a significantly higher fluorescence than the non-induced chips. This again shows that the constructs work as intended: The TEV protease cuts the linker so that the fusion protein is separated into GFP and a dark quencher, disabling the quenching. GFP has a clear fluorescence emission after the fusion protein has been successfully cut into two pieces by the TEV protease. <br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319002 K1319002]==<br />
<span class="anchor" id="partsK1319002"></span><br />
<br />
===RFC [25] - compatible dark quencher based on K1319000 (E0030) called REACh2===<br />
<br />
This part is a RFC[25] dark quencher that is based upon K1319000 (the RFC[25] version of E0030/EYFP) called REACh2. <br />
<br />
Three mutations were introduced that eliminated fluorescence: <br />
* L90I <br />
* Y145W <br />
* H148R<br />
<br />
===References===<br />
* Ganesan S, Ameer-Beg SM, Ng TT, Vojnovic B, Wouters FS. A dark yellow fluorescent protein (YFP)-based Resonance Energy-Accepting Chromoprotein (REACh) for Förster resonance energy transfer with GFP. Proc Natl Acad Sci U S A. 2006 Mar 14;103(11):4089-94. Epub 2006 Mar 6. PubMed PMID: 16537489; PubMed Central PMCID: PMC1449651. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1449651/?report=classic PubMed Central]<br />
<br />
===Usage and Biology===<br />
This protein is designed to be a dark quencher for GFP ([http://parts.igem.org/Part:BBa_E0040 E0040]) in a FRET system. When used in a fusion protein with GFP it reduces the observed fluorescence of GFP drastically. In the biobrick [http://parts.igem.org/Part:BBa_K1319014 K1319014] this is realized and the proteins are fused with the linker [http://parts.igem.org/Part:BBa_K1319016 K1319016] which includes a specific TEV protease (available as [http://parts.igem.org/Part:BBa_K1319004 K1319004]) cleavage site. The fusion of the proteins brings GFP and REACh2 in proximity to each other which allows GFP and REACh2 to act as donors and acceptors in a FRET (Förster Energy Transfer System) system. GFPs emission energy is thereby taken up by REACh2 and released as thermal energy instead of visible light. This eliminates the GFP fluorescence and allows a release of a strong fluorescence signal if a TEV protease is expressed and the linker is cut. The cutting separates GFP and REACh2 cancelling the FRET interaction and providing a GFP fluorescence response.<br />
<br />
===Characterization===<br />
<br />
In order to characterize K1319002 it was expressed as a fusion protein together with GFP to show its quenching ability. This fusion protein, produced by K1319014, was located on the plasmid backbone pSB3K3. To show that the reduced fluorescence was due to the quenching ability of K1319002 and not based on a faulty expression, K1319008 was also introduced into the cells on a pSB1C3 backbone to allow for a double plasmid system. Both constructs were put into ''E. coli'' BL21 (DE3) and compared to [http://parts.igem.org/Part:BBa_I20260 I20260] and [http://parts.igem.org/Part:BBa_B0015 B0015]. B0015 was used as a negative control and I20260 was chosen as a positive control due to the expressed GFP being identical to the GFP being expressed in the fusion protein of K1319014 and having the same promoter, RBS, Terminator and plasmid backbone. <br />
<br />
The double plasmid system, B0015 and I20260 were measured in biological triplicates and after 2 h one set of triplicates of each of the three systems were induced with 50 µl 100mM IPTG in their 50 ml culture (500 ml shake flasks). Fluorescence measurement was performed with the [http://www.biotek.com/products/microplate_detection/synergymx_monochromator_based_multimode_microplate_reader.html Synergy Mx from BioTek] with an excitation wavelength of 496 ± 9 nm and emission wavelength of 516 ± 9 nm. The following graph shows the resulting fluorescence adjusted for the measured optical density to account for difference in growth of the cultures and to only show the fluorescence per cell.<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|800px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/3/3e/Aachen_Graph2_14.PNG" width="800px"></html><br />
|-<br />
|'''{{{title|Comparison of the fluorescence adjusted for OD of I20260, B0015 and the double plasmid system K1319014 + K1319008}}}'''<br />{{{subtitle|After induction with IPTG after 2 h the double plasmid system produced a fast fluorescence response with an over 10-fold increase compared to the non induced state. I20260 served as positive control and B0015 as negative control.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
The strong fluorescence response (10-fold increase) after induction with IPTG shows the functionality of GFP inside the fusion protein. After the induction a TEV protease is produced which is specifically cutting the recognition sequence build inside the linker (K1319016) between GFP and K1319002. The cutting results in a separation of GFP and REACh2 resulting in a collapse of the FRET system between the two. This results in a fluorescence signal of GFP due to the fact that the emission is no longer absorbed by REACh2 and emitted as heat but rather as fluorescence with a wavelength of 511 nm. The overall fluorescence of the double plasmid system reaches the fluorescence level of the positive control indicating a total clavage of all fusion proteins by the produced TEV protease. <br />
<br />
The very low fluorescence in the non induced double plasmid system of K1319014 and K1319008 shows the functionality of K1319002. As established before, the GFP is being expressed correctly inside the fusion protein, therefore the reduction in fluorescence in the non induced double plasmid system is a direct result of the quenching ability of K1319002. The quenching, after subtraction of the background fluorescence, reduced the fluorescence of GFP by a factor of 10. This also includes the slight leakiness of the TEV protease.<br />
<br />
To eliminate the effect of the leakiness of the K1319008 construct in determining the quenching ability of K1319002, K1319014 was also compared against I20260 and B0015 on its own under the same condition as above (again in a biological triplicate).<br />
<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|800px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/6/61/Aachen_K1319002_characterization_positive_negative.PNG" width="800px"></html><br />
|-<br />
|'''{{{title|Comparison of the fluorescence adjusted for OD of I20260, B0015 and K1319014}}}'''<br />{{{subtitle|The expressed fusion protein K1319014 exhibits a fluorescence more than nearly 25-fold smaller as the positive control of I20260.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
<br />
This experiments shows that the fluorescence of the fusion protein GFP-REACh2 is nearly 25-fold lower than normal GFP expression under identical circumstances (same backbone, promoter, RBS, terminator and cultivation circumstances). This demonstrates a quenching percentage of GFP of ~96 %!<br />
<br />
To prove that the measured constructs were the same as assumed the plasmids were tested with specially designed Check PCRs with one primer binding upstream on the plasmid backbone and one primer binding specifically inside the insert. The following results were obtained for K1319014.<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|500px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/0/08/Aachen_K1319014_Gel_Check_PCR.png" width="500px"></html><br />
|-<br />
|'''{{{title|Check PCR for K1319014}}}'''<br />{{{subtitle|The length of the PCR product matches the length of the control plasmid.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
<br />
The PCR clearly shows that the used plasmid had the correct insert. After a positive identification of K1319014 the double plasmid system was also checked for the correct TEV protease plasmid K1319008.<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|500px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/0/0c/Aachen_K1319008_Gel_Check_PCR.png" width="500px"></html><br />
|-<br />
|'''{{{title|Check PCR for K1319008}}}'''<br />{{{subtitle|The length of the PCR product matches the length of the control plasmid.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
<br />
This PCR also positively identified K1319008. Additionally both parts were sequenced. The sequencing data for [http://parts.igem.org/Part:BBa_K1319008 K1319008] and [http://parts.igem.org/Part:BBa_K1319014 K1319014] can be found in the parts registry.<br />
<br />
===Characterization of K1319001 with the iGEM Team Aachen [https://2014.igem.org/Team:Aachen/Project/2D_Biosensor 2D Biosensor]===<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 40px; border:{{{border|0px solid #aaa}}};width:{{{width|500px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/6/6d/Aachen_K1319014%2B8_and_K1319013%2B8_pixelig_minus_bg.gif" width="500px"></html><br />
|-<br />
|'''{{{title|K1319013 + K1319008 and K1319014 + K1319008 non-induced (top) and induced (bottom) in sensor cells }}}'''<br />{{{subtitle|The induced double plasmid systems K1319013 + K1319008 and K1319014&nbsp;+&nbsp;K1319008 exhibit a clear fluorescence response in our sensor cells which in response to induction with 2 µl IPTG. }}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
To further characterize the REACh construct, they were introduced into the sensor cells which were then induced with 2&nbsp;µL IPTG with a concentration of 100&nbsp;mM. Subsequently, we took fluorescence measurement read-outs (GFP, excitation 496&nbsp;±&nbsp;9&nbsp;nm, emission&nbsp;516&nbsp;±&nbsp;9&nbsp;nm) roughly every 10&nbsp;min in the plate reader. The results were plotted in the heatmap shown on the left. <br />
<br />
The heatmap shows an increase of fluorescence from blue (no fluorescence) to red (high fluorescence). It is clearly visible that the induced chips are exhibiting a significantly higher fluorescence than the non-induced chips. This again shows that the constructs work as intended: The TEV protease cuts the linker so that the fusion protein is separated into GFP and a dark quencher, disabling the quenching. GFP has a clear fluorescence emission after the fusion protein has been successfully cut into two pieces by the TEV protease. <br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319003 K1319003]==<br />
<span class="anchor" id="partsK1319003"></span><br />
<br />
===Human galectin-3, codon-optimized for ''E.&nbsp;coli''===<br />
<br />
Galectin-3 is a 26 kDa protein that binds certain LPS patterns. It especially bind the O-section of the LPS.<br />
<br />
Galectins are proteins of the lectin family, which posess '''carbonhydrate recognition domains''' binding specifically to β-galactoside sugar residues. In humans, 10 different galectines have been identified, among which is galectin-3. <br />
<br />
Galectin-3 has a size of about 31&nbsp;kDA and is encoded by a single gene, LGALS3. It has many physiological functions, such as '''cell adhesion, cell growth and differentiation,''' and contributes to the development of '''cancer, inflammation, fibrosis and others'''.<br />
<br />
Human galectin-3 is a protein of the lectin-family that was shown to bind the LPS of multiple human pathogens.<br />
Some of them, including ''Pseudomonas&nbsp;aeruginosa'' protect themselves against the human immune system by mimicking the lipopolysaccharides (LPS) present on human erythrocytes.<br />
<br />
===Usage and Biology===<br />
<br />
K1319003 was used to create [http://parts.igem.org/Part:BBa_K1319020 K1319020], a Galectin-mRFP fusion protein with a C-terminal His tag in the [https://2014.igem.org/Team:Heidelberg/Team/Collaborations Heidelberger expression vector] pSBX1A3.<br />
<br />
We also cloned our K1319003 into the pET17 expression vector and expressed all combinations of fusion proteins in E.&nbsp;coli&nbsp;BL21(DE3). An SDS-PAGE showed that all fusion proteins were fully translated:<br />
<center><br />
<div class="figure" style="float:{{{align|center}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|800px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/thumb/5/52/Aachen_14-10-04_Expression_Pellets_iMO.png/425px-Aachen_14-10-04_Expression_Pellets_iMO.png" width="400px"></html><br />
|-<br />
|'''{{{title|Pellets of different fusion protein expressions}}}'''<br />{{{subtitle|Expression in the pET17 vector was much more leaky than the expression in the pSBX vectors.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
<center><br />
<div class="figure" style="float:{{{align|center}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|800px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/thumb/6/62/Aachen_Gal3_Expression.png/425px-Aachen_Gal3_Expression.png" width="400px"></html><br />
|-<br />
|'''{{{title|SDS-PAGE of K1319020 expression}}}'''<br />{{{subtitle|The fusion protein was fully translated to the correct molecular mass of 74&nbsp;kDa.}}}<br />
|}<br />
</div><br />
</center><br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319004 K1319004]==<br />
<span class="anchor" id="partsK1319004"></span><br />
<br />
===TEV protease with anti-self cleavage mutation S219V, codon optimized for ''E. coli''===<br />
<br />
This part is a TEV protease in RFC25 that was optimized for expression in E. coli. The part contains the S219V anti-self cleavage mutation.<br />
<br />
The TEV Protease (also known as Tobaco Edge Virus nuclear inclusion a endopeptidase) is a highly sequence specific cysteine protease from the Tobacco Edge Virus (TEV). The protease is highly sequence specific. The consensus sequence for the cut is ENLYFQ\S with \ denoting the cleaved peptide bond. This sequence can be found in the part [http://parts.igem.org/Part:BBa_K1319016 K1319016]. <br />
<br />
ENLYFQ\S is the optimal cleavage site with the highest activity but the protease is also active to a greater or lesser extent on a range of substrates. The highest cleavage is of sequences closest to the consensus EXLYΦQ\φ where X is any residue, Φ is any large or medium hydrophobic amino acid and φ is any small hydrophobic amino acid.<br />
<br />
The TEV protease is commonly used as a biochemical tool to cleave affinity tags from purified proteins like [http://parts.igem.org/Part:BBa_K1319007 His-Tags]. The high specifity makes the protease relatively non-toxic ''in vitro'' and ''in vivo''. The molecular weight of the TEV protease is 27 kDa.<br />
<br />
===Usage and Biology===<br />
<br />
The TEV Protease was used and characterizes in the [http://parts.igem.org/Part:BBa_K1319008 K1319008] construct.<br />
<br />
To characterize the TEV protease we used the fusion protein [http://parts.igem.org/Part:BBa_K1319014 K1319014]. This fusion protein contains GFP ([http://parts.igem.org/Part:BBa_E0040 E0040]) bound to a dark quencher ([http://parts.igem.org/Part:BBa_K1319002 REACh2/K1319002]) over a [http://parts.igem.org/Part:BBa_K1319016 linker] which includes the TEV protease cleavage site. If the TEV protease successfully cuts the linker, GFP and its quencher would separate and the FRET (Förster Resonance Energy Transfer) system would be shut down. This would result in an increased GFP fluorescence.<br />
<br />
To demonstrate this behaviour a double plasmid system was designed using the biobrick K1319013 in a pSB3K3 backbone and K1319008 in a pSB1C3 backbone. Also [http://parts.igem.org/Part:BBa_I20260 I20260] was used as a positive control because it produces the same GFP as used in the fusion protein and is regulated by the same promoter, RBS and Terminator on the same plasmid backbone. [http://parts.igem.org/Part:BBa_B0015 B0015] was used as negative control. Induction of the double plasmid constructs occured after 2 h with 50 µl of 100mM IPTG in a 50 ml shake flask culture. <br />
<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|800px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/3/3e/Aachen_Graph2_14.PNG" width="800px"></html><br />
|-<br />
|'''{{{title|Comparison of the fluorescence adjusted for OD of I20260, B0015 and the double plasmid system K1319014 + K1319008}}}'''<br />{{{subtitle|After induction with IPTG after 2 h the double plasmid system produced a fast fluorescence response with an over 10-fold increase compared to the non induced state. I20260 served as positive control and B0015 as negative control.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
<br />
The increase in fluorescence after induction with IPTG is clear sign of funtional expression of the TEV protease. The difference between not induced and induced plasmid is proof that the increase in fluorescence is only attributed to the successful cleavage of the linker. Therefore this is proof of a functional expression of the TEV protease after induction with IPTG.<br />
<br />
Furthermore we validated the used plasmid with a Check PCR with one primer binding upstream on the plasmid backbone and one specifically in the insert.<br />
<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|500px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/0/0c/Aachen_K1319008_Gel_Check_PCR.png" width="500px"></html><br />
|-<br />
|'''{{{title|Check PCR for K1319008}}}'''<br />{{{subtitle|The length of the PCR product matches the length of the control plasmid.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
<br />
This validates that the construct is indeed the TEV protease and thereby the functionality of the TEV protease is established. The construct K1319008 was also sequenced. The sequencing data can be seen in the parts registry [http://parts.igem.org/Part:BBa_K1319008 here].<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319008 K1319008]==<br />
<span class="anchor" id="partsK1319008"></span><br />
<br />
=== IPTG-induced and T7-driven expression of TEV protease ===<br />
<br />
This protein generator produces TEV protease when induced with IPTG in a DE3 strain.<br />
<br />
===Usage and Biology===<br />
More information about the characterization of this part is available on the parts registry page for [http://parts.igem.org/Part:BBa_K1319004 K1319004]. This part was alos used in the validation and characterization of the parts [http://parts.igem.org/Part:BBa_K1319001 K1319001] and [http://parts.igem.org/Part:BBa_K1319001 K1319001].<br />
<br />
This biobrick is used in our [https://2014.igem.org/Team:Aachen/Project/FRET_Reporter REACh construct].<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319009 K1319009]==<br />
<br />
===mRFP-gal3-his fusion protein CDS===<br />
<span class="anchor" id="partsK1319009"></span><br />
<br />
This is a fusion protein created from E1010, K1319003 and K1319007.<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319010 K1319010]==<br />
<span class="anchor" id="partsK1319010"></span><br />
<br />
=== Constitutive expression of K1319000 ===<br />
<br />
This part expresses [http://parts.igem.org/Part:BBa_K1319000 K1319000] behind a [http://parts.igem.org/Part:BBa_J23101 J23101] constitutive promoter.<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319011 K1319011]==<br />
<span class="anchor" id="partsK1319011"></span><br />
<br />
=== Constitutive expression of K1319001 ===<br />
<br />
This part expresses [http://parts.igem.org/Part:BBa_K1319001 K1319001] behind a [http://parts.igem.org/Part:BBa_J23101 J23101] constitutive promoter.<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
== [http://parts.igem.org/Part:BBa_K1319012 K1319012] ==<br />
<span class="anchor" id="partsK1319012"></span><br />
<br />
=== Constitutive expression of K1319002 ===<br />
<br />
This part expresses [http://parts.igem.org/Part:BBa_K1319002 K1319002] behind a [http://parts.igem.org/Part:BBa_J23101 J23101] constitutive promoter.<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
== [http://parts.igem.org/Part:BBa_K1319013 K1319013] ==<br />
<span class="anchor" id="partsK1319013"></span><br />
<br />
=== Constitutive expression of GFP-REACh1 fusion protein ===<br />
<br />
This part expresses a [http://parts.igem.org/Part:BBa_E0040 E0040].[http://parts.igem.org/Part:BBa_K1319001 K1319001] fusion protein (GFP-REACh1) behind a [http://parts.igem.org/Part:BBa_J23101 J23101] promoter. <br />
<br />
===Usage and Biology===<br />
More information about the characterization of this part can be found on the part page of [http://parts.igem.org/Part:BBa_K1319001 K1319001].<br />
<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
== [http://parts.igem.org/Part:BBa_K1319014 K1319014] ==<br />
<span class="anchor" id="partsK1319014"></span><br />
<br />
===Constitutive expression of GFP-REACh2 fusion protein===<br />
<br />
This part expresses a [http://parts.igem.org/Part:BBa_E0040 E0040].[http://parts.igem.org/Part:BBa_K1319002 K1319002] fusion protein (GFP-REACh1) behind a [http://parts.igem.org/Part:BBa_J23101 J23101] promoter. <br />
[http://parts.igem.org/Part:BBa_K1319002 K1319002] is a '''dark quencher''' that eliminates the fluorescence of the GFP-domain by Förster Resonance Energy Transfer (FRET), but does not exhibit strong fluorescence itself.<br />
<br />
===Usage and Biology===<br />
The two domains can be separated from each other via a TEV protease cleavage site in the linker.<br />
<br />
More information about the characterization of this part can be found on the part page of [http://parts.igem.org/Part:BBa_K1319002 K1319002].<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
== [http://parts.igem.org/Part:BBa_K1319015 K1319015] ==<br />
<span class="anchor" id="partsK1319015"></span><br />
<br />
===Constitutive expression of GFP-EYFP fusion protein===<br />
<br />
This part expresses a [http://parts.igem.org/Part:BBa_E0040 E0040].[http://parts.igem.org/Part:BBa_K1319000 K1319000] fusion protein (GFP-EYFP) behind a [http://parts.igem.org/Part:BBa_J23101 J23101] promoter.<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
== [http://parts.igem.org/Part:BBa_K1319016 K1319016] ==<br />
<span class="anchor" id="partsK1319016"></span><br />
<br />
===TEV protease cleavage site===<br />
<br />
This sequence codes for a [http://parts.igem.org/Part:BBa_K1319004 TEV protease] cleavage site.<br />
<br />
ENLYFQ\S is the optimal cleavage site with the highest activity but the protease is also active to a greater or lesser extent on a range of substrates. The highest cleavage is of sequences closest to the consensus EXLYΦQ\φ where X is any residue, Φ is any large or medium hydrophobic amino acid and φ is any small hydrophobic amino acid.<br />
<br />
===Usage and Biology===<br />
<br />
A TEV protease is available codon optimised for ''E. coli'' with the part [http://parts.igem.org/Part:BBa_K1319004 K1319004].<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319017 K1319017]==<br />
<span class="anchor" id="partsK1319017"></span><br />
<br />
=== LasI induced iLOV ===<br />
<br />
This device produces iLOV (K660004) in response to a quorum sensing input.<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319020 K1319020]==<br />
<span class="anchor" id="partsK1319020"></span><br />
<br />
===Translational unit of mRFP-galectin3-His===<br />
<br />
This part is a translational unit of a mRFP-galectin-3-his (B0032.E1010.K1319003.K1319016.B0015)<br />
<br />
===Usage and Biology===<br />
<br />
For more information about the characterization of this part check out [http://parts.igem.org/Part:BBa_K1319003 K1319003].<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319042 K1319042]==<br />
<span class="anchor" id="partsK1319042"></span><br />
<br />
===IPTG inducible iLOV===<br />
<br />
This part can be used for IPTG-induced expression of K660004 (iLOV).<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
===iGEM Team Aachen Biobrick overview===<br />
<br />
<groupparts>iGEM14 Aachen</groupparts><br />
<br />
{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/PartsTeam:Aachen/Parts2014-10-18T03:30:34Z<p>Nbailly: /* characterization of K1319001 with the iGEM Team Aachen 2D Biosensor */</p>
<hr />
<div>__NOTOC__<br />
{{Team:Aachen/Header}}<br />
= iGEM Team Aachen BioBricks =<br />
<br />
This page lists the collection of BioBricks developed by our team for the project ''Cellock Holmes - A Case of Identity''.<br />
<br />
<center><br />
{| class="wikitable"<br />
! BioBrick !! Description<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319000 K1319000]<br />
||RFC[25] version of E0030<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319001 K1319001]<br />
||RFC[25]-compatible dark quencher based on K1319000 (E0030)<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319002 K1319002]<br />
||RFC[25]-compatible dark quencher based on K1319000 (E0030)<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319003 K1319003]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319004 K1319004]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319008 K1319008]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319009 K1319009]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319010 K1319010]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319011 K1319011]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319012 K1319012]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319013 K1319013]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319014 K1319014]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319015 K1319015]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319016 K1319016]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319017 K1319017]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319020 K1319020]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319042 K1319042]<br />
|} </center><br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319000 K1319000]==<br />
<span class="anchor" id="partsK1319000"></span><br />
<br />
===RFC &#91;25&#93; Version of E0020===<br />
<br />
This part is an RFC [25]-compatible version of BBa_E0030. The start and stop codons have been removed to make it RFC [25]-compatible and the part is flanked by the RFC [25] prefix- and suffix-sequences.<br />
<br />
The coding sequence encodes EYFP (enhanced yellow fluorescent protein) which is derived from ''A. victoria'' GFP. The excitation is 512&nbsp;nm and the emission is 534 nm. This part was used to create the parts K1319001 and K1319002. It can also be used in a fusion protein instead of E0030 due to its RFC[25] compability.<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319001 K1319001]==<br />
<span class="anchor" id="partsK1319001"></span><br />
<br />
===RFC [25] - compatible dark quencher based on K1319000 (E0030) called REACh1===<br />
<br />
This part is a RFC[25] dark quencher that is based upon K1319000 (the RFC[25] version of E0030/EYFP) called REACh1.<br />
<br />
Two mutations were introduced that eliminated fluorescence:<br />
* L90I<br />
* Y145W<br />
<br />
===References===<br />
* Ganesan S, Ameer-Beg SM, Ng TT, Vojnovic B, Wouters FS. A dark yellow fluorescent protein (YFP)-based Resonance Energy-Accepting Chromoprotein (REACh) for Förster resonance energy transfer with GFP. Proc Natl Acad Sci U S A. 2006 Mar 14;103(11):4089-94. Epub 2006 Mar 6. PubMed PMID: 16537489; PubMed Central PMCID: PMC1449651. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1449651/?report=classic PubMed Central]<br />
<br />
<br />
===Usage and Biology===<br />
This protein is designed to be a dark quencher for GFP ([http://parts.igem.org/Part:BBa_E0040 E0040]) in a FRET system. When used in a fusion protein with GFP it reduces the observed fluorescence of GFP drastically. In the biobrick [http://parts.igem.org/Part:BBa_K1319013 K1319013] this is realized and the proteins are fused together with the linker [http://parts.igem.org/Part:BBa_K1319016 K1319016] which includes a specific TEV protease (available as [http://parts.igem.org/Part:BBa_K1319004 K1319004]) cleavage site. The fusion of the proteins bring GFP and REACh1 in proximity to each other which allows GFP and REACh1 to act as donors and acceptors in a FRET (Förster Energy Transfer System) system. GFPs emission energy is thereby taken up by REACh1 and released as thermal energy instead of visible light. This eliminates the GFP fluorescence and allows the release of a strong fluorescence signal, if a TEV protease is expressed and the linker is cut. The cutting separates GFP and REACh1 cancelling the FRET interaction and providing a GFP fluorescence response.<br />
<br />
===Characterization===<br />
<br />
In order to characterize K1319001 it was expressed as a fusion protein together with GFP to show its quenching ability. This fusion protein, produced by K1319013, was located on the plasmid backbone pSB3K3. To show that the reduced fluorescence was due to the quenching ability of K1319001 and not based on a faulty expression, K1319008 was also introduced into the cells on a pSB1C3 backbone to create a double plasmid system. Both constructs were put into ''E. coli'' BL21 (DE3) and compared to [http://parts.igem.org/Part:BBa_I20260 I20260] and [http://parts.igem.org/Part:BBa_B0015 B0015]. B0015 was used as a negative control and I20260 was chosen as a positive control due to the expressed GFP being identical to the GFP being expressed in the fusion protein of K1319013 and having the same promoter, RBS, Terminator and plasmid backbone. <br />
<br />
The double plasmid system, B0015 and I20260 were measured in biological triplicates and after 2 h one set of triplicates of each of the three systems were induced with 50 µl 100mM IPTG in their 50 ml culture (500 ml shake flasks). Fluorescence measurement was performed with the [http://www.biotek.com/products/microplate_detection/synergymx_monochromator_based_multimode_microplate_reader.html Synergy Mx from BioTek] with an excitation wavelength of 496 ± 9 nm and emission wavelength of 516 ± 9 nm. The following graph shows the resulting fluorescence adjusted for the measured optical density to account for difference in growth of the cultures and to only show the fluorescence per cell.<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|800px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/5/56/Aachen_Graph2_13.PNG" width="800px"></html><br />
|-<br />
|'''{{{title|Comparison of the fluorescence adjusted for OD of I20260, B0015 and the double plasmid system K1319013 + K1319008}}}'''<br />{{{subtitle|After induction with IPTG after 2 h the double plasmid system produced a fast fluorescence response with an over 9-fold increase compared to the non induced state. I20260 served as positive control and B0015 as negative control.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
The strong fluorescence response (9-fold increase) after induction with IPTG shows the functionality of GFP inside the fusion protein. After the induction a TEV protease is produced which is specifically cutting the recognition sequence build inside the linker (K1319016) between GFP and K1319001. The cutting results in a separation of GFP and REACh1 collapsing the FRET system between the two. This results in a fluorescence signal of GFP due to the fact that the emission is no longer absorbed by REACh1 and emitted as heat but rather as fluorescence with a wavelength of 511 nm.<br />
<br />
The very low fluorescence in the non induced double plasmid system of K1319013 and K1319008 shows the functionality of K1319001. As established before, the GFP is being expressed correctly inside the fusion protein, therefore the reduction in fluorescence in the non induced double plasmid system is a direct result of the quenching ability of K1319001. The quenching, after subtraction of the background fluorescence, reduced the fluorescence of GFP by a factor of 12,5. This also includes the slight leakiness of the TEV protease.<br />
<br />
To eliminate the effect of the leakiness of the K1319008 construct in determining the quenching ability of K1319001, K1319013 was also compared against I20260 and B0015 on its own under the same condition as above (again in a biological triplicate).<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|800px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/b/bb/Aachen_K1319001_comparison_positive_negative.PNG" width="800px"></html><br />
|-<br />
|'''{{{title|Comparison of the fluorescence adjusted for OD of I20260, B0015 and K1319013}}}'''<br />{{{subtitle|The expressed fusion protein K1319013 exhibits a fluorescence more than 30 fold smaller as the positive control of I20260.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
<br />
This experiments shows that the fluorescence of the fusion protein GFP-REACh1 is more than 30-fold lower than normal GFP expression under identical circumstances (same backbone, promoter, RBS, terminator and cultivation circumstances). This demonstrates a quenching percentage of GFP of >96 %!<br />
<br />
To prove that the measured constructs were the same as assumed the plasmids were tested with specially designed Check PCRs with one primer binding upstream on the plasmid backbone and one primer binding specifically inside the insert. The following results were obtained for K1319013.<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|500px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/7/74/Aachen_K1319013_Gel_Check_PCR.png" width="500px"></html><br />
|-<br />
|'''{{{title|Check PCR for K1319013}}}'''<br />{{{subtitle|The length of the PCR product matches the length of the control plasmid.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
<br />
The PCR clearly shows that the used plasmid had the correct insert. After a positive identification of K1319013 the double plasmid system was also checked for the correct TEV protease plasmid K1319008.<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|500px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/0/0c/Aachen_K1319008_Gel_Check_PCR.png" width="500px"></html><br />
|-<br />
|'''{{{title|Check PCR for K1319008}}}'''<br />{{{subtitle|The length of the PCR product matches the length of the control plasmid.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
<br />
This PCR also positively identified K1319008. Additionally both parts were sequenced. The sequencing data for [http://parts.igem.org/Part:BBa_K1319008 K1319008] and [http://parts.igem.org/Part:BBa_K1319013 K1319013] can be found in the parts registry.<br />
<br />
===Characterization of K1319001 with the iGEM Team Aachen [https://2014.igem.org/Team:Aachen/Project/2D_Biosensor 2D Biosensor]===<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 40px; border:{{{border|0px solid #aaa}}};width:{{{width|500px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/6/6d/Aachen_K1319014%2B8_and_K1319013%2B8_pixelig_minus_bg.gif" width="500px"></html><br />
|-<br />
|'''{{{title|K1319013 + K1319008 and K1319014 + K1319008 non-induced (top) and induced (bottom) in sensor cells }}}'''<br />{{{subtitle|The induced double plasmid systems K1319013 + K1319008 and K1319014&nbsp;+&nbsp;K1319008 exhibit a clear fluorescence response in our sensor cells which in response to induction with 2 µl IPTG. }}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
To further characterize the REACh construct, they were introduced into the sensor cells which were then induced with 2&nbsp;µL IPTG with a concentration of 100&nbsp;mM. Subsequently, we took fluorescence measurement read-outs (GFP, excitation 496&nbsp;±&nbsp;9&nbsp;nm, emission&nbsp;516&nbsp;±&nbsp;9&nbsp;nm) roughly every 10&nbsp;min in the plate reader. The results were plotted in the heatmap shown on the left. <br />
<br />
The heatmap shows an increase of fluorescence from blue (no fluorescence) to red (high fluorescence). It is clearly visible that the induced chips are exhibiting a significantly higher fluorescence than the non-induced chips. This again shows that the constructs work as intended: The TEV protease cuts the linker so that the fusion protein is separated into GFP and a dark quencher, disabling the quenching. GFP has a clear fluorescence emission after the fusion protein has been successfully cut into two pieces by the TEV protease. <br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319002 K1319002]==<br />
<span class="anchor" id="partsK1319002"></span><br />
<br />
===RFC [25] - compatible dark quencher based on K1319000 (E0030) called REACh2===<br />
<br />
This part is a RFC[25] dark quencher that is based upon K1319000 (the RFC[25] version of E0030/EYFP) called REACh2. <br />
<br />
Three mutations were introduced that eliminated fluorescence: <br />
* L90I <br />
* Y145W <br />
* H148R<br />
<br />
===References===<br />
* Ganesan S, Ameer-Beg SM, Ng TT, Vojnovic B, Wouters FS. A dark yellow fluorescent protein (YFP)-based Resonance Energy-Accepting Chromoprotein (REACh) for Förster resonance energy transfer with GFP. Proc Natl Acad Sci U S A. 2006 Mar 14;103(11):4089-94. Epub 2006 Mar 6. PubMed PMID: 16537489; PubMed Central PMCID: PMC1449651. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1449651/?report=classic PubMed Central]<br />
<br />
===Usage and Biology===<br />
This protein is designed to be a dark quencher for GFP ([http://parts.igem.org/Part:BBa_E0040 E0040]) in a FRET system. When used in a fusion protein with GFP it reduces the observed fluorescence of GFP drastically. In the biobrick [http://parts.igem.org/Part:BBa_K1319014 K1319014] this is realized and the proteins are fused with the linker [http://parts.igem.org/Part:BBa_K1319016 K1319016] which includes a specific TEV protease (available as [http://parts.igem.org/Part:BBa_K1319004 K1319004]) cleavage site. The fusion of the proteins brings GFP and REACh2 in proximity to each other which allows GFP and REACh2 to act as donors and acceptors in a FRET (Förster Energy Transfer System) system. GFPs emission energy is thereby taken up by REACh2 and released as thermal energy instead of visible light. This eliminates the GFP fluorescence and allows a release of a strong fluorescence signal if a TEV protease is expressed and the linker is cut. The cutting separates GFP and REACh2 cancelling the FRET interaction and providing a GFP fluorescence response.<br />
<br />
===Characterization===<br />
<br />
In order to characterize K1319002 it was expressed as a fusion protein together with GFP to show its quenching ability. This fusion protein, produced by K1319014, was located on the plasmid backbone pSB3K3. To show that the reduced fluorescence was due to the quenching ability of K1319002 and not based on a faulty expression, K1319008 was also introduced into the cells on a pSB1C3 backbone to allow for a double plasmid system. Both constructs were put into ''E. coli'' BL21 (DE3) and compared to [http://parts.igem.org/Part:BBa_I20260 I20260] and [http://parts.igem.org/Part:BBa_B0015 B0015]. B0015 was used as a negative control and I20260 was chosen as a positive control due to the expressed GFP being identical to the GFP being expressed in the fusion protein of K1319014 and having the same promoter, RBS, Terminator and plasmid backbone. <br />
<br />
The double plasmid system, B0015 and I20260 were measured in biological triplicates and after 2 h one set of triplicates of each of the three systems were induced with 50 µl 100mM IPTG in their 50 ml culture (500 ml shake flasks). Fluorescence measurement was performed with the [http://www.biotek.com/products/microplate_detection/synergymx_monochromator_based_multimode_microplate_reader.html Synergy Mx from BioTek] with an excitation wavelength of 496 ± 9 nm and emission wavelength of 516 ± 9 nm. The following graph shows the resulting fluorescence adjusted for the measured optical density to account for difference in growth of the cultures and to only show the fluorescence per cell.<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|800px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/3/3e/Aachen_Graph2_14.PNG" width="800px"></html><br />
|-<br />
|'''{{{title|Comparison of the fluorescence adjusted for OD of I20260, B0015 and the double plasmid system K1319014 + K1319008}}}'''<br />{{{subtitle|After induction with IPTG after 2 h the double plasmid system produced a fast fluorescence response with an over 10-fold increase compared to the non induced state. I20260 served as positive control and B0015 as negative control.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
The strong fluorescence response (10-fold increase) after induction with IPTG shows the functionality of GFP inside the fusion protein. After the induction a TEV protease is produced which is specifically cutting the recognition sequence build inside the linker (K1319016) between GFP and K1319002. The cutting results in a separation of GFP and REACh2 resulting in a collapse of the FRET system between the two. This results in a fluorescence signal of GFP due to the fact that the emission is no longer absorbed by REACh2 and emitted as heat but rather as fluorescence with a wavelength of 511 nm. The overall fluorescence of the double plasmid system reaches the fluorescence level of the positive control indicating a total clavage of all fusion proteins by the produced TEV protease. <br />
<br />
The very low fluorescence in the non induced double plasmid system of K1319014 and K1319008 shows the functionality of K1319002. As established before, the GFP is being expressed correctly inside the fusion protein, therefore the reduction in fluorescence in the non induced double plasmid system is a direct result of the quenching ability of K1319002. The quenching, after subtraction of the background fluorescence, reduced the fluorescence of GFP by a factor of 10. This also includes the slight leakiness of the TEV protease.<br />
<br />
To eliminate the effect of the leakiness of the K1319008 construct in determining the quenching ability of K1319002, K1319014 was also compared against I20260 and B0015 on its own under the same condition as above (again in a biological triplicate).<br />
<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|800px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/6/61/Aachen_K1319002_characterization_positive_negative.PNG" width="800px"></html><br />
|-<br />
|'''{{{title|Comparison of the fluorescence adjusted for OD of I20260, B0015 and K1319014}}}'''<br />{{{subtitle|The expressed fusion protein K1319014 exhibits a fluorescence more than nearly 25-fold smaller as the positive control of I20260.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
<br />
This experiments shows that the fluorescence of the fusion protein GFP-REACh2 is nearly 25-fold lower than normal GFP expression under identical circumstances (same backbone, promoter, RBS, terminator and cultivation circumstances). This demonstrates a quenching percentage of GFP of ~96 %!<br />
<br />
To prove that the measured constructs were the same as assumed the plasmids were tested with specially designed Check PCRs with one primer binding upstream on the plasmid backbone and one primer binding specifically inside the insert. The following results were obtained for K1319014.<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|500px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/0/08/Aachen_K1319014_Gel_Check_PCR.png" width="500px"></html><br />
|-<br />
|'''{{{title|Check PCR for K1319014}}}'''<br />{{{subtitle|The length of the PCR product matches the length of the control plasmid.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
<br />
The PCR clearly shows that the used plasmid had the correct insert. After a positive identification of K1319014 the double plasmid system was also checked for the correct TEV protease plasmid K1319008.<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|500px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/0/0c/Aachen_K1319008_Gel_Check_PCR.png" width="500px"></html><br />
|-<br />
|'''{{{title|Check PCR for K1319008}}}'''<br />{{{subtitle|The length of the PCR product matches the length of the control plasmid.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
<br />
This PCR also positively identified K1319008. Additionally both parts were sequenced. The sequencing data for [http://parts.igem.org/Part:BBa_K1319008 K1319008] and [http://parts.igem.org/Part:BBa_K1319014 K1319014] can be found in the parts registry.<br />
<br />
===Characterization of K1319001 with the iGEM Team Aachen [https://2014.igem.org/Team:Aachen/Project/2D_Biosensor 2D Biosensor]===<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 40px; border:{{{border|0px solid #aaa}}};width:{{{width|500px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/6/6d/Aachen_K1319014%2B8_and_K1319013%2B8_pixelig_minus_bg.gif" width="500px"></html><br />
|-<br />
|'''{{{title|K1319013 + K1319008 and K1319014 + K1319008 non-induced (top) and induced (bottom) in sensor cells }}}'''<br />{{{subtitle|The induced double plasmid systems K1319013 + K1319008 and K1319014&nbsp;+&nbsp;K1319008 exhibit a clear fluorescence response in our sensor cells which in response to induction with 2 µl IPTG. }}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
To further characterize the REACh construct, they were introduced into the sensor cells which were then induced with 2&nbsp;µL IPTG with a concentration of 100&nbsp;mM. Subsequently, we took fluorescence measurement read-outs (GFP, excitation 496&nbsp;±&nbsp;9&nbsp;nm, emission&nbsp;516&nbsp;±&nbsp;9&nbsp;nm) roughly every 10&nbsp;min in the plate reader. The results were plotted in the heatmap shown on the left. <br />
<br />
The heatmap shows an increase of fluorescence from blue (no fluorescence) to red (high fluorescence). It is clearly visible that the induced chips are exhibiting a significantly higher fluorescence than the non-induced chips. This again shows that the constructs work as intended: The TEV protease cuts the linker so that the fusion protein is separated into GFP and a dark quencher, disabling the quenching. GFP has a clear fluorescence emission after the fusion protein has been successfully cut into two pieces by the TEV protease. <br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319003 K1319003]==<br />
<span class="anchor" id="partsK1319003"></span><br />
<br />
===human galectin-3, codon optimized for ''E. coli''===<br />
<br />
Galectin-3 is a 26 kDa protein that binds certain LPS patterns. It especially bind the O-section of the LPS.<br />
<br />
Galectins are proteins of the lectin family, which posess '''carbonhydrate recognition domains''' binding specifically to β-galactoside sugar residues. In humans, 10 different galectines have been identified, among which is galectin-3. <br />
<br />
Galectin-3 has a size of about 31&nbsp;kDA and is encoded by a single gene, LGALS3. It has many physiological functions, such as '''cell adhesion, cell growth and differentiation,''' and contributes to the development of '''cancer, inflammation, fibrosis and others'''.<br />
<br />
Human galectin-3 is a protein of the lectin-family that was shown to bind the LPS of multiple human pathogens.<br />
Some of them, including ''Pseudomonas&nbsp;aeruginosa'' protect themselves against the human immune system by mimicking the lipopolysaccharides (LPS) present on human erythrocytes. <br />
<br />
===Usage and Biology===<br />
<br />
K1319003 was used to create [http://parts.igem.org/Part:BBa_K1319020 K1319020], a Galectin-mRFP fusion protein with a C-terminal His tag in the [https://2014.igem.org/Team:Heidelberg/Team/Collaborations Heidelberger expression vector] pSBX1A3.<br />
<br />
We also cloned our K1319003 into the pET17 expression vector and expressed all combinations of fusion proteins in E.&nbsp;coli&nbsp;BL21(DE3). An SDS-PAGE showed that all fusion proteins were fully translated:<br />
<center><br />
<div class="figure" style="float:{{{align|center}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|800px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/thumb/5/52/Aachen_14-10-04_Expression_Pellets_iMO.png/425px-Aachen_14-10-04_Expression_Pellets_iMO.png" width="400px"></html><br />
|-<br />
|'''{{{title|Pellets of different fusion protein expressions}}}'''<br />{{{subtitle|Expression in the pET17 vector was much more leaky than the expression in the pSBX vectors.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
<center><br />
<div class="figure" style="float:{{{align|center}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|800px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/thumb/6/62/Aachen_Gal3_Expression.png/425px-Aachen_Gal3_Expression.png" width="400px"></html><br />
|-<br />
|'''{{{title|SDS-PAGE of K1319020 expression}}}'''<br />{{{subtitle|The fusion protein was fully translated to the correct molecular mass of 74&nbsp;kDa.}}}<br />
|}<br />
</div><br />
</center><br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319004 K1319004]==<br />
<span class="anchor" id="partsK1319004"></span><br />
<br />
===TEV protease with anti-self cleavage mutation S219V, codon optimized for ''E. coli''===<br />
<br />
This part is a TEV protease in RFC25 that was optimized for expression in E. coli. The part contains the S219V anti-self cleavage mutation.<br />
<br />
The TEV Protease (also known as Tobaco Edge Virus nuclear inclusion a endopeptidase) is a highly sequence specific cysteine protease from the Tobacco Edge Virus (TEV). The protease is highly sequence specific. The consensus sequence for the cut is ENLYFQ\S with \ denoting the cleaved peptide bond. This sequence can be found in the part [http://parts.igem.org/Part:BBa_K1319016 K1319016]. <br />
<br />
ENLYFQ\S is the optimal cleavage site with the highest activity but the protease is also active to a greater or lesser extent on a range of substrates. The highest cleavage is of sequences closest to the consensus EXLYΦQ\φ where X is any residue, Φ is any large or medium hydrophobic amino acid and φ is any small hydrophobic amino acid.<br />
<br />
The TEV protease is commonly used as a biochemical tool to cleave affinity tags from purified proteins like [http://parts.igem.org/Part:BBa_K1319007 His-Tags]. The high specifity makes the protease relatively non-toxic ''in vitro'' and ''in vivo''. The molecular weight of the TEV protease is 27 kDa.<br />
<br />
===Usage and Biology===<br />
<br />
The TEV Protease was used and characterizes in the [http://parts.igem.org/Part:BBa_K1319008 K1319008] construct.<br />
<br />
To characterize the TEV protease we used the fusion protein [http://parts.igem.org/Part:BBa_K1319014 K1319014]. This fusion protein contains GFP ([http://parts.igem.org/Part:BBa_E0040 E0040]) bound to a dark quencher ([http://parts.igem.org/Part:BBa_K1319002 REACh2/K1319002]) over a [http://parts.igem.org/Part:BBa_K1319016 linker] which includes the TEV protease cleavage site. If the TEV protease successfully cuts the linker, GFP and its quencher would separate and the FRET (Förster Resonance Energy Transfer) system would be shut down. This would result in an increased GFP fluorescence.<br />
<br />
To demonstrate this behaviour a double plasmid system was designed using the biobrick K1319013 in a pSB3K3 backbone and K1319008 in a pSB1C3 backbone. Also [http://parts.igem.org/Part:BBa_I20260 I20260] was used as a positive control because it produces the same GFP as used in the fusion protein and is regulated by the same promoter, RBS and Terminator on the same plasmid backbone. [http://parts.igem.org/Part:BBa_B0015 B0015] was used as negative control. Induction of the double plasmid constructs occured after 2 h with 50 µl of 100mM IPTG in a 50 ml shake flask culture. <br />
<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|800px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/3/3e/Aachen_Graph2_14.PNG" width="800px"></html><br />
|-<br />
|'''{{{title|Comparison of the fluorescence adjusted for OD of I20260, B0015 and the double plasmid system K1319014 + K1319008}}}'''<br />{{{subtitle|After induction with IPTG after 2 h the double plasmid system produced a fast fluorescence response with an over 10-fold increase compared to the non induced state. I20260 served as positive control and B0015 as negative control.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
<br />
The increase in fluorescence after induction with IPTG is clear sign of funtional expression of the TEV protease. The difference between not induced and induced plasmid is proof that the increase in fluorescence is only attributed to the successful cleavage of the linker. Therefore this is proof of a functional expression of the TEV protease after induction with IPTG.<br />
<br />
Furthermore we validated the used plasmid with a Check PCR with one primer binding upstream on the plasmid backbone and one specifically in the insert.<br />
<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|500px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/0/0c/Aachen_K1319008_Gel_Check_PCR.png" width="500px"></html><br />
|-<br />
|'''{{{title|Check PCR for K1319008}}}'''<br />{{{subtitle|The length of the PCR product matches the length of the control plasmid.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
<br />
This validates that the construct is indeed the TEV protease and thereby the functionality of the TEV protease is established. The construct K1319008 was also sequenced. The sequencing data can be seen in the parts registry [http://parts.igem.org/Part:BBa_K1319008 here].<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319008 K1319008]==<br />
<span class="anchor" id="partsK1319008"></span><br />
<br />
=== IPTG-induced and T7-driven expression of TEV protease ===<br />
<br />
This protein generator produces TEV protease when induced with IPTG in a DE3 strain.<br />
<br />
===Usage and Biology===<br />
More information about the characterization of this part is available on the parts registry page for [http://parts.igem.org/Part:BBa_K1319004 K1319004]. This part was alos used in the validation and characterization of the parts [http://parts.igem.org/Part:BBa_K1319001 K1319001] and [http://parts.igem.org/Part:BBa_K1319001 K1319001].<br />
<br />
This biobrick is used in our [https://2014.igem.org/Team:Aachen/Project/FRET_Reporter REACh construct].<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319009 K1319009]==<br />
<br />
===mRFP-gal3-his fusion protein CDS===<br />
<span class="anchor" id="partsK1319009"></span><br />
<br />
This is a fusion protein created from E1010, K1319003 and K1319007.<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319010 K1319010]==<br />
<span class="anchor" id="partsK1319010"></span><br />
<br />
=== Constitutive expression of K1319000 ===<br />
<br />
This part expresses [http://parts.igem.org/Part:BBa_K1319000 K1319000] behind a [http://parts.igem.org/Part:BBa_J23101 J23101] constitutive promoter.<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319011 K1319011]==<br />
<span class="anchor" id="partsK1319011"></span><br />
<br />
=== Constitutive expression of K1319001 ===<br />
<br />
This part expresses [http://parts.igem.org/Part:BBa_K1319001 K1319001] behind a [http://parts.igem.org/Part:BBa_J23101 J23101] constitutive promoter.<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
== [http://parts.igem.org/Part:BBa_K1319012 K1319012] ==<br />
<span class="anchor" id="partsK1319012"></span><br />
<br />
=== Constitutive expression of K1319002 ===<br />
<br />
This part expresses [http://parts.igem.org/Part:BBa_K1319002 K1319002] behind a [http://parts.igem.org/Part:BBa_J23101 J23101] constitutive promoter.<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
== [http://parts.igem.org/Part:BBa_K1319013 K1319013] ==<br />
<span class="anchor" id="partsK1319013"></span><br />
<br />
=== Constitutive expression of GFP-REACh1 fusion protein ===<br />
<br />
This part expresses a [http://parts.igem.org/Part:BBa_E0040 E0040].[http://parts.igem.org/Part:BBa_K1319001 K1319001] fusion protein (GFP-REACh1) behind a [http://parts.igem.org/Part:BBa_J23101 J23101] promoter. <br />
<br />
===Usage and Biology===<br />
More information about the characterization of this part can be found on the part page of [http://parts.igem.org/Part:BBa_K1319001 K1319001].<br />
<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
== [http://parts.igem.org/Part:BBa_K1319014 K1319014] ==<br />
<span class="anchor" id="partsK1319014"></span><br />
<br />
===Constitutive expression of GFP-REACh2 fusion protein===<br />
<br />
This part expresses a [http://parts.igem.org/Part:BBa_E0040 E0040].[http://parts.igem.org/Part:BBa_K1319002 K1319002] fusion protein (GFP-REACh1) behind a [http://parts.igem.org/Part:BBa_J23101 J23101] promoter. <br />
[http://parts.igem.org/Part:BBa_K1319002 K1319002] is a '''dark quencher''' that eliminates the fluorescence of the GFP-domain by Förster Resonance Energy Transfer (FRET), but does not exhibit strong fluorescence itself.<br />
<br />
===Usage and Biology===<br />
The two domains can be separated from each other via a TEV protease cleavage site in the linker.<br />
<br />
More information about the characterization of this part can be found on the part page of [http://parts.igem.org/Part:BBa_K1319002 K1319002].<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
== [http://parts.igem.org/Part:BBa_K1319015 K1319015] ==<br />
<span class="anchor" id="partsK1319015"></span><br />
<br />
===Constitutive expression of GFP-EYFP fusion protein===<br />
<br />
This part expresses a [http://parts.igem.org/Part:BBa_E0040 E0040].[http://parts.igem.org/Part:BBa_K1319000 K1319000] fusion protein (GFP-EYFP) behind a [http://parts.igem.org/Part:BBa_J23101 J23101] promoter.<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
== [http://parts.igem.org/Part:BBa_K1319016 K1319016] ==<br />
<span class="anchor" id="partsK1319016"></span><br />
<br />
===TEV protease cleavage site===<br />
<br />
This sequence codes for a [http://parts.igem.org/Part:BBa_K1319004 TEV protease] cleavage site.<br />
<br />
ENLYFQ\S is the optimal cleavage site with the highest activity but the protease is also active to a greater or lesser extent on a range of substrates. The highest cleavage is of sequences closest to the consensus EXLYΦQ\φ where X is any residue, Φ is any large or medium hydrophobic amino acid and φ is any small hydrophobic amino acid.<br />
<br />
===Usage and Biology===<br />
<br />
A TEV protease is available codon optimised for ''E. coli'' with the part [http://parts.igem.org/Part:BBa_K1319004 K1319004].<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319017 K1319017]==<br />
<span class="anchor" id="partsK1319017"></span><br />
<br />
=== LasI induced iLOV ===<br />
<br />
This device produces iLOV (K660004) in response to a quorum sensing input.<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319020 K1319020]==<br />
<span class="anchor" id="partsK1319020"></span><br />
<br />
===Translational unit of mRFP-galectin3-His===<br />
<br />
This part is a translational unit of a mRFP-galectin-3-his (B0032.E1010.K1319003.K1319016.B0015)<br />
<br />
===Usage and Biology===<br />
<br />
For more information about the characterization of this part check out [http://parts.igem.org/Part:BBa_K1319003 K1319003].<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319042 K1319042]==<br />
<span class="anchor" id="partsK1319042"></span><br />
<br />
===IPTG inducible iLOV===<br />
<br />
This part can be used for IPTG-induced expression of K660004 (iLOV).<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
===iGEM Team Aachen Biobrick overview===<br />
<br />
<groupparts>iGEM14 Aachen</groupparts><br />
<br />
{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/PartsTeam:Aachen/Parts2014-10-18T03:29:36Z<p>Nbailly: /* characterization of K1319001 with the iGEM Team Aachen 2D Biosensor */</p>
<hr />
<div>__NOTOC__<br />
{{Team:Aachen/Header}}<br />
= iGEM Team Aachen BioBricks =<br />
<br />
This page lists the collection of BioBricks developed by our team for the project ''Cellock Holmes - A Case of Identity''.<br />
<br />
<center><br />
{| class="wikitable"<br />
! BioBrick !! Description<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319000 K1319000]<br />
||RFC[25] version of E0030<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319001 K1319001]<br />
||RFC[25]-compatible dark quencher based on K1319000 (E0030)<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319002 K1319002]<br />
||RFC[25]-compatible dark quencher based on K1319000 (E0030)<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319003 K1319003]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319004 K1319004]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319008 K1319008]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319009 K1319009]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319010 K1319010]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319011 K1319011]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319012 K1319012]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319013 K1319013]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319014 K1319014]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319015 K1319015]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319016 K1319016]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319017 K1319017]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319020 K1319020]<br />
|-<br />
| [https://2014.igem.org/Team:Aachen/Parts#partsK1319042 K1319042]<br />
|} </center><br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319000 K1319000]==<br />
<span class="anchor" id="partsK1319000"></span><br />
<br />
===RFC &#91;25&#93; Version of E0020===<br />
<br />
This part is an RFC [25]-compatible version of BBa_E0030. The start and stop codons have been removed to make it RFC [25]-compatible and the part is flanked by the RFC [25] prefix- and suffix-sequences.<br />
<br />
The coding sequence encodes EYFP (enhanced yellow fluorescent protein) which is derived from ''A. victoria'' GFP. The excitation is 512&nbsp;nm and the emission is 534 nm. This part was used to create the parts K1319001 and K1319002. It can also be used in a fusion protein instead of E0030 due to its RFC[25] compability.<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319001 K1319001]==<br />
<span class="anchor" id="partsK1319001"></span><br />
<br />
===RFC [25] - compatible dark quencher based on K1319000 (E0030) called REACh1===<br />
<br />
This part is a RFC[25] dark quencher that is based upon K1319000 (the RFC[25] version of E0030/EYFP) called REACh1.<br />
<br />
Two mutations were introduced that eliminated fluorescence:<br />
* L90I<br />
* Y145W<br />
<br />
===References===<br />
* Ganesan S, Ameer-Beg SM, Ng TT, Vojnovic B, Wouters FS. A dark yellow fluorescent protein (YFP)-based Resonance Energy-Accepting Chromoprotein (REACh) for Förster resonance energy transfer with GFP. Proc Natl Acad Sci U S A. 2006 Mar 14;103(11):4089-94. Epub 2006 Mar 6. PubMed PMID: 16537489; PubMed Central PMCID: PMC1449651. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1449651/?report=classic PubMed Central]<br />
<br />
<br />
===Usage and Biology===<br />
This protein is designed to be a dark quencher for GFP ([http://parts.igem.org/Part:BBa_E0040 E0040]) in a FRET system. When used in a fusion protein with GFP it reduces the observed fluorescence of GFP drastically. In the biobrick [http://parts.igem.org/Part:BBa_K1319013 K1319013] this is realized and the proteins are fused together with the linker [http://parts.igem.org/Part:BBa_K1319016 K1319016] which includes a specific TEV protease (available as [http://parts.igem.org/Part:BBa_K1319004 K1319004]) cleavage site. The fusion of the proteins bring GFP and REACh1 in proximity to each other which allows GFP and REACh1 to act as donors and acceptors in a FRET (Förster Energy Transfer System) system. GFPs emission energy is thereby taken up by REACh1 and released as thermal energy instead of visible light. This eliminates the GFP fluorescence and allows the release of a strong fluorescence signal, if a TEV protease is expressed and the linker is cut. The cutting separates GFP and REACh1 cancelling the FRET interaction and providing a GFP fluorescence response.<br />
<br />
===Characterization===<br />
<br />
In order to characterize K1319001 it was expressed as a fusion protein together with GFP to show its quenching ability. This fusion protein, produced by K1319013, was located on the plasmid backbone pSB3K3. To show that the reduced fluorescence was due to the quenching ability of K1319001 and not based on a faulty expression, K1319008 was also introduced into the cells on a pSB1C3 backbone to create a double plasmid system. Both constructs were put into ''E. coli'' BL21 (DE3) and compared to [http://parts.igem.org/Part:BBa_I20260 I20260] and [http://parts.igem.org/Part:BBa_B0015 B0015]. B0015 was used as a negative control and I20260 was chosen as a positive control due to the expressed GFP being identical to the GFP being expressed in the fusion protein of K1319013 and having the same promoter, RBS, Terminator and plasmid backbone. <br />
<br />
The double plasmid system, B0015 and I20260 were measured in biological triplicates and after 2 h one set of triplicates of each of the three systems were induced with 50 µl 100mM IPTG in their 50 ml culture (500 ml shake flasks). Fluorescence measurement was performed with the [http://www.biotek.com/products/microplate_detection/synergymx_monochromator_based_multimode_microplate_reader.html Synergy Mx from BioTek] with an excitation wavelength of 496 ± 9 nm and emission wavelength of 516 ± 9 nm. The following graph shows the resulting fluorescence adjusted for the measured optical density to account for difference in growth of the cultures and to only show the fluorescence per cell.<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|800px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/5/56/Aachen_Graph2_13.PNG" width="800px"></html><br />
|-<br />
|'''{{{title|Comparison of the fluorescence adjusted for OD of I20260, B0015 and the double plasmid system K1319013 + K1319008}}}'''<br />{{{subtitle|After induction with IPTG after 2 h the double plasmid system produced a fast fluorescence response with an over 9-fold increase compared to the non induced state. I20260 served as positive control and B0015 as negative control.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
The strong fluorescence response (9-fold increase) after induction with IPTG shows the functionality of GFP inside the fusion protein. After the induction a TEV protease is produced which is specifically cutting the recognition sequence build inside the linker (K1319016) between GFP and K1319001. The cutting results in a separation of GFP and REACh1 collapsing the FRET system between the two. This results in a fluorescence signal of GFP due to the fact that the emission is no longer absorbed by REACh1 and emitted as heat but rather as fluorescence with a wavelength of 511 nm.<br />
<br />
The very low fluorescence in the non induced double plasmid system of K1319013 and K1319008 shows the functionality of K1319001. As established before, the GFP is being expressed correctly inside the fusion protein, therefore the reduction in fluorescence in the non induced double plasmid system is a direct result of the quenching ability of K1319001. The quenching, after subtraction of the background fluorescence, reduced the fluorescence of GFP by a factor of 12,5. This also includes the slight leakiness of the TEV protease.<br />
<br />
To eliminate the effect of the leakiness of the K1319008 construct in determining the quenching ability of K1319001, K1319013 was also compared against I20260 and B0015 on its own under the same condition as above (again in a biological triplicate).<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|800px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/b/bb/Aachen_K1319001_comparison_positive_negative.PNG" width="800px"></html><br />
|-<br />
|'''{{{title|Comparison of the fluorescence adjusted for OD of I20260, B0015 and K1319013}}}'''<br />{{{subtitle|The expressed fusion protein K1319013 exhibits a fluorescence more than 30 fold smaller as the positive control of I20260.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
<br />
This experiments shows that the fluorescence of the fusion protein GFP-REACh1 is more than 30-fold lower than normal GFP expression under identical circumstances (same backbone, promoter, RBS, terminator and cultivation circumstances). This demonstrates a quenching percentage of GFP of >96 %!<br />
<br />
To prove that the measured constructs were the same as assumed the plasmids were tested with specially designed Check PCRs with one primer binding upstream on the plasmid backbone and one primer binding specifically inside the insert. The following results were obtained for K1319013.<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|500px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/7/74/Aachen_K1319013_Gel_Check_PCR.png" width="500px"></html><br />
|-<br />
|'''{{{title|Check PCR for K1319013}}}'''<br />{{{subtitle|The length of the PCR product matches the length of the control plasmid.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
<br />
The PCR clearly shows that the used plasmid had the correct insert. After a positive identification of K1319013 the double plasmid system was also checked for the correct TEV protease plasmid K1319008.<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|500px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/0/0c/Aachen_K1319008_Gel_Check_PCR.png" width="500px"></html><br />
|-<br />
|'''{{{title|Check PCR for K1319008}}}'''<br />{{{subtitle|The length of the PCR product matches the length of the control plasmid.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
<br />
This PCR also positively identified K1319008. Additionally both parts were sequenced. The sequencing data for [http://parts.igem.org/Part:BBa_K1319008 K1319008] and [http://parts.igem.org/Part:BBa_K1319013 K1319013] can be found in the parts registry.<br />
<br />
===Characterization of K1319001 with the iGEM Team Aachen [https://2014.igem.org/Team:Aachen/Project/2D_Biosensor 2D Biosensor]===<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 40px; border:{{{border|0px solid #aaa}}};width:{{{width|500px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/6/6d/Aachen_K1319014%2B8_and_K1319013%2B8_pixelig_minus_bg.gif" width="500px"></html><br />
|-<br />
|'''{{{title|K1319013 + K1319008 and K1319014 + K1319008 non-induced (top) and induced (bottom) in sensor cells }}}'''<br />{{{subtitle|The induced double plasmid systems K1319013 + K1319008 and K1319014&nbsp;+&nbsp;K1319008 exhibit a clear fluorescence response in our sensor cells which in response to induction with 2 µl IPTG. }}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
To further characterize the REACh construct, they were introduced into the sensor cells which were then induced with 2&nbsp;µL IPTG with a concentration of 100&nbsp;mM. Subsequently, we took fluorescence measurement read-outs (GFP, excitation 496&nbsp;±&nbsp;9&nbsp;nm, emission&nbsp;516&nbsp;±&nbsp;9&nbsp;nm) roughly every 10&nbsp;min in the plate reader. The results were plotted in the heatmap shown on the left. <br />
<br />
The heatmap shows an increase of fluorescence from blue (no fluorescence) to red (high fluorescence). It is clearly visible that the induced chips are exhibiting a significantly higher fluorescence than the non-induced chips. This again shows that the constructs work as intended: The TEV protease cuts the linker so that the fusion protein is separated into GFP and a dark quencher, disabling the quenching. GFP has a clear fluorescence emission after the fusion protein has been successfully cut into two pieces by the TEV protease. <br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319002 K1319002]==<br />
<span class="anchor" id="partsK1319002"></span><br />
<br />
===RFC [25] - compatible dark quencher based on K1319000 (E0030) called REACh2===<br />
<br />
This part is a RFC[25] dark quencher that is based upon K1319000 (the RFC[25] version of E0030/EYFP) called REACh2. <br />
<br />
Three mutations were introduced that eliminated fluorescence: <br />
* L90I <br />
* Y145W <br />
* H148R<br />
<br />
===References===<br />
* Ganesan S, Ameer-Beg SM, Ng TT, Vojnovic B, Wouters FS. A dark yellow fluorescent protein (YFP)-based Resonance Energy-Accepting Chromoprotein (REACh) for Förster resonance energy transfer with GFP. Proc Natl Acad Sci U S A. 2006 Mar 14;103(11):4089-94. Epub 2006 Mar 6. PubMed PMID: 16537489; PubMed Central PMCID: PMC1449651. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1449651/?report=classic PubMed Central]<br />
<br />
===Usage and Biology===<br />
This protein is designed to be a dark quencher for GFP ([http://parts.igem.org/Part:BBa_E0040 E0040]) in a FRET system. When used in a fusion protein with GFP it reduces the observed fluorescence of GFP drastically. In the biobrick [http://parts.igem.org/Part:BBa_K1319014 K1319014] this is realized and the proteins are fused with the linker [http://parts.igem.org/Part:BBa_K1319016 K1319016] which includes a specific TEV protease (available as [http://parts.igem.org/Part:BBa_K1319004 K1319004]) cleavage site. The fusion of the proteins brings GFP and REACh2 in proximity to each other which allows GFP and REACh2 to act as donors and acceptors in a FRET (Förster Energy Transfer System) system. GFPs emission energy is thereby taken up by REACh2 and released as thermal energy instead of visible light. This eliminates the GFP fluorescence and allows a release of a strong fluorescence signal if a TEV protease is expressed and the linker is cut. The cutting separates GFP and REACh2 cancelling the FRET interaction and providing a GFP fluorescence response.<br />
<br />
===Characterization===<br />
<br />
In order to characterize K1319002 it was expressed as a fusion protein together with GFP to show its quenching ability. This fusion protein, produced by K1319014, was located on the plasmid backbone pSB3K3. To show that the reduced fluorescence was due to the quenching ability of K1319002 and not based on a faulty expression, K1319008 was also introduced into the cells on a pSB1C3 backbone to allow for a double plasmid system. Both constructs were put into ''E. coli'' BL21 (DE3) and compared to [http://parts.igem.org/Part:BBa_I20260 I20260] and [http://parts.igem.org/Part:BBa_B0015 B0015]. B0015 was used as a negative control and I20260 was chosen as a positive control due to the expressed GFP being identical to the GFP being expressed in the fusion protein of K1319014 and having the same promoter, RBS, Terminator and plasmid backbone. <br />
<br />
The double plasmid system, B0015 and I20260 were measured in biological triplicates and after 2 h one set of triplicates of each of the three systems were induced with 50 µl 100mM IPTG in their 50 ml culture (500 ml shake flasks). Fluorescence measurement was performed with the [http://www.biotek.com/products/microplate_detection/synergymx_monochromator_based_multimode_microplate_reader.html Synergy Mx from BioTek] with an excitation wavelength of 496 ± 9 nm and emission wavelength of 516 ± 9 nm. The following graph shows the resulting fluorescence adjusted for the measured optical density to account for difference in growth of the cultures and to only show the fluorescence per cell.<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|800px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/3/3e/Aachen_Graph2_14.PNG" width="800px"></html><br />
|-<br />
|'''{{{title|Comparison of the fluorescence adjusted for OD of I20260, B0015 and the double plasmid system K1319014 + K1319008}}}'''<br />{{{subtitle|After induction with IPTG after 2 h the double plasmid system produced a fast fluorescence response with an over 10-fold increase compared to the non induced state. I20260 served as positive control and B0015 as negative control.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
The strong fluorescence response (10-fold increase) after induction with IPTG shows the functionality of GFP inside the fusion protein. After the induction a TEV protease is produced which is specifically cutting the recognition sequence build inside the linker (K1319016) between GFP and K1319002. The cutting results in a separation of GFP and REACh2 resulting in a collapse of the FRET system between the two. This results in a fluorescence signal of GFP due to the fact that the emission is no longer absorbed by REACh2 and emitted as heat but rather as fluorescence with a wavelength of 511 nm. The overall fluorescence of the double plasmid system reaches the fluorescence level of the positive control indicating a total clavage of all fusion proteins by the produced TEV protease. <br />
<br />
The very low fluorescence in the non induced double plasmid system of K1319014 and K1319008 shows the functionality of K1319002. As established before, the GFP is being expressed correctly inside the fusion protein, therefore the reduction in fluorescence in the non induced double plasmid system is a direct result of the quenching ability of K1319002. The quenching, after subtraction of the background fluorescence, reduced the fluorescence of GFP by a factor of 10. This also includes the slight leakiness of the TEV protease.<br />
<br />
To eliminate the effect of the leakiness of the K1319008 construct in determining the quenching ability of K1319002, K1319014 was also compared against I20260 and B0015 on its own under the same condition as above (again in a biological triplicate).<br />
<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|800px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/6/61/Aachen_K1319002_characterization_positive_negative.PNG" width="800px"></html><br />
|-<br />
|'''{{{title|Comparison of the fluorescence adjusted for OD of I20260, B0015 and K1319014}}}'''<br />{{{subtitle|The expressed fusion protein K1319014 exhibits a fluorescence more than nearly 25-fold smaller as the positive control of I20260.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
<br />
This experiments shows that the fluorescence of the fusion protein GFP-REACh2 is nearly 25-fold lower than normal GFP expression under identical circumstances (same backbone, promoter, RBS, terminator and cultivation circumstances). This demonstrates a quenching percentage of GFP of ~96 %!<br />
<br />
To prove that the measured constructs were the same as assumed the plasmids were tested with specially designed Check PCRs with one primer binding upstream on the plasmid backbone and one primer binding specifically inside the insert. The following results were obtained for K1319014.<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|500px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/0/08/Aachen_K1319014_Gel_Check_PCR.png" width="500px"></html><br />
|-<br />
|'''{{{title|Check PCR for K1319014}}}'''<br />{{{subtitle|The length of the PCR product matches the length of the control plasmid.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
<br />
The PCR clearly shows that the used plasmid had the correct insert. After a positive identification of K1319014 the double plasmid system was also checked for the correct TEV protease plasmid K1319008.<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|500px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/0/0c/Aachen_K1319008_Gel_Check_PCR.png" width="500px"></html><br />
|-<br />
|'''{{{title|Check PCR for K1319008}}}'''<br />{{{subtitle|The length of the PCR product matches the length of the control plasmid.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
<br />
This PCR also positively identified K1319008. Additionally both parts were sequenced. The sequencing data for [http://parts.igem.org/Part:BBa_K1319008 K1319008] and [http://parts.igem.org/Part:BBa_K1319014 K1319014] can be found in the parts registry.<br />
<br />
===characterization of K1319001 with the iGEM Team Aachen [https://2014.igem.org/Team:Aachen/Project/2D_Biosensor 2D Biosensor]===<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 40px; border:{{{border|0px solid #aaa}}};width:{{{width|500px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/6/6d/Aachen_K1319014%2B8_and_K1319013%2B8_pixelig_minus_bg.gif" width="500px"></html><br />
|-<br />
|'''{{{title|K1319013 + K1319008 and K1319014 + K1319008 non-induced (top) and induced (bottom) in sensor cells }}}'''<br />{{{subtitle|The induced double plasmid systems K1319013 + K1319008 and K1319014&nbsp;+&nbsp;K1319008 exhibit a clear fluorescence response in our sensor cells which in response to induction with 2 µl IPTG. }}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
To further characterize the REACh construct, they were introduced into the sensor cells which were then induced with 2&nbsp;µL IPTG with a concentration of 100&nbsp;mM. Subsequently, we took fluorescence measurement read-outs (GFP, excitation 496&nbsp;±&nbsp;9&nbsp;nm, emission&nbsp;516&nbsp;±&nbsp;9&nbsp;nm) roughly every 10&nbsp;min in the plate reader. The results were plotted in the heatmap shown on the left. <br />
<br />
The heatmap shows an increase of fluorescence from blue (no fluorescence) to red (high fluorescence). It is clearly visible that the induced chips are exhibiting a significantly higher fluorescence than the non-induced chips. This again shows that the constructs work as intended: The TEV protease cuts the linker so that the fusion protein is separated into GFP and a dark quencher, disabling the quenching. GFP has a clear fluorescence emission after the fusion protein has been successfully cut into two pieces by the TEV protease. <br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319003 K1319003]==<br />
<span class="anchor" id="partsK1319003"></span><br />
<br />
===human galectin-3, codon optimized for ''E. coli''===<br />
<br />
Galectin-3 is a 26 kDa protein that binds certain LPS patterns. It especially bind the O-section of the LPS.<br />
<br />
Galectins are proteins of the lectin family, which posess '''carbonhydrate recognition domains''' binding specifically to β-galactoside sugar residues. In humans, 10 different galectines have been identified, among which is galectin-3. <br />
<br />
Galectin-3 has a size of about 31&nbsp;kDA and is encoded by a single gene, LGALS3. It has many physiological functions, such as '''cell adhesion, cell growth and differentiation,''' and contributes to the development of '''cancer, inflammation, fibrosis and others'''.<br />
<br />
Human galectin-3 is a protein of the lectin-family that was shown to bind the LPS of multiple human pathogens.<br />
Some of them, including ''Pseudomonas&nbsp;aeruginosa'' protect themselves against the human immune system by mimicking the lipopolysaccharides (LPS) present on human erythrocytes. <br />
<br />
===Usage and Biology===<br />
<br />
K1319003 was used to create [http://parts.igem.org/Part:BBa_K1319020 K1319020], a Galectin-mRFP fusion protein with a C-terminal His tag in the [https://2014.igem.org/Team:Heidelberg/Team/Collaborations Heidelberger expression vector] pSBX1A3.<br />
<br />
We also cloned our K1319003 into the pET17 expression vector and expressed all combinations of fusion proteins in E.&nbsp;coli&nbsp;BL21(DE3). An SDS-PAGE showed that all fusion proteins were fully translated:<br />
<center><br />
<div class="figure" style="float:{{{align|center}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|800px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/thumb/5/52/Aachen_14-10-04_Expression_Pellets_iMO.png/425px-Aachen_14-10-04_Expression_Pellets_iMO.png" width="400px"></html><br />
|-<br />
|'''{{{title|Pellets of different fusion protein expressions}}}'''<br />{{{subtitle|Expression in the pET17 vector was much more leaky than the expression in the pSBX vectors.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
<center><br />
<div class="figure" style="float:{{{align|center}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|800px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/thumb/6/62/Aachen_Gal3_Expression.png/425px-Aachen_Gal3_Expression.png" width="400px"></html><br />
|-<br />
|'''{{{title|SDS-PAGE of K1319020 expression}}}'''<br />{{{subtitle|The fusion protein was fully translated to the correct molecular mass of 74&nbsp;kDa.}}}<br />
|}<br />
</div><br />
</center><br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319004 K1319004]==<br />
<span class="anchor" id="partsK1319004"></span><br />
<br />
===TEV protease with anti-self cleavage mutation S219V, codon optimized for ''E. coli''===<br />
<br />
This part is a TEV protease in RFC25 that was optimized for expression in E. coli. The part contains the S219V anti-self cleavage mutation.<br />
<br />
The TEV Protease (also known as Tobaco Edge Virus nuclear inclusion a endopeptidase) is a highly sequence specific cysteine protease from the Tobacco Edge Virus (TEV). The protease is highly sequence specific. The consensus sequence for the cut is ENLYFQ\S with \ denoting the cleaved peptide bond. This sequence can be found in the part [http://parts.igem.org/Part:BBa_K1319016 K1319016]. <br />
<br />
ENLYFQ\S is the optimal cleavage site with the highest activity but the protease is also active to a greater or lesser extent on a range of substrates. The highest cleavage is of sequences closest to the consensus EXLYΦQ\φ where X is any residue, Φ is any large or medium hydrophobic amino acid and φ is any small hydrophobic amino acid.<br />
<br />
The TEV protease is commonly used as a biochemical tool to cleave affinity tags from purified proteins like [http://parts.igem.org/Part:BBa_K1319007 His-Tags]. The high specifity makes the protease relatively non-toxic ''in vitro'' and ''in vivo''. The molecular weight of the TEV protease is 27 kDa.<br />
<br />
===Usage and Biology===<br />
<br />
The TEV Protease was used and characterizes in the [http://parts.igem.org/Part:BBa_K1319008 K1319008] construct.<br />
<br />
To characterize the TEV protease we used the fusion protein [http://parts.igem.org/Part:BBa_K1319014 K1319014]. This fusion protein contains GFP ([http://parts.igem.org/Part:BBa_E0040 E0040]) bound to a dark quencher ([http://parts.igem.org/Part:BBa_K1319002 REACh2/K1319002]) over a [http://parts.igem.org/Part:BBa_K1319016 linker] which includes the TEV protease cleavage site. If the TEV protease successfully cuts the linker, GFP and its quencher would separate and the FRET (Förster Resonance Energy Transfer) system would be shut down. This would result in an increased GFP fluorescence.<br />
<br />
To demonstrate this behaviour a double plasmid system was designed using the biobrick K1319013 in a pSB3K3 backbone and K1319008 in a pSB1C3 backbone. Also [http://parts.igem.org/Part:BBa_I20260 I20260] was used as a positive control because it produces the same GFP as used in the fusion protein and is regulated by the same promoter, RBS and Terminator on the same plasmid backbone. [http://parts.igem.org/Part:BBa_B0015 B0015] was used as negative control. Induction of the double plasmid constructs occured after 2 h with 50 µl of 100mM IPTG in a 50 ml shake flask culture. <br />
<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|800px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/3/3e/Aachen_Graph2_14.PNG" width="800px"></html><br />
|-<br />
|'''{{{title|Comparison of the fluorescence adjusted for OD of I20260, B0015 and the double plasmid system K1319014 + K1319008}}}'''<br />{{{subtitle|After induction with IPTG after 2 h the double plasmid system produced a fast fluorescence response with an over 10-fold increase compared to the non induced state. I20260 served as positive control and B0015 as negative control.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
<br />
The increase in fluorescence after induction with IPTG is clear sign of funtional expression of the TEV protease. The difference between not induced and induced plasmid is proof that the increase in fluorescence is only attributed to the successful cleavage of the linker. Therefore this is proof of a functional expression of the TEV protease after induction with IPTG.<br />
<br />
Furthermore we validated the used plasmid with a Check PCR with one primer binding upstream on the plasmid backbone and one specifically in the insert.<br />
<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|500px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/0/0c/Aachen_K1319008_Gel_Check_PCR.png" width="500px"></html><br />
|-<br />
|'''{{{title|Check PCR for K1319008}}}'''<br />{{{subtitle|The length of the PCR product matches the length of the control plasmid.}}}<br />
|}<br />
</div><br />
</center><br />
<html></br></html><br />
<br />
This validates that the construct is indeed the TEV protease and thereby the functionality of the TEV protease is established. The construct K1319008 was also sequenced. The sequencing data can be seen in the parts registry [http://parts.igem.org/Part:BBa_K1319008 here].<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319008 K1319008]==<br />
<span class="anchor" id="partsK1319008"></span><br />
<br />
=== IPTG-induced and T7-driven expression of TEV protease ===<br />
<br />
This protein generator produces TEV protease when induced with IPTG in a DE3 strain.<br />
<br />
===Usage and Biology===<br />
More information about the characterization of this part is available on the parts registry page for [http://parts.igem.org/Part:BBa_K1319004 K1319004]. This part was alos used in the validation and characterization of the parts [http://parts.igem.org/Part:BBa_K1319001 K1319001] and [http://parts.igem.org/Part:BBa_K1319001 K1319001].<br />
<br />
This biobrick is used in our [https://2014.igem.org/Team:Aachen/Project/FRET_Reporter REACh construct].<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319009 K1319009]==<br />
<br />
===mRFP-gal3-his fusion protein CDS===<br />
<span class="anchor" id="partsK1319009"></span><br />
<br />
This is a fusion protein created from E1010, K1319003 and K1319007.<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319010 K1319010]==<br />
<span class="anchor" id="partsK1319010"></span><br />
<br />
=== Constitutive expression of K1319000 ===<br />
<br />
This part expresses [http://parts.igem.org/Part:BBa_K1319000 K1319000] behind a [http://parts.igem.org/Part:BBa_J23101 J23101] constitutive promoter.<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319011 K1319011]==<br />
<span class="anchor" id="partsK1319011"></span><br />
<br />
=== Constitutive expression of K1319001 ===<br />
<br />
This part expresses [http://parts.igem.org/Part:BBa_K1319001 K1319001] behind a [http://parts.igem.org/Part:BBa_J23101 J23101] constitutive promoter.<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
== [http://parts.igem.org/Part:BBa_K1319012 K1319012] ==<br />
<span class="anchor" id="partsK1319012"></span><br />
<br />
=== Constitutive expression of K1319002 ===<br />
<br />
This part expresses [http://parts.igem.org/Part:BBa_K1319002 K1319002] behind a [http://parts.igem.org/Part:BBa_J23101 J23101] constitutive promoter.<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
== [http://parts.igem.org/Part:BBa_K1319013 K1319013] ==<br />
<span class="anchor" id="partsK1319013"></span><br />
<br />
=== Constitutive expression of GFP-REACh1 fusion protein ===<br />
<br />
This part expresses a [http://parts.igem.org/Part:BBa_E0040 E0040].[http://parts.igem.org/Part:BBa_K1319001 K1319001] fusion protein (GFP-REACh1) behind a [http://parts.igem.org/Part:BBa_J23101 J23101] promoter. <br />
<br />
===Usage and Biology===<br />
More information about the characterization of this part can be found on the part page of [http://parts.igem.org/Part:BBa_K1319001 K1319001].<br />
<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
== [http://parts.igem.org/Part:BBa_K1319014 K1319014] ==<br />
<span class="anchor" id="partsK1319014"></span><br />
<br />
===Constitutive expression of GFP-REACh2 fusion protein===<br />
<br />
This part expresses a [http://parts.igem.org/Part:BBa_E0040 E0040].[http://parts.igem.org/Part:BBa_K1319002 K1319002] fusion protein (GFP-REACh1) behind a [http://parts.igem.org/Part:BBa_J23101 J23101] promoter. <br />
[http://parts.igem.org/Part:BBa_K1319002 K1319002] is a '''dark quencher''' that eliminates the fluorescence of the GFP-domain by Förster Resonance Energy Transfer (FRET), but does not exhibit strong fluorescence itself.<br />
<br />
===Usage and Biology===<br />
The two domains can be separated from each other via a TEV protease cleavage site in the linker.<br />
<br />
More information about the characterization of this part can be found on the part page of [http://parts.igem.org/Part:BBa_K1319002 K1319002].<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
== [http://parts.igem.org/Part:BBa_K1319015 K1319015] ==<br />
<span class="anchor" id="partsK1319015"></span><br />
<br />
===Constitutive expression of GFP-EYFP fusion protein===<br />
<br />
This part expresses a [http://parts.igem.org/Part:BBa_E0040 E0040].[http://parts.igem.org/Part:BBa_K1319000 K1319000] fusion protein (GFP-EYFP) behind a [http://parts.igem.org/Part:BBa_J23101 J23101] promoter.<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
== [http://parts.igem.org/Part:BBa_K1319016 K1319016] ==<br />
<span class="anchor" id="partsK1319016"></span><br />
<br />
===TEV protease cleavage site===<br />
<br />
This sequence codes for a [http://parts.igem.org/Part:BBa_K1319004 TEV protease] cleavage site.<br />
<br />
ENLYFQ\S is the optimal cleavage site with the highest activity but the protease is also active to a greater or lesser extent on a range of substrates. The highest cleavage is of sequences closest to the consensus EXLYΦQ\φ where X is any residue, Φ is any large or medium hydrophobic amino acid and φ is any small hydrophobic amino acid.<br />
<br />
===Usage and Biology===<br />
<br />
A TEV protease is available codon optimised for ''E. coli'' with the part [http://parts.igem.org/Part:BBa_K1319004 K1319004].<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319017 K1319017]==<br />
<span class="anchor" id="partsK1319017"></span><br />
<br />
=== LasI induced iLOV ===<br />
<br />
This device produces iLOV (K660004) in response to a quorum sensing input.<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319020 K1319020]==<br />
<span class="anchor" id="partsK1319020"></span><br />
<br />
===Translational unit of mRFP-galectin3-His===<br />
<br />
This part is a translational unit of a mRFP-galectin-3-his (B0032.E1010.K1319003.K1319016.B0015)<br />
<br />
===Usage and Biology===<br />
<br />
For more information about the characterization of this part check out [http://parts.igem.org/Part:BBa_K1319003 K1319003].<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==[http://parts.igem.org/Part:BBa_K1319042 K1319042]==<br />
<span class="anchor" id="partsK1319042"></span><br />
<br />
===IPTG inducible iLOV===<br />
<br />
This part can be used for IPTG-induced expression of K660004 (iLOV).<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
===iGEM Team Aachen Biobrick overview===<br />
<br />
<groupparts>iGEM14 Aachen</groupparts><br />
<br />
{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/OD/F_deviceTeam:Aachen/OD/F device2014-10-18T03:29:10Z<p>Nbailly: /* Outlook */</p>
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<div>__NOTOC__<br />
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{{Team:Aachen/Stylesheet}}<br />
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<br />
= OD/F Device =<br />
<br />
Measuring '''Optical Density''' (OD) or absorbance is one of the key and indispensable element in the field of microbiology. One question that has to be answered often is '''how many cells are in a suspension'''? Here, the OD can give a hint. However, the commercially available [http://www.laboratory-equipment.com/laboratory-equipment/cell-density-meter.php OD meters] are expensive and limit its application and usage in low budget institutions.<br />
<br />
Therefore, here we present our OD/F Device. The device is specifically designed for biohackspaces, Do It Yourself (DIY), community laboratories and schools. With our OD/F Device, we aim to enable precise and inexpensive science research at a low cost.<br />
<br />
Further, in Synthetic Biology, the task of measuring OD and fluorescence are often performed at the same time. Hence, here we present a device that can be configured to '''simultaneously measure both fluorescence and OD'''. With such a configuration of the OD/F Device, the production of fluorescence signal can be correlated to cell growth using a single and a portable device.<br />
<br />
<html><br />
<center><br />
<ul class="menusmall-grid"><br />
<!-- Overview --><br />
<br />
<li><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/OD/F_device#odfmeasuringprinciple" style="color:black"><br />
<div class="menusmall-item menusmall-info" ><div class="menukachel" style="top:25%; line-height:1.5em;">Measuring Principle</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/0/0f/Aachen_14-10-10_ODF_Button_ipo.png); norepeat scroll 0% 0% transparent; background-size:100%"><br />
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<br />
<li><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/OD/F_device#odfapplication" style="color:black"><br />
<div class="menusmall-item menusmall-info" ><div class="menukachel" style="top:35%; line-height:1.5em;">Application</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/5/55/Aachen_17-10-14_Glowing_cuvette-ipo.png); norepeat scroll 0% 0% transparent; background-size:100%"><br />
</div><br />
</a><br />
</li><br />
<br />
<li><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/OD/F_device#odfachievements" style="color:black"><br />
<div class="menusmall-item menusmall-info" ><div class="menukachel" style="top:25%; line-height:1.5em;">Achieve-<br/>ments</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/e/ef/Aachen_14-10-15_Medal_Cellocks_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%"><br />
</div><br />
</a><br />
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<br />
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<a class="menulink" href="https://2014.igem.org/Team:Aachen/OD/F_device#odfoutlook" style="color:black"><br />
<div class="menusmall-item menusmall-info" ><div class="menukachel" style="top:35%; line-height:1.5em;">Outlook</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/6/67/Aachen_14-10-16_Outlook_Cellocks_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%"><br />
</div><br />
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</li><br />
<br />
</ul><br />
</center><br />
</html><br />
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<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_14-10-10_ODF_Button_ipo.png|right|150px]]<br />
<br />
== Measuring Principle ==<br />
<span class="anchor" id="odfmeasuringprinciple"></span><br />
<br />
Measuring Principle<br />
The measuring principle for both optical density (OD) and fluorescence measurement is shown below. For OD measurement, the sample is illuminated with an LED and a fixed slit width. A filter blocks any light less than 600 nm. In this way, the sensor mainly senses the 600 nm light which is needed for OD{{sub|600}} measurement.<br />
<br />
For the fluorescence measurement, a similar approach is followed. The filter, again, is used to block the exciting light from being sensed. In this way, only the emitted light from the fluorescence protein is detected and measured.<br />
<br />
Further details about selecting filters, code, a construction manual and evaluation can be found [https://2014.igem.org/Team:Aachen/Notebook/Engineering/ODF here].<br />
<br />
<center><br />
{{Team:Aachen/Figure|Aachen_odf_schemes.png|title=Measuring principle for OD/F Device|subtitle=The left image shows the measurement approach for the optical density. The light shines through the sample with a fixed width. The right image shows the fluorescence measurement approach, exciting the fluorescence proteins from below and measuring from the side.|width=500px}}<br />
</center><br />
<br />
<center><br />
{{Team:Aachen/Figure|Aachen_ODF_7.JPG|title=The combined OD/F Device for optical density and fluorescence measurement.|subtitle= |width=650px}}<br />
</center><br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen 17-10-14 Glowing cuvette-ipo.png|right|150px]]<br />
<br />
== ''Modus operandi'' of the OD/F Device==<br />
<span class="anchor" id="odfapplication"></span><br />
<br />
The device is constructed to make it easy-to-handle for the end users. The standard operating procedure to operate and measure optical density or fluorescence is schematically shown in the figure below.<br />
<br />
{{Team:Aachen/Figure|Aachen 14-10-09 Flowsheet OD-device ipo.png|title=|subtitle=|width=1000px}}<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_14-10-15_Medal_Cellocks_iNB.png|right|150px]]<br />
<br />
== Achievements ==<br />
<span class="anchor" id="odfachievements"></span><br />
<br />
When building the OD/F Device, '''our goal''' was to develop a system that<br />
<br />
* easy-to-handle<br />
* precise, stable, and reproducible results<br />
* portable<br />
* easy to build from Open Source parts<br />
* combined measurement of optical density and fluorescence<br />
* low cost<br />
<br />
Commercially available equipment uses lasers and a set of two fine filters, one between laser and sample and one between sample and sensor. To reduce the cost, our OD/F Device uses a simpler measuring principle: it is designed with one low-cost filter, between sample and sensor, and illuminates with an LED instead of a laser. Nevertheless, one main goal was to produce an inexpensive device. Given that, we therefore had to compromise some of the measurement quality, were we still able to produce stable, precise and good data?<br />
<br />
{{Team:Aachen/Figure|Aachen_ODallstrains1.png|title=Transmission of different cell types at OD-values from 0.001-1|subtitle=The transmittance data of NIH&nbsp;3T3 (mouse fibroblasts) cells align with the transmittance of ''P.&nbsp;putida'' and ''S.&nbsp;cerevisiae'' strains, even though the measured optical densities are lower by 1-2 orders of magnitude.|width=800px}}<br />
<br />
The answer is: Yes! With the optimal design of our cuvette holder we achieved good-quality results albeit using the cheap filter. The transmission to true OD conversion is stable for all cell types as expected.<br />
<br />
Have we been re-inventing the wheel? No!<br />
In fact, you can find some DIY posts for turbidity meters such as [http://www.thingiverse.com/thing:74415 turbidity sensors]. However, a proper assessment of their linearity as well as a calculated OD-value are missing. <br />
<br />
Regarding fluorescence, we are also not re-inventing the wheel. The [https://2010.igem.org/Team:Cambridge 2010 iGEM Cambridge] team actually built a very similar device, the [https://2010.igem.org/Team:Cambridge/Tools/Eglometer E.glometer]. However, there's no data available showing an actual comparison of the data from their device and some proven commercial system to, for example, assess linearity of the measurement.<br />
<br />
We made a commercial assessment of the OD/F Device that results in a total cost of 60 $. The unit is built from acrylic glass for the casing. The compact design results in a weight which is less than 200g. The device can be easily connected to any power adapter via USB. The technical details and a construction manual of OD/F Device is [https://2014.igem.org/Team:Aachen/Notebook/Engineering/ODF#diy published] on our engineering page.<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_14-10-16_Outlook_Cellocks_iNB.png|right|150px]]<br />
<br />
== Outlook ==<br />
<span class="anchor" id="odfoutlook"></span><br />
<br />
We have proven that our device is capable of delivering good results, even in hard conditions as low cell concentrations.<br />
Yet there is room for improvement.<br />
The calibration process is quite intensive work. An application to do this automatically would help for this process.<br />
For the ease of use and to prevent data loss from noting down measured values manually, a smartphone application that can directly correlate OD and fluorescence values would be a great addition. This addition will be implemented in the next version.<br />
<br />
<br />
{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/OD/F_deviceTeam:Aachen/OD/F device2014-10-18T03:28:56Z<p>Nbailly: /* Measuring Principle */</p>
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= OD/F Device =<br />
<br />
Measuring '''Optical Density''' (OD) or absorbance is one of the key and indispensable element in the field of microbiology. One question that has to be answered often is '''how many cells are in a suspension'''? Here, the OD can give a hint. However, the commercially available [http://www.laboratory-equipment.com/laboratory-equipment/cell-density-meter.php OD meters] are expensive and limit its application and usage in low budget institutions.<br />
<br />
Therefore, here we present our OD/F Device. The device is specifically designed for biohackspaces, Do It Yourself (DIY), community laboratories and schools. With our OD/F Device, we aim to enable precise and inexpensive science research at a low cost.<br />
<br />
Further, in Synthetic Biology, the task of measuring OD and fluorescence are often performed at the same time. Hence, here we present a device that can be configured to '''simultaneously measure both fluorescence and OD'''. With such a configuration of the OD/F Device, the production of fluorescence signal can be correlated to cell growth using a single and a portable device.<br />
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<a class="menulink" href="https://2014.igem.org/Team:Aachen/OD/F_device#odfmeasuringprinciple" style="color:black"><br />
<div class="menusmall-item menusmall-info" ><div class="menukachel" style="top:25%; line-height:1.5em;">Measuring Principle</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/0/0f/Aachen_14-10-10_ODF_Button_ipo.png); norepeat scroll 0% 0% transparent; background-size:100%"><br />
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<a class="menulink" href="https://2014.igem.org/Team:Aachen/OD/F_device#odfapplication" style="color:black"><br />
<div class="menusmall-item menusmall-info" ><div class="menukachel" style="top:35%; line-height:1.5em;">Application</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/5/55/Aachen_17-10-14_Glowing_cuvette-ipo.png); norepeat scroll 0% 0% transparent; background-size:100%"><br />
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<a class="menulink" href="https://2014.igem.org/Team:Aachen/OD/F_device#odfachievements" style="color:black"><br />
<div class="menusmall-item menusmall-info" ><div class="menukachel" style="top:25%; line-height:1.5em;">Achieve-<br/>ments</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/e/ef/Aachen_14-10-15_Medal_Cellocks_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%"><br />
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<a class="menulink" href="https://2014.igem.org/Team:Aachen/OD/F_device#odfoutlook" style="color:black"><br />
<div class="menusmall-item menusmall-info" ><div class="menukachel" style="top:35%; line-height:1.5em;">Outlook</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/6/67/Aachen_14-10-16_Outlook_Cellocks_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%"><br />
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[[File:Aachen_14-10-10_ODF_Button_ipo.png|right|150px]]<br />
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== Measuring Principle ==<br />
<span class="anchor" id="odfmeasuringprinciple"></span><br />
<br />
Measuring Principle<br />
The measuring principle for both optical density (OD) and fluorescence measurement is shown below. For OD measurement, the sample is illuminated with an LED and a fixed slit width. A filter blocks any light less than 600 nm. In this way, the sensor mainly senses the 600 nm light which is needed for OD{{sub|600}} measurement.<br />
<br />
For the fluorescence measurement, a similar approach is followed. The filter, again, is used to block the exciting light from being sensed. In this way, only the emitted light from the fluorescence protein is detected and measured.<br />
<br />
Further details about selecting filters, code, a construction manual and evaluation can be found [https://2014.igem.org/Team:Aachen/Notebook/Engineering/ODF here].<br />
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<center><br />
{{Team:Aachen/Figure|Aachen_odf_schemes.png|title=Measuring principle for OD/F Device|subtitle=The left image shows the measurement approach for the optical density. The light shines through the sample with a fixed width. The right image shows the fluorescence measurement approach, exciting the fluorescence proteins from below and measuring from the side.|width=500px}}<br />
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{{Team:Aachen/Figure|Aachen_ODF_7.JPG|title=The combined OD/F Device for optical density and fluorescence measurement.|subtitle= |width=650px}}<br />
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[[File:Aachen 17-10-14 Glowing cuvette-ipo.png|right|150px]]<br />
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== ''Modus operandi'' of the OD/F Device==<br />
<span class="anchor" id="odfapplication"></span><br />
<br />
The device is constructed to make it easy-to-handle for the end users. The standard operating procedure to operate and measure optical density or fluorescence is schematically shown in the figure below.<br />
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{{Team:Aachen/Figure|Aachen 14-10-09 Flowsheet OD-device ipo.png|title=|subtitle=|width=1000px}}<br />
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[[File:Aachen_14-10-15_Medal_Cellocks_iNB.png|right|150px]]<br />
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== Achievements ==<br />
<span class="anchor" id="odfachievements"></span><br />
<br />
When building the OD/F Device, '''our goal''' was to develop a system that<br />
<br />
* easy-to-handle<br />
* precise, stable, and reproducible results<br />
* portable<br />
* easy to build from Open Source parts<br />
* combined measurement of optical density and fluorescence<br />
* low cost<br />
<br />
Commercially available equipment uses lasers and a set of two fine filters, one between laser and sample and one between sample and sensor. To reduce the cost, our OD/F Device uses a simpler measuring principle: it is designed with one low-cost filter, between sample and sensor, and illuminates with an LED instead of a laser. Nevertheless, one main goal was to produce an inexpensive device. Given that, we therefore had to compromise some of the measurement quality, were we still able to produce stable, precise and good data?<br />
<br />
{{Team:Aachen/Figure|Aachen_ODallstrains1.png|title=Transmission of different cell types at OD-values from 0.001-1|subtitle=The transmittance data of NIH&nbsp;3T3 (mouse fibroblasts) cells align with the transmittance of ''P.&nbsp;putida'' and ''S.&nbsp;cerevisiae'' strains, even though the measured optical densities are lower by 1-2 orders of magnitude.|width=800px}}<br />
<br />
The answer is: Yes! With the optimal design of our cuvette holder we achieved good-quality results albeit using the cheap filter. The transmission to true OD conversion is stable for all cell types as expected.<br />
<br />
Have we been re-inventing the wheel? No!<br />
In fact, you can find some DIY posts for turbidity meters such as [http://www.thingiverse.com/thing:74415 turbidity sensors]. However, a proper assessment of their linearity as well as a calculated OD-value are missing. <br />
<br />
Regarding fluorescence, we are also not re-inventing the wheel. The [https://2010.igem.org/Team:Cambridge 2010 iGEM Cambridge] team actually built a very similar device, the [https://2010.igem.org/Team:Cambridge/Tools/Eglometer E.glometer]. However, there's no data available showing an actual comparison of the data from their device and some proven commercial system to, for example, assess linearity of the measurement.<br />
<br />
We made a commercial assessment of the OD/F Device that results in a total cost of 60 $. The unit is built from acrylic glass for the casing. The compact design results in a weight which is less than 200g. The device can be easily connected to any power adapter via USB. The technical details and a construction manual of OD/F Device is [https://2014.igem.org/Team:Aachen/Notebook/Engineering/ODF#diy published] on our engineering page.<br />
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[[File:Aachen_14-10-16_Outlook_Cellocks_iNB.png|right|150px]]<br />
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== Outlook ==<br />
<span class="anchor" id="odfoutlook"></span><br />
<br />
We have proven that our device is capable of delivering good results, even in hard conditions as low cell concentrations.<br />
Yet there is room for improvement.<br />
The calibration process is quite intensive work. An application to do this automatically would help for this process.<br />
For the ease of use and to prevent data loss from noting down measured values manually, a smartphone application that can directly correlate OD and fluorescence values would be a great addition. This addition will be implemented in the next version.<br />
<br />
{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/Project/Gal3Team:Aachen/Project/Gal32014-10-18T03:28:26Z<p>Nbailly: </p>
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{{CSS/Main}}<br />
{{Team:Aachen/Stylesheet}}<br />
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= Galectin-3 =<br />
<br />
We are committed to constantly improve our detection methods. While the current method uses the quorum sensing system, it is thus limited to bacteria that secrete autoinducers. Hence, we present an alternative approach for the detection of pathogens. In our alternative detection system, biomolecules are tagged with a fluorescent reporter that bind to the surface of the cell and reveal its presence.<br />
<br />
<html><br />
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<ul class="team-grid" style="width:inherit;"><br />
<!-- Overview --><br />
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<li><a href="https://2014.igem.org/Team:Aachen/Project/Gal3#naturalfunctions" style="color:black"><br />
<div class="team-item team-info" ><br />
<div class="menukachel" style="line-height:1.5em;top:40%;">Natural Functions</div><br />
<!-- <br/><br/><br />
<b>Principle of Operation</br><br />
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click for more information --><br />
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<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/7/76/Aachen_14-10-13_Galectin-3_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%"> </div></a><br />
</li><br />
<br />
<li><a href="https://2014.igem.org/Team:Aachen/Project/Gal3#alternativesensing" style="color:black"><br />
<div class="team-item team-info" ><br />
<div class="menukachel" style="line-height:1.5em;top:32%;">An Alternative Sensing Molecule</div><br />
<!-- <br/><br/><br />
<b>Principle of Operation</br><br />
<br/><br/><br />
click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/7/74/Aachen_14-10-13_Galectin-3-YFP_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%"> </div></a><br />
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<li><a href="https://2014.igem.org/Team:Aachen/Project/Gal3#gal3achievements" style="color:black"><br />
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<div class="menukachel" style="line-height:1.5em;top:40%;">Achievements</div><br />
<!-- <br/><br/><br />
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[[File:Aachen_14-10-13_Galectin-3_iNB.png|150px|right]]<br />
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= Natural Functions of Galectin-3 =<br />
<span class="anchor" id="naturalfunctions"></span><br />
<br />
Galectins are proteins of the lectin family, which possess carbohydrate recognition domains '''binding specifically to β-galactoside sugar residues'''. In humans, 10 different galantines have been identified, among which is galectin-3. <br />
Galectin-3 has a size of about 31&nbsp;kDA and is encoded by a single gene, LGALS3. It is found to have many physiological functions, such as '''cell adhesion, cell growth and differentiation''', and contributes to the development of '''cancer, inflammation, fibrosis''' and others.<br />
<br />
Human galectin-3 is a protein of the lectin-family that was shown to bind the LPS of multiple human pathogens. Some of them, including ''P.&nbsp;aeruginosa'' protect themselves against the human immune system by mimicking the lipopolysaccharides (LPS) present on human erythrocytes.<br />
By making fusion proteins of galectin-3 with fluorescent reporter proteins, pathogens can be labelled and '''made visible by fluorescence-microscopy'''.<br />
<br />
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{{Team:Aachen/BlockSeparator}}<br />
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[[File:Aachen_14-10-13_Galectin-3-YFP_iNB.png|150px|right]]<br />
<br />
== An Alternative Sensing Molecule ==<br />
<span class="anchor" id="alternativesensing"></span><br />
<br />
{{Team:Aachen/FigureFloat|Aachen_14-10-09_Pseudomonas_LPS_iNB.png|title=Cell wall composition of ''P.&nbsp;aeruginosa''|subtitle=Gram-negative bacteria have two cell membranes. The LPS are embedded in the outer membrane and are composed of a lipid and an O polysaccharide.|width=420px}}<br />
<br />
Characteristic parts of the '''lipopolysaccharide structure (LPS)''' of ''P.&nbsp;aeruginosa'' can be bound by galectin-3. Specifically, the O polysaccharide (see figure on the left) of the LPS is recognized by galectin-3. Therefore, this specific binding of galectin-3 enables the construction of a fluorescent based detection system. A fusion protein of galectin-3 and a reporter protein, such as a fluorescent protein, can be built and applied in the detection of ''P.&nbsp;aeruginosa''.<br />
<br />
In our approach, a '''galectin-3-YFP fusion protein''' is built and expressed in ''E.&nbsp;coli'', including a his-tag and a snap-tag for purification. The fusion protein can then be incorporated into a '''cell-free biosensor system'''. These biosensors have many advantages over systems that use living cells such as an uncomplicated storage. Furthermore, from a [https://2014.igem.org/Team:Aachen/Safety biosafety] and social acceptance point of view, it is advantageous if the sensor system does not contain alive genetically modified organisms.<br />
<br />
{{Team:Aachen/FigureFloatRight|align=center|Aachen_14-10-09_Cell_Free_Biosensor_iNB.png|width=500px}}<br />
<br />
<br />
<br />
<br />
To detect ''P.&nbsp;aeruginosa'' cells, an agar chip could be used to sample a solid surface. However, other materials but agar can be considered to collect pathogens. The cells stick to the sampling chip which is then immersed in a detection buffer containing the galectin-3-YFP fusion protein. Excess protein is removed during washing in a suitable buffer. The galectin-3 remains bound to the pathogen and '''illumination with 514&nbsp;nm''', the excitation wavelength of YFP, in a modified version of our measurement device reveals the location of the cells. The picture taken by the measurement device can then be analyzed by our software ''Measurarty''.<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
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[[File:Aachen_14-10-15_Medal_Cellocks_iNB.png|right|150px]]<br />
<br />
= Achievements =<br />
<span class="anchor" id="gal3achievements"></span><br />
<br />
Due to the generous support of Sophia Böcker and Prof.&nbsp;Dr.&nbsp;Elling of the Helmholtz Institute for Biomedical Engineering in Aachen, we got access to a pET17-derived expression plasmid for a His- and SNAP-tagged YFP-galectin-3 fusion protein. We transformed the fusion protein into ''E.&nbsp;coli''&nbsp;Rosetta cells and conducted a batch fermentation to obtain large amounts of protein.<br />
<br />
With the help of David Schönauer and Alan Mertens from the RWTH Aachen Institute of Biotechnolgy we then purified the fusion protein using FPLC.<br />
Subsequently, we aimed to test the binding of the Gal-3 fusion protein to the LPS of ''P.&nbsp;aeruginosa'' as shown previously (Kupper, Böcker, Liu et al., 2013). Apparently, because of insufficient sensitivity of the used fluorescence microscope, this could not be confirmed and would require further experiments, idealy using other detection methods.<br />
<br />
After we received the collection of [https://2014.igem.org/Team:Aachen/Collaborations/Heidelberg pSBX-expression vectors] from Team Heidelberg, we used Gibson assembly to make K1319020 from K1319003 and pSBX1A3, which is the translational unit for an mRFP-Gal3 fusion protein with a C-terminal 6xHis tag:<br />
<center>{{Team:Aachen/Figure|Aachen_K1319020.png|width=800px|title=K1319009|subtitle=This BioBrick is a construction intermediate of K1319003 (gal3), E1010 (mRFP), K1319007 (6xHis tag) to K1319020 (translational unit of the fusion protein).}}</center><br />
<br />
In addition, we cloned our BioBrick K1319003 into the pET17 expression vector and expressed all combinations of fusion proteins in E.&nbsp;coli&nbsp;BL21(DE3). An SDS-PAGE showed that all fusion proteins were fully translated:<br />
<br />
{{Team:Aachen/FigureDual<br />
|Aachen_14-10-04_Expression_Pellets_iMO.png|Aachen_Gal3_Expression.png|title1=Pellets of different fusion protein expressions|title2=SDS-PAGE of K1319020 expression|subtitle1=Expression in the pET17 vector was much more leaky than the expression in the pSBX vectors.|subtitle2=The fusion protein was fully translated to the correct molecular mass of 74&nbsp;kDa.|width=425px}} <br />
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== References ==<br />
Kupper, C. E., Böcker, S., Elling, L., Lui, H., Adamzyk, C., de Kamp, J. v., et al. (2013). Fluorescent SNAP-tag galectin fusion proteins as novel tools in glycobiology. Current Pharmaceutical Design, 19(30), 5457-67. Available at: http://www.ncbi.nlm.nih.gov/pubmed/23431989. <br />
<br />
<br />
{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/Project/Gal3Team:Aachen/Project/Gal32014-10-18T03:27:10Z<p>Nbailly: /* Achievements */</p>
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<div>__NOTOC__<br />
{{CSS/Main}}<br />
{{Team:Aachen/Stylesheet}}<br />
{{Team:Aachen/Header}}<br />
<br />
= Galectin-3 =<br />
<br />
We are committed to constantly improve our detection methods. While the current method uses the quorum sensing system, it is thus limited to bacteria that secrete autoinducers. Hence, we present an alternative approach for the detection of pathogens. In our alternative detection system, biomolecules are tagged with a fluorescent reporter that bind to the surface of the cell and reveal its presence.<br />
<br />
<html><br />
<center><br />
<ul class="team-grid" style="width:inherit;"><br />
<!-- Overview --><br />
<br />
<li><a href="https://2014.igem.org/Team:Aachen/Project/Gal3#naturalfunctions" style="color:black"><br />
<div class="team-item team-info" ><br />
<div class="menukachel" style="line-height:1.5em;top:40%;">Natural Functions</div><br />
<!-- <br/><br/><br />
<b>Principle of Operation</br><br />
<br/><br/><br />
click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/7/76/Aachen_14-10-13_Galectin-3_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%"> </div></a><br />
</li><br />
<br />
<li><a href="https://2014.igem.org/Team:Aachen/Project/Gal3#alternativesensing" style="color:black"><br />
<div class="team-item team-info" ><br />
<div class="menukachel" style="line-height:1.5em;top:32%;">An Alternative Sensing Molecule</div><br />
<!-- <br/><br/><br />
<b>Principle of Operation</br><br />
<br/><br/><br />
click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/7/74/Aachen_14-10-13_Galectin-3-YFP_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%"> </div></a><br />
</li><br />
<br />
<li><a href="https://2014.igem.org/Team:Aachen/Project/Gal3#gal3achievements" style="color:black"><br />
<div class="team-item team-info" ><br />
<div class="menukachel" style="line-height:1.5em;top:40%;">Achievements</div><br />
<!-- <br/><br/><br />
<b>Principle of Operation</br><br />
<br/><br/><br />
click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/e/ef/Aachen_14-10-15_Medal_Cellocks_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%"> </div></a><br />
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</ul><br />
</center><br />
</html><br />
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<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_14-10-13_Galectin-3_iNB.png|150px|right]]<br />
<br />
= Natural Functions of Galectin-3 =<br />
<span class="anchor" id="naturalfunctions"></span><br />
<br />
Galectins are proteins of the lectin family, which possess carbohydrate recognition domains '''binding specifically to β-galactoside sugar residues'''. In humans, 10 different galantines have been identified, among which is galectin-3. <br />
Galectin-3 has a size of about 31&nbsp;kDA and is encoded by a single gene, LGALS3. It is found to have many physiological functions, such as '''cell adhesion, cell growth and differentiation''', and contributes to the development of '''cancer, inflammation, fibrosis''' and others.<br />
<br />
Human galectin-3 is a protein of the lectin-family that was shown to bind the LPS of multiple human pathogens. Some of them, including ''P.&nbsp;aeruginosa'' protect themselves against the human immune system by mimicking the lipopolysaccharides (LPS) present on human erythrocytes.<br />
By making fusion proteins of galectin-3 with fluorescent reporter proteins, pathogens can be labelled and '''made visible by fluorescence-microscopy'''.<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_14-10-13_Galectin-3-YFP_iNB.png|150px|right]]<br />
<br />
== An Alternative Sensing Molecule ==<br />
<span class="anchor" id="alternativesensing"></span><br />
<br />
{{Team:Aachen/FigureFloat|Aachen_14-10-09_Pseudomonas_LPS_iNB.png|title=Cell wall composition of ''P. aeruginosa''|subtitle=Gram-negative bacteria have two cell membranes. The LPS are embedded in the outer membrane and are composed of a lipid and an O polysaccharide.|width=420px}}<br />
<br />
Characteristic parts of the '''lipopolysaccharide structure (LPS)''' of ''P. aeruginosa'' can be bound by galectin-3. Specifically, the O polysaccharide (see figure on the left) of the LPS is recognized by galectin-3. Therefore, this specific binding of galectin-3 enables the construction of a fluorescent based detection system. A fusion protein of galectin-3 and a reporter protein, such as a fluorescent protein, can be built and applied in the detection of ''P.&nbsp;aeruginosa''.<br />
<br />
In our approach, a '''galectin-3-YFP fusion protein''' is built and expressed in ''E.&nbsp;coli'', including a his-tag and a snap-tag for purification. The fusion protein can then be incorporated into a '''cell-free biosensor system'''. These biosensors have many advantages over systems that use living cells such as an uncomplicated storage. Furthermore, from a [https://2014.igem.org/Team:Aachen/Safety biosafety] and social acceptance point of view, it is advantageous if the sensor system does not contain alive genetically modified organisms.<br />
<br />
{{Team:Aachen/FigureFloatRight|align=center|Aachen_14-10-09_Cell_Free_Biosensor_iNB.png|width=500px}}<br />
<br />
<br />
<br />
<br />
To detect ''P.&nbsp;aeruginosa'' cells, an agar chip could be used to sample a solid surface. However, other materials but agar can be considered to collect pathogens. The cells stick to the sampling chip which is then immersed in a detection buffer containing the galectin-3-YFP fusion protein. Excess protein is removed during washing in a suitable buffer. The galectin-3 remains bound to the pathogen and '''illumination with 514&nbsp;nm''', the excitation wavelength of YFP, in a modified version of our measurement device reveals the location of the cells. The picture taken by the measurement device can then be analyzed by our software ''Measurarty''.<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_14-10-15_Medal_Cellocks_iNB.png|right|150px]]<br />
<br />
= Achievements =<br />
<span class="anchor" id="gal3achievements"></span><br />
<br />
Due to the generous support of Sophia Böcker and Prof.&nbsp;Dr.&nbsp;Elling of the Helmholtz Institute for Biomedical Engineering in Aachen, we got access to a pET17-derived expression plasmid for a His- and SNAP-tagged YFP-galectin-3 fusion protein. We transformed the fusion protein into ''E.&nbsp;coli''&nbsp;Rosetta cells and conducted a batch fermentation to obtain large amounts of protein.<br />
<br />
With the help of David Schönauer and Alan Mertens from the RWTH Aachen Institute of Biotechnolgy we then purified the fusion protein using FPLC.<br />
Subsequently, we aimed to test the binding of the Gal-3 fusion protein to the LPS of ''P. aeruginosa'' as shown previously (Kupper, Böcker, Liu et al., 2013). Apparently, because of insufficient sensitivity of the used fluorescence microscope, this could not be confirmed and would require further experiments, idealy using other detection methods.<br />
<br />
After we received the collection of [https://2014.igem.org/Team:Aachen/Collaborations/Heidelberg pSBX-expression vectors] from Team Heidelberg, we used Gibson assembly to make K1319020 from K1319003 and pSBX1A3, which is the translational unit for an mRFP-Gal3 fusion protein with a C-terminal 6xHis tag:<br />
<center>{{Team:Aachen/Figure|Aachen_K1319020.png|width=800px|title=K1319009|subtitle=This BioBrick is a construction intermediate of K1319003 (gal3), E1010 (mRFP), K1319007 (6xHis tag) to K1319020 (translational unit of the fusion protein).}}</center><br />
<br />
In addition, we cloned our BioBrick K1319003 into the pET17 expression vector and expressed all combinations of fusion proteins in E.&nbsp;coli&nbsp;BL21(DE3). An SDS-PAGE showed that all fusion proteins were fully translated:<br />
<br />
{{Team:Aachen/FigureDual<br />
|Aachen_14-10-04_Expression_Pellets_iMO.png|Aachen_Gal3_Expression.png|title1=Pellets of different fusion protein expressions|title2=SDS-PAGE of K1319020 expression|subtitle1=Expression in the pET17 vector was much more leaky than the expression in the pSBX vectors.|subtitle2=The fusion protein was fully translated to the correct molecular mass of 74&nbsp;kDa.|width=425px}} <br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
== References ==<br />
Kupper, C. E., Böcker, S., Elling, L., Lui, H., Adamzyk, C., de Kamp, J. v., et al. (2013). Fluorescent SNAP-tag galectin fusion proteins as novel tools in glycobiology. Current Pharmaceutical Design, 19(30), 5457-67. Available at: http://www.ncbi.nlm.nih.gov/pubmed/23431989. <br />
<br />
<br />
{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/Project/Gal3Team:Aachen/Project/Gal32014-10-18T03:26:22Z<p>Nbailly: </p>
<hr />
<div>__NOTOC__<br />
{{CSS/Main}}<br />
{{Team:Aachen/Stylesheet}}<br />
{{Team:Aachen/Header}}<br />
<br />
= Galectin-3 =<br />
<br />
We are committed to constantly improve our detection methods. While the current method uses the quorum sensing system, it is thus limited to bacteria that secrete autoinducers. Hence, we present an alternative approach for the detection of pathogens. In our alternative detection system, biomolecules are tagged with a fluorescent reporter that bind to the surface of the cell and reveal its presence.<br />
<br />
<html><br />
<center><br />
<ul class="team-grid" style="width:inherit;"><br />
<!-- Overview --><br />
<br />
<li><a href="https://2014.igem.org/Team:Aachen/Project/Gal3#naturalfunctions" style="color:black"><br />
<div class="team-item team-info" ><br />
<div class="menukachel" style="line-height:1.5em;top:40%;">Natural Functions</div><br />
<!-- <br/><br/><br />
<b>Principle of Operation</br><br />
<br/><br/><br />
click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/7/76/Aachen_14-10-13_Galectin-3_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%"> </div></a><br />
</li><br />
<br />
<li><a href="https://2014.igem.org/Team:Aachen/Project/Gal3#alternativesensing" style="color:black"><br />
<div class="team-item team-info" ><br />
<div class="menukachel" style="line-height:1.5em;top:32%;">An Alternative Sensing Molecule</div><br />
<!-- <br/><br/><br />
<b>Principle of Operation</br><br />
<br/><br/><br />
click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/7/74/Aachen_14-10-13_Galectin-3-YFP_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%"> </div></a><br />
</li><br />
<br />
<li><a href="https://2014.igem.org/Team:Aachen/Project/Gal3#gal3achievements" style="color:black"><br />
<div class="team-item team-info" ><br />
<div class="menukachel" style="line-height:1.5em;top:40%;">Achievements</div><br />
<!-- <br/><br/><br />
<b>Principle of Operation</br><br />
<br/><br/><br />
click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/e/ef/Aachen_14-10-15_Medal_Cellocks_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%"> </div></a><br />
</li><br />
</ul><br />
</center><br />
</html><br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_14-10-13_Galectin-3_iNB.png|150px|right]]<br />
<br />
= Natural Functions of Galectin-3 =<br />
<span class="anchor" id="naturalfunctions"></span><br />
<br />
Galectins are proteins of the lectin family, which possess carbohydrate recognition domains '''binding specifically to β-galactoside sugar residues'''. In humans, 10 different galantines have been identified, among which is galectin-3. <br />
Galectin-3 has a size of about 31&nbsp;kDA and is encoded by a single gene, LGALS3. It is found to have many physiological functions, such as '''cell adhesion, cell growth and differentiation''', and contributes to the development of '''cancer, inflammation, fibrosis''' and others.<br />
<br />
Human galectin-3 is a protein of the lectin-family that was shown to bind the LPS of multiple human pathogens. Some of them, including ''P.&nbsp;aeruginosa'' protect themselves against the human immune system by mimicking the lipopolysaccharides (LPS) present on human erythrocytes.<br />
By making fusion proteins of galectin-3 with fluorescent reporter proteins, pathogens can be labelled and '''made visible by fluorescence-microscopy'''.<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_14-10-13_Galectin-3-YFP_iNB.png|150px|right]]<br />
<br />
== An Alternative Sensing Molecule ==<br />
<span class="anchor" id="alternativesensing"></span><br />
<br />
{{Team:Aachen/FigureFloat|Aachen_14-10-09_Pseudomonas_LPS_iNB.png|title=Cell wall composition of ''P. aeruginosa''|subtitle=Gram-negative bacteria have two cell membranes. The LPS are embedded in the outer membrane and are composed of a lipid and an O polysaccharide.|width=420px}}<br />
<br />
Characteristic parts of the '''lipopolysaccharide structure (LPS)''' of ''P. aeruginosa'' can be bound by galectin-3. Specifically, the O polysaccharide (see figure on the left) of the LPS is recognized by galectin-3. Therefore, this specific binding of galectin-3 enables the construction of a fluorescent based detection system. A fusion protein of galectin-3 and a reporter protein, such as a fluorescent protein, can be built and applied in the detection of ''P.&nbsp;aeruginosa''.<br />
<br />
In our approach, a '''galectin-3-YFP fusion protein''' is built and expressed in ''E.&nbsp;coli'', including a his-tag and a snap-tag for purification. The fusion protein can then be incorporated into a '''cell-free biosensor system'''. These biosensors have many advantages over systems that use living cells such as an uncomplicated storage. Furthermore, from a [https://2014.igem.org/Team:Aachen/Safety biosafety] and social acceptance point of view, it is advantageous if the sensor system does not contain alive genetically modified organisms.<br />
<br />
{{Team:Aachen/FigureFloatRight|align=center|Aachen_14-10-09_Cell_Free_Biosensor_iNB.png|width=500px}}<br />
<br />
<br />
<br />
<br />
To detect ''P.&nbsp;aeruginosa'' cells, an agar chip could be used to sample a solid surface. However, other materials but agar can be considered to collect pathogens. The cells stick to the sampling chip which is then immersed in a detection buffer containing the galectin-3-YFP fusion protein. Excess protein is removed during washing in a suitable buffer. The galectin-3 remains bound to the pathogen and '''illumination with 514&nbsp;nm''', the excitation wavelength of YFP, in a modified version of our measurement device reveals the location of the cells. The picture taken by the measurement device can then be analyzed by our software ''Measurarty''.<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_14-10-15_Medal_Cellocks_iNB.png|right|150px]]<br />
<br />
= Achievements =<br />
<span class="anchor" id="gal3achievements"></span><br />
<br />
Due to the generous support of Sophia Böcker and Prof.&nbsp;Dr.&nbsp;Elling of the Helmholtz Institute for Biomedical Engineering in Aachen, we got access to a pET17-derived expression plasmid for a His- and SNAP-tagged YFP-galectin-3 fusion protein. We transformed the fusion protein into ''E.&nbsp;coli''&nbsp;Rosetta cells and conducted a batch fermentation to obtain large amounts of protein.<br />
<br />
With the help of David Schönauer and Alan Mertens from the RWTH Aachen Institute of Biotechnolgy we then purified the fusion protein using FPLC.<br />
Subsequently, we aimed to test the binding of the Gal-3 fusion protein to the LPS of ''P. aeruginosa'' as shown previously [1]. Apparently, because of insufficient sensitivity of the used fluorescence microscope, this could not be confirmed and would require further experiments, idealy using other detection methods.<br />
<br />
After we received the collection of [https://2014.igem.org/Team:Aachen/Collaborations/Heidelberg pSBX-expression vectors] from Team Heidelberg, we used Gibson assembly to make K1319020 from K1319003 and pSBX1A3, which is the translational unit for an mRFP-Gal3 fusion protein with a C-terminal 6xHis tag:<br />
<center>{{Team:Aachen/Figure|Aachen_K1319020.png|width=800px|title=K1319009|subtitle=This BioBrick is a construction intermediate of K1319003 (gal3), E1010 (mRFP), K1319007 (6xHis tag) to K1319020 (translational unit of the fusion protein).}}</center><br />
<br />
In addition, we cloned our BioBrick K1319003 into the pET17 expression vector and expressed all combinations of fusion proteins in E.&nbsp;coli&nbsp;BL21(DE3). An SDS-PAGE showed that all fusion proteins were fully translated:<br />
<br />
{{Team:Aachen/FigureDual<br />
|Aachen_14-10-04_Expression_Pellets_iMO.png|Aachen_Gal3_Expression.png|title1=Pellets of different fusion protein expressions|title2=SDS-PAGE of K1319020 expression|subtitle1=Expression in the pET17 vector was much more leaky than the expression in the pSBX vectors.|subtitle2=The fusion protein was fully translated to the correct molecular mass of 74&nbsp;kDa.|width=425px}} <br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
== References ==<br />
Kupper, C. E., Böcker, S., Elling, L., Lui, H., Adamzyk, C., de Kamp, J. v., et al. (2013). Fluorescent SNAP-tag galectin fusion proteins as novel tools in glycobiology. Current Pharmaceutical Design, 19(30), 5457-67. Available at: http://www.ncbi.nlm.nih.gov/pubmed/23431989. <br />
<br />
<br />
{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/Project/Gal3Team:Aachen/Project/Gal32014-10-18T03:22:26Z<p>Nbailly: /* Natural Functions of Galectin-3 */</p>
<hr />
<div>__NOTOC__<br />
{{CSS/Main}}<br />
{{Team:Aachen/Stylesheet}}<br />
{{Team:Aachen/Header}}<br />
<br />
= Galectin-3 =<br />
<br />
We are committed to constantly improve our detection methods. While the current method uses the quorum sensing system, it is thus limited to bacteria that secrete autoinducers. Hence, we present an alternative approach for the detection of pathogens. In our alternative detection system, biomolecules are tagged with a fluorescent reporter that bind to the surface of the cell and reveal its presence.<br />
<br />
<html><br />
<center><br />
<ul class="team-grid" style="width:inherit;"><br />
<!-- Overview --><br />
<br />
<li><a href="https://2014.igem.org/Team:Aachen/Project/Gal3#naturalfunctions" style="color:black"><br />
<div class="team-item team-info" ><br />
<div class="menukachel" style="line-height:1.5em;top:40%;">Natural Functions</div><br />
<!-- <br/><br/><br />
<b>Principle of Operation</br><br />
<br/><br/><br />
click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/7/76/Aachen_14-10-13_Galectin-3_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%"> </div></a><br />
</li><br />
<br />
<li><a href="https://2014.igem.org/Team:Aachen/Project/Gal3#alternativesensing" style="color:black"><br />
<div class="team-item team-info" ><br />
<div class="menukachel" style="line-height:1.5em;top:32%;">An Alternative Sensing Molecule</div><br />
<!-- <br/><br/><br />
<b>Principle of Operation</br><br />
<br/><br/><br />
click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/7/74/Aachen_14-10-13_Galectin-3-YFP_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%"> </div></a><br />
</li><br />
<br />
<li><a href="https://2014.igem.org/Team:Aachen/Project/Gal3#gal3achievements" style="color:black"><br />
<div class="team-item team-info" ><br />
<div class="menukachel" style="line-height:1.5em;top:40%;">Achievements</div><br />
<!-- <br/><br/><br />
<b>Principle of Operation</br><br />
<br/><br/><br />
click for more information --><br />
</div><br />
<div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/e/ef/Aachen_14-10-15_Medal_Cellocks_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%"> </div></a><br />
</li><br />
</ul><br />
</center><br />
</html><br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_14-10-13_Galectin-3_iNB.png|150px|right]]<br />
<br />
= Natural Functions of Galectin-3 =<br />
<span class="anchor" id="naturalfunctions"></span><br />
<br />
Galectins are proteins of the lectin family, which possess carbohydrate recognition domains '''binding specifically to β-galactoside sugar residues'''. In humans, 10 different galantines have been identified, among which is galectin-3. <br />
Galectin-3 has a size of about 31&nbsp;kDA and is encoded by a single gene, LGALS3. It is found to have many physiological functions, such as '''cell adhesion, cell growth and differentiation''', and contributes to the development of '''cancer, inflammation, fibrosis''' and others.<br />
<br />
Human galectin-3 is a protein of the lectin-family that was shown to bind the LPS of multiple human pathogens. Some of them, including ''P.&nbsp;aeruginosa'' protect themselves against the human immune system by mimicking the lipopolysaccharides (LPS) present on human erythrocytes.<br />
By making fusion proteins of galectin-3 with fluorescent reporter proteins, pathogens can be labelled and '''made visible by fluorescence-microscopy'''.<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_14-10-13_Galectin-3-YFP_iNB.png|150px|right]]<br />
<br />
== An Alternative Sensing Molecule ==<br />
<span class="anchor" id="alternativesensing"></span><br />
<br />
{{Team:Aachen/FigureFloat|Aachen_14-10-09_Pseudomonas_LPS_iNB.png|title=Cell wall composition of ''P. aeruginosa''|subtitle=Gram-negative bacteria have two cell membranes. The LPS are embedded in the outer membrane and are composed of a lipid and an O polysaccharide.|width=420px}}<br />
<br />
Characteristic parts of the '''lipopolysaccharide structure (LPS)''' of ''P. aeruginosa'' can be bound by galectin-3. Specifically, the O polysaccharide (see figure on the left) of the LPS is recognized by galectin-3. Therefore, this specific binding of galectin-3 enables the construction of a fluorescent based detection system. A fusion protein of galectin-3 and a reporter protein, such as a fluorescent protein, can be built and applied in the detection of ''P.&nbsp;aeruginosa''.<br />
<br />
In our approach, a '''galectin-3-YFP fusion protein''' is built and expressed in ''E.&nbsp;coli'', including a his-tag and a snap-tag for purification. The fusion protein can then be incorporated into a '''cell-free biosensor system'''. These biosensors have many advantages over systems that use living cells such as an uncomplicated storage. Furthermore, from a [https://2014.igem.org/Team:Aachen/Safety biosafety] and social acceptance point of view, it is advantageous if the sensor system does not contain alive genetically modified organisms.<br />
<br />
{{Team:Aachen/FigureFloatRight|align=center|Aachen_14-10-09_Cell_Free_Biosensor_iNB.png|width=500px}}<br />
<br />
<br />
<br />
<br />
To detect ''P.&nbsp;aeruginosa'' cells, an agar chip could be used to sample a solid surface. However, other materials but agar can be considered to collect pathogens. The cells stick to the sampling chip which is then immersed in a detection buffer containing the galectin-3-YFP fusion protein. Excess protein is removed during washing in a suitable buffer. The galectin-3 remains bound to the pathogen and '''illumination with 514&nbsp;nm''', the excitation wavelength of YFP, in a modified version of our measurement device reveals the location of the cells. The picture taken by the measurement device can then be analyzed by our software ''Measurarty''.<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_14-10-15_Medal_Cellocks_iNB.png|right|150px]]<br />
<br />
= Achievements =<br />
<span class="anchor" id="gal3achievements"></span><br />
<br />
Due to the generous support of Sophia Böcker and Prof.&nbsp;Dr.&nbsp;Elling of the Helmholtz Institute for Biomedical Engineering in Aachen, we got access to a pET17-derived expression plasmid for a His- and SNAP-tagged YFP-galectin-3 fusion protein. We transformed the fusion protein into ''E.&nbsp;coli''&nbsp;Rosetta cells and conducted a batch fermentation to obtain large amounts of protein.<br />
<br />
With the help of David Schönauer and Alan Mertens from the RWTH Aachen Institute of Biotechnolgy we then purified the fusion protein using FPLC.<br />
Subsequently, we aimed to test the binding of the Gal-3 fusion protein to the LPS of ''P. aeruginosa'' as shown previously [1]. Apparently, because of insufficient sensitivity of the used fluorescence microscope, this could not be confirmed and would require further experiments, idealy using other detection methods.<br />
<br />
After we received the collection of [https://2014.igem.org/Team:Aachen/Collaborations/Heidelberg pSBX-expression vectors] from Team Heidelberg, we used Gibson assembly to make K1319020 from K1319003 and pSBX1A3, which is the translational unit for an mRFP-Gal3 fusion protein with a C-terminal 6xHis tag:<br />
<center>{{Team:Aachen/Figure|Aachen_K1319020.png|width=800px|title=K1319009|subtitle=This BioBrick is a construction intermediate of K1319003 (gal3), E1010 (mRFP), K1319007 (6xHis tag) to K1319020 (translational unit of the fusion protein).}}</center><br />
<br />
In addition, we cloned our BioBrick K1319003 into the pET17 expression vector and expressed all combinations of fusion proteins in E.&nbsp;coli&nbsp;BL21(DE3). An SDS-PAGE showed that all fusion proteins were fully translated:<br />
<br />
{{Team:Aachen/FigureDual<br />
|Aachen_14-10-04_Expression_Pellets_iMO.png|Aachen_Gal3_Expression.png|title1=Pellets of different fusion protein expressions|title2=SDS-PAGE of K1319020 expression|subtitle1=Expression in the pET17 vector was much more leaky than the expression in the pSBX vectors.|subtitle2=The fusion protein was fully translated to the correct molecular mass of 74&nbsp;kDa.|width=425px}} <br />
<br />
== References ==<br />
[1] Kupper CE, Böcker S, Liu H, et al. Fluorescent SNAP-tag galectin fusion proteins as novel tools in glycobiology. Curr Pharm Des. 2013;19(30):5457-67. Available at: http://www.ncbi.nlm.nih.gov/pubmed/23431989. <br />
{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/Project/Measurement_DeviceTeam:Aachen/Project/Measurement Device2014-10-18T03:22:11Z<p>Nbailly: /* Achievements */</p>
<hr />
<div>__NOTOC__<br />
{{CSS/Main}}<br />
{{Team:Aachen/Stylesheet}}<br />
{{Team:Aachen/Header}}<br />
<br />
= ''WatsOn'' =<br />
<br />
<html><br></html><br />
{{Team:Aachen/FigureFloatRight|Aachen_Device_11.jpg|title=''WatsOn''|subtitle= |width=360px}}<br />
The ''WatsOn'' device aims to answer the central question "What's on the chip?". The device is designed to incubate the sensing cells and capture images. <html><br></html>The interactive ''WatsOn'' software enables the end user not only to take images and time lapse shootings, but also analyzes the images and visualizes the presence/absence of a pathogen.<br />
<br />
The construction manual and the technical details are published on the [https://2014.igem.org/Team:Aachen/Notebook/Engineering/WatsOn#watsondiy engineering page].<br />
<br />
<html><br />
<center><br />
<ul class="menusmall-grid"><br />
<br />
<li style="margin-bottom:5px;margin-top:5px;"><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/Project/Measurement_Device#watsonapplication" style="color:black"><br />
<div class="menusmall-item menusmall-info" ><div class="menukachel" style="top:25%; line-height:1.5em;">Modus Operandi</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/c/c7/Aachen_WatsOn_easy.png); norepeat scroll 0% 0% transparent; background-size:100%"><br />
</div><br />
</a><br />
</li><br />
<br />
<li style="margin-bottom:5px;margin-top:5px;"><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/Project/Measurement_Device#watsonhardware" style="color:black"><br />
<div class="menusmall-item menusmall-info" ><div class="menukachel" style="top:35%; line-height:1.5em;">Hardware</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/5/59/Aachen_14-10-16_Hardware_button_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%"><br />
</div><br />
</a><br />
</li><br />
</ul><br />
<br />
<ul class="menusmall-grid"><br />
<li><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/Project/Measurement_Device#watsonsoftware" style="color:black"><br />
<div class="menusmall-item menusmall-info" ><div class="menukachel" style="top:35%; line-height:1.5em;">Software</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/1/13/Aachen_14-10-16_Software_button_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%"><br />
</div><br />
</a><br />
</li><br />
<br />
<li><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/Project/Measurement_Device#watsonmeasurarty" style="color:black"><br />
<div class="menusmall-item menusmall-info" ><div class="menukachel" style="top:35%; line-height:1.5em;">Measurarty</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/2/21/Aachen_14-10-16_Measurarty_button_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%"><br />
</div><br />
</a><br />
</li><br />
<br />
<li><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/Project/Measurement_Device#watsonachievements" style="color:black"><br />
<div class="menusmall-item menusmall-info" ><div class="menukachel" style="top:25%; line-height:1.5em;">Achieve-<br/>ments</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/e/ef/Aachen_14-10-15_Medal_Cellocks_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%"><br />
</div><br />
</a><br />
</li><br />
<br />
</ul><br />
</center><br />
</html><br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
== Modus Operandi ==<br />
<span class="anchor" id="watsonapplication"></span><br />
<br />
{{Team:Aachen/Figure|How_two_use_watsOn_flowsheet_V7_ipo.png|title=How to use ''WatsOn''|subtitle=This scheme illustrates handling ''WatsOn'' when testing the 2D biosensor chip for a fluorescent signal.|width=1000px}}<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_14-10-16_Hardware_button_iNB.png|right|150px]]<br />
<br />
== Hardware ==<br />
<span class="anchor" id="watsonhardware"></span><br />
<br />
{{Team:Aachen/Figure|Aachen_Device_Elektronikkomponenten1.jpg|title=Hardware components|subtitle=From top left to bottom right: Arduino, Peltier element, Raspberry Pi, relay, cables, MOSFET, temperature display, camera, LEDs and resistors.|width=520px}}<br />
<br />
Our hardware consists of the casing and the electronical components. The casing which can be seen in the first section was build from laser cutted acrylic glass.<br />
<br />
The electronic circuit is a combination of the components displayed in the image above. We combined the Raspberry Pi - a small single-board computer running a Linux operating system - and an Arduino board which is a programmable microcontroller. The Arduino operates the excitation LEDs and a Peltier heater for incubation. For taking images of the sensor chips we used the Raspberry Pi camera module which is directly connected to the board.<br />
<br />
''WatsOn'' is designed such that it can be easily copied. Our work heavily emphasizes the Open Source concept. Therefore a detailed description of all components and the wiring can be found in the [https://2014.igem.org/Team:Aachen/Notebook/Engineering/WatsOn#watsonhardware Engineering section of our Notebook].<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
<!-- [[File:Aachen_14-10-16_Software_button_iNB.png|right|150px]] --><br />
<br />
== Software ==<br />
<span class="anchor" id="watsonsoftware"></span><br />
<br />
{{Team:Aachen/FigureDual|Aachen_WatsOn_igem_GUI_originalImage.png|Aachen_WatsOn_igem_GUI_analyzedImage.png|title1=image taken with the camera |title2=analyzed image |subtitle1= |subtitle2= |width=500px}}<br />
<br />
The ''WatsOn'' software is responsible for presenting a user interface on the display of the device and to take images with the LED wavelength selected by the user. Therefore, it is separated into three single components: the graphical user interface (GUI) with a backend script running on the Raspberry Pi and the code on the Arduino board.<html><br/></html><br />
The GUI (left image) provides the user with the option to take a single image or a time lapse shooting and specify parameters for the camera and the wavelength of the LEDs. The wavelength used in our device are 480nm for GFP and 450nm for iLOV. Furthermore, the images are analysed for the presence or absence of P. aeruginosa by processing the image with [https://2014.igem.org/Team:Aachen/Project/Measurement_Device#watsonmeasurarty Measurarty] and providing the user with a visual feedback (right image). All taken images can be saved to disk manually for single images and automatically for time lapse shootings.<html><br/></html><br />
Further details on the software including the backend which gives the possibility of using the GUI remotely on a different device (e.g. notebook) in the same local network can be found here [https://2014.igem.org/Team:Aachen/Notebook/Engineering/WatsOn#watsonsoftware].<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_14-10-16_Measurarty_button_iNB.png|right|150px]]<br />
<br />
== ''Measurarty'' ==<br />
<span class="anchor" id="watsonmeasurarty"></span><br />
<center><br />
{{Team:Aachen/Figure|Aachen_srm_regions_3.PNG|title=SRM component of our image analysis component ''Measurarty''|subtitle=SRM is one of the core components of our image analysis approach. This image shows the different regions created.|width=500px}}<br />
</center><br />
<br />
''Measurarty'' is the '''image analysis software''' of our device and is designed to allow an automatic segmentation and classification of our '''agar chip pictures'''.<br />
Therefore, it accepts an image from ''WatsOn'' as an input and produces an output image with pathogenic regions marked in red.<br />
<br />
This component mainly focuses on recognizing pathogens early, such that pure thresholding is not necessary.<br />
We therefore designed a pipeline and established a smoothness index to make statements about the pathogenity of a chip as early as possible, but also with as much certainty as possible.<br />
<br />
A sample output of the segmentation is presented below, showing that the pipeline works as intended.<html><br/></html><br />
The left half shows the original images from the device and the right half shows the same pictures with the detected pathogenic region analyzed by ''Measurarty''.<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|960px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/f/fc/Aachen_Measurarty_combined_slow.gif" width="960px"></html><br />
|-<br />
|'''{{{title|Detecting ''P. aeroginosa'' with K131026}}}'''<br />{{{subtitle|Measurarty identifies the fluorescence signal given by K131026 in response to ''P. aeruginosa''.}}}<br />
|}<br />
</div><br />
</center><br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_14-10-15_Medal_Cellocks_iNB.png|right|150px]]<br />
<br />
== Achievements ==<br />
<span class="anchor" id="watsonachievements"></span><br />
<br />
We developed ''WatsOn'' to meet the following requirements: i.e. the device<br />
*incubates the sensing cells and the sampling chip <br />
*illuminates the chip with the right excitation wavelength for our fluorescence proteins<br />
*takes pictures and time lapse shootings of the chips<br />
*uses cheap filter slides to block the light emitted from the LEDs<br />
*analyzes the fluorescence signal<br />
*gives feedback to the user about the presence or absence of P. aeruginosa through a GUI (graphical user interface)<br />
*prevents escape of potentially sampled pathogens and our genetically engineered cells<br />
*is portable and fast in analyzing the images<br />
<br />
With our final device we achieved all of the above mentioned goal. ''WatsOn'' is housed in a closed casing and is able to take images and time lapse shooting using LEDs with required wavelengths, analyze the image and visualize the result.<br />
<br />
All technical details including laser cutting plans, the list of needed components, source codes for the different software and a building instruction are open-source and available on our [https://2014.igem.org/Team:Aachen/Notebook/Engineering/WatsOn engineering page].<br />
<br />
<br />
{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/Project/Measurement_DeviceTeam:Aachen/Project/Measurement Device2014-10-18T03:21:17Z<p>Nbailly: /* Achievements */</p>
<hr />
<div>__NOTOC__<br />
{{CSS/Main}}<br />
{{Team:Aachen/Stylesheet}}<br />
{{Team:Aachen/Header}}<br />
<br />
= ''WatsOn'' =<br />
<br />
<html><br></html><br />
{{Team:Aachen/FigureFloatRight|Aachen_Device_11.jpg|title=''WatsOn''|subtitle= |width=360px}}<br />
The ''WatsOn'' device aims to answer the central question "What's on the chip?". The device is designed to incubate the sensing cells and capture images. <html><br></html>The interactive ''WatsOn'' software enables the end user not only to take images and time lapse shootings, but also analyzes the images and visualizes the presence/absence of a pathogen.<br />
<br />
The construction manual and the technical details are published on the [https://2014.igem.org/Team:Aachen/Notebook/Engineering/WatsOn#watsondiy engineering page].<br />
<br />
<html><br />
<center><br />
<ul class="menusmall-grid"><br />
<br />
<li style="margin-bottom:5px;margin-top:5px;"><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/Project/Measurement_Device#watsonapplication" style="color:black"><br />
<div class="menusmall-item menusmall-info" ><div class="menukachel" style="top:25%; line-height:1.5em;">Modus Operandi</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/c/c7/Aachen_WatsOn_easy.png); norepeat scroll 0% 0% transparent; background-size:100%"><br />
</div><br />
</a><br />
</li><br />
<br />
<li style="margin-bottom:5px;margin-top:5px;"><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/Project/Measurement_Device#watsonhardware" style="color:black"><br />
<div class="menusmall-item menusmall-info" ><div class="menukachel" style="top:35%; line-height:1.5em;">Hardware</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/5/59/Aachen_14-10-16_Hardware_button_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%"><br />
</div><br />
</a><br />
</li><br />
</ul><br />
<br />
<ul class="menusmall-grid"><br />
<li><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/Project/Measurement_Device#watsonsoftware" style="color:black"><br />
<div class="menusmall-item menusmall-info" ><div class="menukachel" style="top:35%; line-height:1.5em;">Software</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/1/13/Aachen_14-10-16_Software_button_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%"><br />
</div><br />
</a><br />
</li><br />
<br />
<li><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/Project/Measurement_Device#watsonmeasurarty" style="color:black"><br />
<div class="menusmall-item menusmall-info" ><div class="menukachel" style="top:35%; line-height:1.5em;">Measurarty</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/2/21/Aachen_14-10-16_Measurarty_button_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%"><br />
</div><br />
</a><br />
</li><br />
<br />
<li><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/Project/Measurement_Device#watsonachievements" style="color:black"><br />
<div class="menusmall-item menusmall-info" ><div class="menukachel" style="top:25%; line-height:1.5em;">Achieve-<br/>ments</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/e/ef/Aachen_14-10-15_Medal_Cellocks_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%"><br />
</div><br />
</a><br />
</li><br />
<br />
</ul><br />
</center><br />
</html><br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
== Modus Operandi ==<br />
<span class="anchor" id="watsonapplication"></span><br />
<br />
{{Team:Aachen/Figure|How_two_use_watsOn_flowsheet_V7_ipo.png|title=How to use ''WatsOn''|subtitle=This scheme illustrates handling ''WatsOn'' when testing the 2D biosensor chip for a fluorescent signal.|width=1000px}}<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_14-10-16_Hardware_button_iNB.png|right|150px]]<br />
<br />
== Hardware ==<br />
<span class="anchor" id="watsonhardware"></span><br />
<br />
{{Team:Aachen/Figure|Aachen_Device_Elektronikkomponenten1.jpg|title=Hardware components|subtitle=From top left to bottom right: Arduino, Peltier element, Raspberry Pi, relay, cables, MOSFET, temperature display, camera, LEDs and resistors.|width=520px}}<br />
<br />
Our hardware consists of the casing and the electronical components. The casing which can be seen in the first section was build from laser cutted acrylic glass.<br />
<br />
The electronic circuit is a combination of the components displayed in the image above. We combined the Raspberry Pi - a small single-board computer running a Linux operating system - and an Arduino board which is a programmable microcontroller. The Arduino operates the excitation LEDs and a Peltier heater for incubation. For taking images of the sensor chips we used the Raspberry Pi camera module which is directly connected to the board.<br />
<br />
''WatsOn'' is designed such that it can be easily copied. Our work heavily emphasizes the Open Source concept. Therefore a detailed description of all components and the wiring can be found in the [https://2014.igem.org/Team:Aachen/Notebook/Engineering/WatsOn#watsonhardware Engineering section of our Notebook].<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
<!-- [[File:Aachen_14-10-16_Software_button_iNB.png|right|150px]] --><br />
<br />
== Software ==<br />
<span class="anchor" id="watsonsoftware"></span><br />
<br />
{{Team:Aachen/FigureDual|Aachen_WatsOn_igem_GUI_originalImage.png|Aachen_WatsOn_igem_GUI_analyzedImage.png|title1=image taken with the camera |title2=analyzed image |subtitle1= |subtitle2= |width=500px}}<br />
<br />
The ''WatsOn'' software is responsible for presenting a user interface on the display of the device and to take images with the LED wavelength selected by the user. Therefore, it is separated into three single components: the graphical user interface (GUI) with a backend script running on the Raspberry Pi and the code on the Arduino board.<html><br/></html><br />
The GUI (left image) provides the user with the option to take a single image or a time lapse shooting and specify parameters for the camera and the wavelength of the LEDs. The wavelength used in our device are 480nm for GFP and 450nm for iLOV. Furthermore, the images are analysed for the presence or absence of P. aeruginosa by processing the image with [https://2014.igem.org/Team:Aachen/Project/Measurement_Device#watsonmeasurarty Measurarty] and providing the user with a visual feedback (right image). All taken images can be saved to disk manually for single images and automatically for time lapse shootings.<html><br/></html><br />
Further details on the software including the backend which gives the possibility of using the GUI remotely on a different device (e.g. notebook) in the same local network can be found here [https://2014.igem.org/Team:Aachen/Notebook/Engineering/WatsOn#watsonsoftware].<br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_14-10-16_Measurarty_button_iNB.png|right|150px]]<br />
<br />
== ''Measurarty'' ==<br />
<span class="anchor" id="watsonmeasurarty"></span><br />
<center><br />
{{Team:Aachen/Figure|Aachen_srm_regions_3.PNG|title=SRM component of our image analysis component ''Measurarty''|subtitle=SRM is one of the core components of our image analysis approach. This image shows the different regions created.|width=500px}}<br />
</center><br />
<br />
''Measurarty'' is the '''image analysis software''' of our device and is designed to allow an automatic segmentation and classification of our '''agar chip pictures'''.<br />
Therefore, it accepts an image from ''WatsOn'' as an input and produces an output image with pathogenic regions marked in red.<br />
<br />
This component mainly focuses on recognizing pathogens early, such that pure thresholding is not necessary.<br />
We therefore designed a pipeline and established a smoothness index to make statements about the pathogenity of a chip as early as possible, but also with as much certainty as possible.<br />
<br />
A sample output of the segmentation is presented below, showing that the pipeline works as intended.<html><br/></html><br />
The left half shows the original images from the device and the right half shows the same pictures with the detected pathogenic region analyzed by ''Measurarty''.<br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|960px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/f/fc/Aachen_Measurarty_combined_slow.gif" width="960px"></html><br />
|-<br />
|'''{{{title|Detecting ''P. aeroginosa'' with K131026}}}'''<br />{{{subtitle|Measurarty identifies the fluorescence signal given by K131026 in response to ''P. aeruginosa''.}}}<br />
|}<br />
</div><br />
</center><br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
[[File:Aachen_14-10-15_Medal_Cellocks_iNB.png|right|150px]]<br />
<br />
== Achievements ==<br />
<span class="anchor" id="watsonachievements"></span><br />
<br />
We developed ''WatsOn'' to meet the following requirements: i.e. the device<br />
*incubates the sensing cells and the sampling chip <br />
*illuminates the chip with the right excitation wavelength for our fluorescence proteins<br />
*takes pictures and time lapse shootings of the chips<br />
*uses cheap filter slides to block the light emitted from the LEDs<br />
*analyzes the fluorescence signal<br />
*gives feedback to the user about the presence or absence of P. aeruginosa through a GUI (graphical user interface)<br />
*prevents escape of potentially sampled pathogens and our genetically engineered cells<br />
*is portable and fast in analyzing the images<br />
<br />
With our final device we achieved all of the above mentioned goal. ''WatsOn'' is housed in a closed casing and is able to take images and time lapse shooting using LEDs with required wavelengths, analyze the image and visualize the result.<br />
<br />
All technical details including laser cutting plans, the list of needed components, source codes for the different software and a building instruction are open-source and available on our https://2014.igem.org/Team:Aachen/Notebook/Engineering/WatsOn engineering page].<br />
<br />
<br />
{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/Project/ModelTeam:Aachen/Project/Model2014-10-18T03:20:46Z<p>Nbailly: /* Modeling */</p>
<hr />
<div>__NOTOC__<br />
{{CSS/Main}}<br />
{{Team:Aachen/Stylesheet}}<br />
{{Team:Aachen/Header}}<br />
<br />
=Modeling=<br />
<center><br />
{{Team:Aachen/FigureFloat|Aachen 14-10-16 REACh approach iFG.png|align=center|title=Molecular approach|subtitle=Expression of the TEV protease is induced by 3-oxo-C<sub>12</sub>-HSL. The protease cleaves the GFP-REACh fusion protein to elicit a fluorescence response.|width=630px}}<br />
</center><br />
<br />
<br />
<center><br />
{{Team:Aachen/FigureFloatRight|Aachen_Modeling_Flowchart.png|title=Modeling Flowchart|align=center|width=300px}}<br />
</center><br />
<br />
For our two-dimensional biosensor, we thought of different methods to generate a faster and stronger fluorescence response from weak promoters. In our molecular approach (left) to detect ''P. aeruginosa'', a fusion protein of GFP and the dark quencher is cleaved by the highly specific TEV protease which is introduced behind the weak quorum sensing promoter. <br />
<br />
To validate our hypothesis, we developed a model of our molecular approach using the CAD tool TinkerCell (Chandran, Bergmann and Sauro, 2009).<br />
<br />
<center><br />
{{Team:Aachen/Figure|Aachen_Model_merged.png|align=center|title=Model of our molecular approach in TinkerCell (A) and the output over time (B)|subtitle=The molecular setup of the novel 3-oxo-C<sub>12</sub>-HSL inducible biosensor (A) yields results indicating a strong and fast fluorescence output after induction (B). A directly inducible system was modeled and added to the plot for comparison.|width=1000px}}<br />
</center><br />
<br />
<br />
In the figure displayed above, we compare the response time of the fluorescence signal between our theoretical system and a traditional biosensor, we included a direct expression of GFP in the same plot. In the results shown above, the strength of the promoter used for the direct GFP expression (traditional approach) is twice as high as the strength of the promoter upstream of the TEV coding sequence in our new approach. Despite a weaker promoter, a '''higher GFP concentration is generated in the model of the novel biosensor''', predicting a quicker response time of our system.<br />
<br />
The model predicted that our approach should be an improvement over the commonly used direct expression, so we proceeded with the clonings and assembled plasmids to test the system.<br />
<br />
Due to the complexity of the quorum sensing circuit, we assembled an IPTG-inducible TEV protease instead of the 3-oxo-C<sub>12</sub>-HSL-inducible version.<br />
Although the modeled and the existing double plasmid system differ regarding the induction mechanism, a '''high correlation between the model predictions and the experimental data''' was observed. However, a new model was designed according to the existing and functional IPTG-inducible system. <br />
<br />
<center><br />
{{Team:Aachen/Figure|Aachen_Model_IPTG_merged.png|align=center|title=Revised model of the molecular approach (A) and output over time (B).|subtitle=This model is for the IPTG-inducible double plasmid system (A) and the calculated output (B). Experimental data was included in the plot for comparison and data validation.|width=1000px}}<br />
</center><br />
<br />
<br />
The model was optimized to fit the data generated from the characterization experiment conducted in shake flasks. Additionally, the data from the characterization experiment of the double plasmid construct (K1319014 + K1319008) in the chip system was included in the plot above. The data was derived from the plate reader output of the four central spots of the chip. The background from the non-induced chip was substracted from the fluorescence response to correct the data and avoid effects from cell growth leading to wrong signal strengths. The development of the fluorescence is presented [https://2014.igem.org/Team:Aachen/Project/FRET_Reporter#reachachievementschip here]. <br />
<br />
It is shown that the fluorescence response in chips develops later than in the characterization experiment in shake flasks. This is because the solid agar chip provides a higher diffusion barrier than liquid medium as used in the shake flasks. Further, a high oxygen transfer rate in shake flask cultivations enhances the rate of fluorescence development, as oxygen is needed for GFP production, in comparison to the cultivation in chips. <br />
<br />
The model predictions correlate with the data generated from the characterization experiments, thus validating our molecular approach. With an iterative cycle of modeling, '''a faster and stronger fluorescence signal could be proven both theoretically and empirically'''. <br />
<br />
<br />
{{Team:Aachen/BlockSeparator}}<br />
<br />
==References==<br />
* Chandran, D., Bergmann, F. T., & Sauro, H. M. (2009). TinkerCell: modular CAD tool for synthetic biology. Journal of biological engineering, 3(1), 19. doi: 10.1186/1754-1611-3-19<br />
<br />
{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/Project/FRET_ReporterTeam:Aachen/Project/FRET Reporter2014-10-18T03:20:07Z<p>Nbailly: /* Producing a GFP-REACh Fusion Protein */</p>
<hr />
<div>__NOTOC__<br />
{{CSS/Main}}<br />
{{Team:Aachen/Stylesheet}}<br />
{{Team:Aachen/Header}}<br />
<br />
=The modified REACh Construct=<br />
<br />
On this page, we present our biosensor on a molecular level. Here you can explore the different parts of our genetic device:<br />
<br />
<html><br />
<center><br />
<ul class="menusmall-grid" style="width:inherit;"><br />
<!-- Overview --><br />
<br />
<li style="width: 156px;margin-left: 8px;margin-right: 8px;"><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/Project/FRET_Reporter#fluorescence" style="color:black"><br />
<div class="menusmall-item menusmall-info" ><div class="menukachel" style="top:32%; line-height:1.5em;">A Faster Answer</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/0/0b/Aachen_14-10-13_GFP_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%"><br />
</div><br />
</a><br />
</li><br />
<br />
<li style="width: 156px;margin-left: 8px;margin-right: 8px;"><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/Project/FRET_Reporter#fret" style="color:black"><br />
<div class="menusmall-item menusmall-info" ><div class="menukachel" style="top:32%; line-height:1.5em;">The FRET System</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/5/54/Aachen_14-10-13_FRET_Arrows_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%"><br />
</div><br />
</a><br />
</li><br />
<br />
<li style="width: 156px;margin-left: 8px;margin-right: 8px;"><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/Project/FRET_Reporter#darkquencher" style="color:black"><br />
<div class="menusmall-item menusmall-info" ><div class="menukachel" style="top:32%; line-height:1.5em;">REACh Quenchers</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/6/65/Aachen_14-10-13_REACh_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%"><br />
</div><br />
</a><br />
</li><br />
<br />
<li style="width: 156px;margin-left: 8px;margin-right: 8px;"><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/Project/FRET_Reporter#gfp-reach" style="color:black"><br />
<div class="menusmall-item menusmall-info" ><div class="menukachel" style="top:32%; line-height:1.5em;">The Fusion Protein</div></div><br />
<div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/0/02/Aachen_14-10-13_Fusion_Protein_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%"><br />
</div><br />
</a><br />
</li><br />
</ul><br />
</center><br />
</html><br />
<br />
<html><br />
<center><br />
<ul class="menusmall-grid" style="width:516px;"><br />
<br />
<li style="width: 156px;margin-left: 8px;margin-right: 8px;"><br />
<a class="menulink" href="https://2014.igem.org/Team:Aachen/Project/FRET_Reporter#tevprotease" style="color:black"><br />
<div class="menusmall-item menusmall-info" ><div class="menukachel" style="top:32%; line-height:1.5em;">TEV Protease</div></div><br />
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<div class="menusmall-item menusmall-info" ><div class="menukachel" style="top:32%; line-height:1.5em;">Achieve-<br/>ments</div></div><br />
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[[File:Aachen_14-10-13_GFP_iNB.png|150px|right]]<br />
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= A Fluorescence Answer Faster than Expression =<br />
<span class="anchor" id="fluorescence"></span><br />
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Biosensor systems are usually based on a reporter gene under the control of a promoter directly induced by the biomolecule of interest. In the case of our 2D biosensor for ''Pseudomonas&nbsp;aeruginosa'', the expression of our fluorescing reporter protein is directly induced by the activity of the bacterium's quorum sensing molecules. However, transcription, translation, folding and post-translational modifications take their time. Since our goal is to detect the pathogen as fast as possible, we aimed to develop a system that provides a fluorescent answer faster than just expressing the fluorescent protein GFP.<br />
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<center><br />
{{Team:Aachen/Figure|Aachen_Traditional_biosensor.png|align=center|title=Schematic model of a traditional biosensor|subtitle=In this model, the expression of GFP is directly controlled by a promoter whose activator binds to a molecule secreted by the pathogen.|width=500px}}<br />
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As an alternative to the traditional approach, we '''constitutively express our reporter gene in a quenched form'''. By fusing GFP to REACh, a mutated variant of EYFP, its original fluorescence is thoroughly suppressed. Thereby, a huge stock of temporary inactivated fluorescence proteins is already available prior to first contact with the pathogen. As a conseqence to the uptake of homoserine lactones of ''P.&nbsp;aeruginosa'' and binding to the LasI promoter in front of the protease gene, these '''autoinducers activate the expression of the TEV protease'''. This way, our biosensor provides a fast increase in fluorescence intensity by cleavage of GFP-REACh-constructs.<br />
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<center><br />
{{Team:Aachen/Figure|Aachen_REACh_approach.png|align=center|title=Schematic model of our novel biosensor|subtitle=Expression of the TEV protease is induced by HSL. The protease cleaves the GFP-REACh fusion protein to elicit a fluorescence response.|width=900px}}<br />
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'''Advantages of the novel biosensor:'''<br />
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* When ''P.&nbsp;aeruginosa'' is detected by our cells, the reporter protein has already been expressed and only waits to be activated. The cleavage reaction catalyzed by the TEV protease is a faster process than expression and correct folding of GFP. Therefore, our sensor cells provide an exceptionally '''early response'''.<br />
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* While a certain concentration of homoserine lactone will produce the same number of gene read-outs and the TEV protease is about the same size as GFP (~27 kDa), one TEV protease can cleave many GFP-REACh constructs per time than fluorescent protein molecules could be expressed and go through fluorescence maturation. Through this additional reaction, we introduce an '''amplification step''' into our system. Hypothetically, the TEV protease will enable the production of '''a much stronger signal''' in a given time interval.<br />
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In parallel, we worked on a [https://2014.igem.org/Team:Aachen/Project/Model model] of the respective biosensor system, in order to confirm our hypothesis of a faster and stronger fluorescence response than a conventional biosensor would povide.<br />
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[[File:Aachen_14-10-13_FRET_Arrows_iNB.png|150px|right]]<br />
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=The FRET (Förster Resonance Energy Transfer) System=<br />
<span class="anchor" id="fret"></span><br />
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Förster resonance energy transfer (FRET), sometimes also called fluorescence resonance energy transfer, is a physical process of energy transfer. In FRET, the energy of a donor chromophore, whose electrons are in an excited state, is passed to a second chromophore, the acceptor. The '''energy is transferred without radiation''' and is therefore not exchanged via emission and absorption of photons. The acceptor then releases the energy received from the donor, for example, as light of a longer wavelength. <br />
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In biochemistry and cell biology fluorescent dyes, which interact via FRET, are applied as "optical nano metering rules", because the intensity of the transfer is dependent on the spacing between donor and acceptor. FRET can be observed over distances of up to 10&nbsp;nm. This way, protein-protein interactions and conformational changes of a variety of tagged biomolecules can be observed. For an efficient FRET to occur, there must be a substantial overlap between the donor fluorescence emission spectrum and the acceptor fluorescence excitation (or absorption) spectrum ([http://www.nature.com/nprot/journal/v8/n2/full/nprot.2012.147.html Broussard et al., 2013]), as described in the figure below.<br />
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{{Team:Aachen/Figure|Aachen_14-10-07_Jablonski_Diagram_and_Absorption_Spectra_iNB.png|align=center|title=FRET betweeen donor and acceptor|subtitle=On the left: Jablonski diagram showing the transfer of energy between donor and acceptor; on the right: For successful FRET, the emission spectrum of of the donor has to overlap with the absorption spectrum of the acceptor.|width=900px}}<br />
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However, '''fluorescence is not an essential requirement for FRET'''. This type of energy transfer can also be observed between donors that are capable of other forms of radiation, such as phosphorescence, bioluminescence or chemiluminescence. Acceptor chromophores do not necessarily emit the energy in form of light, and can lead to quenching instead. Thus, this kind of acceptors are also referred to as '''dark quenchers'''. In our project, we use a FRET system with a dark quencher, namely our '''REACh construct'''.<br />
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[[File:Aachen_14-10-13_REACh_iNB.png|150px|right]]<br />
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=REACh Proteins - Dark Quenchers of GFP=<br />
<span class="anchor" id="darkquencher"></span><br />
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<center><br />
{{Team:Aachen/FigureFloat|Aachen_K1319000.gif|align=center|title=Homology model of REACh.|subtitle=This homology model was created with SWISS-MODEL. We used Chimera to prepare and export a scene that was then rendered into an animation with POV-Ray.|width=256px}}<br />
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In 2006, [http://www.pnas.org/content/103/11/4089.full Ganesan et al.] were the first to present a previously undescribed FRET acceptor, a non-fluorescent yellow fluorescent protein (YFP) mutant called '''REACh (Resonance Energy-Accepting Chromoprotein)'''. YFP can be used as a FRET acceptor in combination with GFP as the donor in FRET microscopy and miscellaneous assays in molecular biology. The ideal FRET couple should possess a large spectral overlap between donor emission and acceptor absorption - as illustrated in previous section - but have separated emission spectra to allow their selective imaging.<br />
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To optimize the spectral overlap of this FRET pair, the group obtained '''a genetically modified YFP acceptor'''. Mutations of amino acid residues that stabilize the excited state of the chromophore in enhanced YFP (EYFP) resulted in a non-fluorescent chromoprotein. Two mutations, H148V and Y145W, reduced the fluorescence emission by 82 and 98%, respectively. Ganesan et al. chose the Y145W mutant and the Y145W/H148V double mutant as FRET acceptors, and named them REACh1 and REACh2, respectively. '''Both REACh1 and REACh2 act as dark quenchers of GFP'''.<br />
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{{Team:Aachen/BlockSeparator}}<br />
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[[File:Aachen_14-10-13_Fusion_Protein_iNB.png|150px|right]]<br />
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=Producing a GFP-REACh Fusion Protein=<br />
<span class="anchor" id="gfp-reach"></span><br />
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In our project, we reproduced the REACh1 and REACh2 proteins by subjecting an RFC-25 compatible version of the BioBrick [http://parts.igem.org/Part:BBa_E0030 E0030] (EYFP) to a '''QuikChange mutation''', creating the BioBricks [http://parts.igem.org/Part:BBa_K1319001 K1319001] and [http://parts.igem.org/Part:BBa_K1319002 K1319002]. Subsequently, we fused each REACh protein with '''GFP (mut3b)''' which is available as BioBrick [http://parts.igem.org/Part:BBa_E0040 E0040]. The protein complex was linked via a '''protease cleavage site''' ([http://parts.igem.org/Part:BBa_K1319016 K1319016]). As constitutive promoter we use [http://parts.igem.org/Part:BBa_J23101 J232101]. When GFP is connected to either REACh quencher, GFP will absorb light but the energy will be transferred to REACh via FRET and is then emitted in the form of heat; the fluorescence is quenched. Our cells also constitutively express the '''[http://parts.igem.org/Part:BBa_C0179 LasR] activator'''. Together with the HSL molecules from ''P. aeruginosa'', LasR binds to the '''[http://parts.igem.org/Part:BBa_J64010 LasI] promoter''' that controls the expression of the TEV protease, which we made available as [http://parts.igem.org/Part:BBa_K1319004 K1319004]. When the fusion protein is cleaved by the TEV protease, REACh will be separated from GFP. The latter will then be able to absorb and emit light as usual.<br />
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{{Team:Aachen/Figure|align=center|Aachen_14-10-08_REACh_approach_with_BioBricks_iNB.png|title= Composition of our biosensor|subtitle=For our biosensor, we use a mix of already available and self-constructed BioBricks.|width=900px}}<br />
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The resulting fusion proteins can be expressed in [http://parts.igem.org/Part:BBa_K1319013 K1319013] (GFP fused with REACh1) and [http://parts.igem.org/Part:BBa_K1319014 K1319014] (GFP fused with REACh2). The linker between the proteins containing a TEV protease cleavage site is labelled as [http://parts.igem.org/Part:BBa_K1319016 K1319016].<br />
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Since we did not have enough time to build the complete system we tested our FRET reporter with an IPTG inducible TEV protease ([http://parts.igem.org/Part:BBa_K1319008 K1319008]). In this way we were able to establish a proof of concept of our reporter system including proper expression of the TEV protease as well as functionality of our GFP-REACh construct.<br />
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{{Team:Aachen/Figure|align=center|Aachen 14-10-17 IPTG REACh iFG.png|title= Composition of our biosensor for IPTG|subtitle=To test the funtionality of our sensor concept, we expressed the TEV protease with a T7 promoter.|width=900px}}<br />
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[[File:Aachen_14-10-13_TEV_Protease_iNB.png|150px|right]]<br />
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=Cutting the Fusion Protein with the TEV Protease=<br />
<span class="anchor" id="tevprotease"></span><br />
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To cleave the GFP-REACh fusion protein, we chose '''Tobacco Etch Virus (TEV) protease''', a highly sequence-specific cysteine protease, that is frequently used for the controlled cleavage of fusion proteins ''in vitro'' and ''in vivo''. The native protease also contains an internal self-cleavage site. This site is slowly cleaved to inactivate the enzyme. The physiological reason for the self-cleavage is unknown, however, undesired for our use. Therefore, our team uses a variant of the native TEV protease containing the mutation S219V which results in an alteration of the cleavage site so that self-inactivation is diminished.<br />
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{{Team:Aachen/Figure|Aachen_TEV_Protease_Model.png|title=TEV protease with a bound peptide|subtitle=This picture shows the TEV protease with a peptide chain bound in the binding pocket ready to be cleaved. The recognition site of the bound peptide chain is located inside the binding pocket of the TEV protease. It was rendered with POV-Ray.|width=800px}}<br />
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Though quite popular in molecular biology, the TEV protease is not avaiable as a BioBrick yet. Hence, the team Aachen introduces the protease with anti-self cleavage mutation S219V and codon optimized for ''E. coli'' [http://parts.igem.org/Part:BBa_K1319004 '''to the Parts Registry this year.''']<br />
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{{Team:Aachen/BlockSeparator}}<br />
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[[File:Aachen_14-10-15_Medal_Cellocks_iNB.png|right|150px]]<br />
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== Achievements ==<br />
<span class="anchor" id="reachachievements"></span> <br />
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===Characterization of GFP-REACh1 and GFP-REACh2 fusion proteins in combination with an IPTG-inducible TEV Protease===<br />
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The characterization of the TEV protease and the REACh1 and REACh2 dark quenchers was performed by introducing both simultaneously into ''E.&nbsp;coli'' Bl21 (DE3). The resulting double plasmid cells therefore contained [http://parts.igem.org/Part:BBa_K1319013 K1319013] (GFP-REACh1 fusion protein) or [http://parts.igem.org/Part:BBa_K1319014 K1319014] (GFP-REACh2 fusion protein) and [http://parts.igem.org/Part:BBa_K1319008 K1319008] (IPTG-inducible TEV protease). K1319013 and K1319014 were located on a pSB3K3 plasmid backbone and K1319008 on a pSB1C3 backbone, two standard iGEM plasmids with different oris allowing simultaneous use in one cell. <br />
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[http://parts.igem.org/Part:BBa_I20260 I20260] was used as a positive control because I20260 contains the same promoter ([http://parts.igem.org/Part:BBa_J23101 J23101]), the same RBS ([http://parts.igem.org/Part:BBa_B0032 B0032]) and the same version of GFP ([http://parts.igem.org/Part:BBa_E0040 E0040]) and is located on the same plasmid backbone pSB3K3. Therefore, it is expected that when all fusion proteins are successfully cut by the TEV protease, the fluorescence level of the double plasmid constructs reaches the same level as the positive control of I20260. As a negative control [http://parts.igem.org/Part:BBa_B0015 B0015] was used, a coding sequence of a terminator which should not show any sign of fluorescence.<br />
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To characterize our REACh1/2 constructs in combination with the TEV protease a growth experiment was conducted. Both of the double plasmid constructs, a constitutive expression of GFP (I20260) as positive control and B0015 as negative control were compared. For each expression IPTG-induced and non-induced cultures were grown in parallel. All measurements were done in a biological triplicate. <br />
<br />
To better evaluate the fluorescence, the observed Optical desity (OD) was taken into account in order to achieve a fluorescence measurement independent of the amount of cells present. This way, the measurement represents the amount of fluorescence per cell only. <br />
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{{Team:Aachen/Figure|Aachen 16-10-14 Graph2iFG.PNG|title=Comparison of K1319013 + K1319008, K1319014 + K1319008, I20260 (positive control) and B0015 (negative control)|subtitle=Both double plasmid construct exhibit a clear fluorescence signal when induced.|width=700px}}<br />
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The negative control B0015 did not exhibit any significant fluorescence. The positive control I20260 showed a steady level of fluorescence as expected due to the constitutive expression of GFP. As expected, the production is also independent of addition of IPTG therefore the triplicates have been merged together.<br />
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Both double plasmid constructs K1319013 + K1319008 and K1319014 + K1319008 did not exhibit a strong fluorescence before induction with IPTG. In the non-induced state, the fluorescence stays low and does not increase over time. It is significantly weaker than the fluorescence reached by the induced constructs or the positive control but was higher than the negative control. The higher base level of fluorescence in the not induced constructs is due to the imperfect quenching and the leakiness of the IPTG inducible promoter. <br />
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The induced double plasmid constructs exhibited a fast rise in fluorescence after induction. '''The signal strenght increased ~ 10-fold over the non-induced constructs.''' K1319014 + K1319008 reached the the same level of fluorescence as I20260, indicating a complete cleavage of the fusion proteins by the TEV protease. K1319013 + K1319008 did not reach a level of fluorescence as high as K1319013 + K1319008, however, the nearly 10-fold increase in fluorescence after induction is a clear indicator for the TEV protease cutting the fusion protein K1319013. The weaker fluorescence signal is probably due to a lower expression level of K1319013 in the cells compared to the expression level of K1319014. <br />
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The fluorescence results of K1319014 + K1319008 was used to fit our [https://2014.igem.org/Team:Aachen/Project/Model model] to experimental data.<br />
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====Summary====<br />
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The double plasmid systems of K1319013 + K1319008 and K1319014 + K1319008 demonstrate the quenching ability of the REACh1 and REACh2 proteins as well as the funcionality of the TEV protease.<br />
The increase in fluorescence after induction with IPTG is based on a functional expression of the TEV protease which proceeds to cut the linker of the fusion protein already produced destroying the FRET system between GFP and its quencher and resulting in a strong fluorescence signal. Combined, this characterization is a '''validation of the functionality of the REACh1 protein ([http://parts.igem.org/Part:BBa_K1319001 K1319001]), the REACh2 protein ([http://parts.igem.org/Part:BBa_K1319002 K1319002]) and the TEV protease ([http://parts.igem.org/Part:BBa_K1319004 K1319004])'''.<br />
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===Determining the Quenching ability of REACh1 and REACh2===<br />
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In order to further evaluate the quenching ability of the REACh1 and REACh2 constructs in the fusion proteins produced by K1319013 and K1319014, they were expressed alone without an IPTG inducible TEV protease. This eliminated the effect of a potential leakiness of the non induced promoter to reliably assess the quenching ability of the REACh1 and REACh2 proteins. <br />
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{{Team:Aachen/Figure|Aachen_K1319001_and_K1319002.PNG|title=Comparison of K1319013 and K1319014 with I20260 and B0015|subtitle=K1319013 and K1319014 show a severely reduced fluorescence compared to the positive control I20260.|width=700px}}<br />
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In the previous experiment it was established that the fusion proteins K1319013 and K1319014 are expressed funtionally. K1319014 reached the same level of fluorescence as the positive control after being cut by the TEV protease. Therefore the reduced fluorescence in this experiment is completely attributable to the quenching of REACh1. The quenching reduces the fluorescence of GFP by a factor ~ 25 which means a '''quenching efficiency of ~ 96%!'''. <br />
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K139013 was not able to reach the same fluorescence level as the positive control in the previous experiment. Therefore a worse rate of functional expression of K1319013 compared to K1319014 is assumed. incorporating this, the difference in fluorescence between induced and not induced is still a factor of ~ 30 resulting in a '''quenching efficiency of ~ 97%'''.<br />
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====Summary ====<br />
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The fusion proteins of GFP combined with REACh1 and REACh2 are not only fully functional but exhibit a great quenching efficiency of ~ 97% for REACh1 and ~ 96% for REACh2.Even though REACh1s quenching ability seems to be slighty superior, the expression level of the K1319014 fusion protein including REACh2 is nearly twice as high as the fusion protein K1319013 containing REACh1 and therefore shows a stronger fluorescence. Ganesan et al. (2006) reported a reduction on emission of GFP of 82% for REACh1 and 98% for REACh2 but with a different Linker between the proteins and on a different vector backbone.<br />
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===Comparing fluorescence kinetics of the GFP-REACh fusion proteins with a Standard lacI-inducible GFP Expression===<br />
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To assess the kinetics of the fusion proteins K1319013 (GFP-REACh1) and K1319014 (GFP-REACh2), the double plasmid systems K1319013 + K1319008 and K1319014 + K1319008 were compared to a standard expression of GFP under the control of a lacI promoter in [http://parts.igem.org/Part:BBa_K731520 K731520], a BioBrick made by the iGEM Team TRENTO in 2012. This tested the hypothesis of achieving a faster fluorescence response with the GFP-REACh fusion proteins compared to a standard expression.<br />
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K731520 and the double plasmid constructs K1319013 + K1319008 and K1319014 + K1319008 were cultivated in ''E. coli'' BL21(DE3), and fluorescence and OD was measured.The fluorescence was adjusted for the OD to show a relative fluorescence on a per cell basis. The difference between the induced and non-induced state, the fluorescence quotient, serves as a better indicator for a system used as a sensor because the difference between an ''on'' and ''off'' state is more important for a clear and unmistakable signal than the overall fluorescence. Hence, the OD-adjusted fluorescence quotient for both double plasmid constructs and K731520 was obtained and plotted in the following graph.<br />
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{{Team:Aachen/Figure|Aachen_16-10-14_GraphQuotient_iFG.PNG|Comparison of K1319013 + K1319008, K1319014 + K1319008 and K731520|subtitle=Fluorescence was normalized to the cell growth. The fluorescence of induced cells was additionally divided by the fluorescence of non-induced cells to obtain the fluorescence quotient.|width=700px}}<br />
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The graph clearly shows the faster response of the cut GFP-REACh fusion protein compared to a standard GFP expression. Both fluorescence signals of the double plasmid constructs achieve a higher difference in fluorescence signal between induced and non-induced state as well as at a faster rate. This proves the hypothesis made earlier about the kinetics of the GFP-REACh fusion protein combined with the TEV protease.<br />
<br />
====Summary====<br />
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The kinetics of the fusion protein combined with the TEV protease exhibits the exact characteristics as predicted. The response is clearly faster than normal expression by accumulating a reservoir of fusion proteins which are not fluorescing due to the dark quencher attached to them. This reservoir is then activated by the induction of the TEV protease expression. Production of the protease results in the cleavage of the fusion protein, releasing GFP from the dark quencher and disturbing the interaction between the FRET pair. This results in the observed faster fluorescence reaction due to the amplificating effect of the TEV protease in which every one TEV protease can account for many fluorescence proteins being activated.<br />
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===Characterizing the GFP-REACh Constructs in Sensor Chips===<br />
To further characterize the REACh construct, they were introduced into the sensor cells which were then induced with 2&nbsp;µL IPTG with a concentration of 100&nbsp;mM. Subsequently, we took fluorescence measurement read-outs (GFP, excitation 496&nbsp;±&nbsp;9&nbsp;nm, emission&nbsp;516&nbsp;±&nbsp;9&nbsp;nm) roughly every 10&nbsp;min in the plate reader. The results were plotted in the heatmap shown here. <br />
<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 40px; border:{{{border|0px solid #aaa}}};width:{{{width|500px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/6/6d/Aachen_K1319014%2B8_and_K1319013%2B8_pixelig_minus_bg.gif" width="500px"></html><br />
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|'''{{{title|K1319014 + K1319008 and K1319013 + K1319008 non-induced (top) and induced (bottom) in sensor cells }}}'''<br />{{{subtitle|The induced double plasmid systems K1319013 + K1319008 and K1319014&nbsp;+&nbsp;K1319008 exhibit a clear fluorescence response in our sensor cells which in response to induction with 2 µl IPTG. }}}<br />
|}<br />
</div><br />
</center><br />
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The heatmap shows an increase of fluorescence from blue (no fluorescence) to red (high fluorescence). It is clearly visible that the induced chips are exhibiting a significantly higher fluorescence than the non-induced chips. This shows that the constructs also work as intended in the sensor chips: The TEV protease cuts the linker so that the fusion protein is separated into GFP and a dark quencher, disabling the quenching. GFP has a clear fluorescence emission after the fusion protein has been successfully cut into two pieces by the TEV protease.<br />
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===Comparing the kinetic of the double plasmid systems K1319013 + K1319008 and K1319014 + K1319008 with standard GFP expression===<br />
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<center><br />
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 40px; border:{{{border|0px solid #aaa}}};width:{{{width|480px}}};padding:10px 10px 0px 0px;"><br />
{|<br />
|<html> <img src="https://static.igem.org/mediawiki/2014/1/1a/Aachen_K13%2B8%2CK14%2B8%2CK731_slower_reduced.gif" width="480px"></html><br />
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|'''{{{title|K1319013 + K1319008, K1319014 + K1319008 and K731520 in an non-induced (top) and induced (bottom) chip}}}'''<br />{{{subtitle|Comparing the factor of fluorescence adjusted for OD between induced (bottom) and not induced (top) sensor chips of the constructs K1319013 + K1319008, K1319014 + K1319008 and K731520.}}}<br />
|}<br />
</div><br />
</center><br />
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The analysis of the different [https://2014.igem.org/Team:Aachen/Project/2D_Biosensor sensor chips] with the three different construct K1319013 + K1319008, K1319014 + K1319008 and K731520 demonstrates the same fluorescence response kinetic as in the shake flask experiments. The double plasmid systems exhibits a faster and stronger fluorescence response compared to a standard GFP expression in K731520. The build up pool of fusion proteins allows for a faster, stronger fluorescence response when the induced TEV protease cleaves the fusion proteins and releases GFP from its dark quencher. <br />
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[[File:Aachen_14-10-16_Outlook_Cellocks_iNB.png|right|150px]]<br />
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== Outlook ==<br />
<span class="anchor" id="reachoutlook"></span><br />
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The system of the GFP-REACh fusion proteins with an inducible TEV protease has been established and shows the desired results of being faster than standard expression. The next step will be to devise an expression of the TEV protease inducible by HSL instead of IPTG and then to incorporate both the HSL inducible TEV protease and the fusion protein into one plasmid backbone. This would also allow us to choose a high copy plasmid for both inserts, instead of a high copy plasmid for the TEV protease and a low to mid copy plasmid for the fusion protein which should yield an overall higher fluorescence readout.<br />
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Subsequently the combined construct will be characterizes the same way as the double plasmid system and other options regarding different fluorescence proteins with different quenchers will be considered to be able to have multiple fluorescence responses readable at the same time while still being faster than normal expression. <br />
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Also finding and testing different promoters which can be used to express the TEV protease is planned to be able to detect not only ''Pseudomonas aeruginosa'' but also other pathogens. Also the technology can be expanded to other molecules enabling other research areas to profit from the faster fluorescence response.<br />
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{{Team:Aachen/BlockSeparator}}<br />
<br />
= References =<br />
* Sundar Ganesan, Simon M. Ameer-Beg, Tony T. C. Ng, Borivoj Vojnovic and Fred S. Wouters. "A dark yellow fluorescent protein (YFP)-based Resonance Energy-Accepting Chromoprotein (REACh) for Förster resonance energy transfer with GFP" Proceedings of the National Academy of Science of the United States of America | March 14,2006 | vol. 103 | no. 11 | 4089 - 4094<br />
<br />
* Broussard, Joshua A, Benjamin Rappaz, Donna J Webb, and Claire M Brown. "Fluorescence resonance energy transfer microscopy as demonstrated by measuring the activation of the serine/threonine kinase Akt." Nature protocols 8.2 (2013): 265-281. doi:10.1038/nprot.2012.147. <br />
<br />
* Broussard, J. A., Rappaz, B., Webb, D. J., & Brown, C. M. (2013). Fluorescence resonance energy transfer microscopy as demonstrated by measuring the activation of the serine/threonine kinase Akt. Nature protocols, 8(2), 265-281. doi: 10.1073/pnas.0509922103 <br />
<br />
'''SWISS-MODEL'''<br />
* Arnold, K., Bordoli, L., Kopp, J., & Schwede, T. (2005). The SWISS-MODEL Workspace: A Web-based Environment For Protein Structure Homology Modelling. Bioinformatics, 22(2), 195-201.<br />
<br />
* Biasini, M., Bienert, S., Schwede, T., Waterhouse, A., Arnold, K., Studer, G., et al. (2014). Nucleic Acids Research. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. doi: 10.1093/nar/gku340.<br />
<br />
* Guex, N., Peitsch, M. C., & Schwede, T. (2009). Automated comparative protein structure modeling with SWISS-MODEL and Swiss-PdbViewer: A historical perspective. Electrophoresis, 30(S1), S162-S173.<br />
<br />
* Kiefer, F., Arnold, K., Kunzli, M., Bordoli, L., & Schwede, T. (2009). The SWISS-MODEL Repository and associated resources. Nucleic Acids Research, 37(Database), D387-D392.<br />
<br />
'''UCSF Chimera'''<br />
* Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt, D. M., Meng, E. C., et al. (2004). UCSF Chimera?A visualization system for exploratory research and analysis. Journal of Computational Chemistry, 25(13), 1605-1612. PubMed PMID: 15264254.<br />
<br />
'''POV-Ray'''<br />
* Persistence of Vision Pty. Ltd. (2004) Persistence of Vision Raytracer (Version 3.7) [Computer software]. Retrieved from http://www.povray.org/download/<br />
<br />
<br />
{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/Project/2D_BiosensorTeam:Aachen/Project/2D Biosensor2014-10-18T03:19:19Z<p>Nbailly: /* References */</p>
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= 2D Biosensor =<br />
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With our 2D biosensor technology we are able to detect the pathogen ''Pseudomonas aeruginosa'' on solid surfaces. The sensor system is comprised of '''two distinct but inseparable modules''', a biological and a technical part:<br />
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* Sensor chips containing '''''Cellocks''''', our '''engineered detective cells''' that fluoresce in the presence of the pathogen, make up the biological part of ''Cellock Holmes''.<br />
* Our '''measurement device [https://2014.igem.org/Team:Aachen/Project/Measurement_Device ''WatsOn'']''' and the complementary '''software [https://2014.igem.org/Team:Aachen/Notebook/Software/Measurarty ''Measurarty'']''' complete our sensing technology on the technical side. <br />
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== Principle of Operation ==<br />
<span class="anchor" id="biosensorpoo"></span><br />
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''Cellock Holmes'' is designed upon a SynBio approach comprising a '''two-dimensional biosensor and a measurement unit'''. The two-dimensional biosensor is devised to recognize quorum sensing molecules secreted by the pathogen cells and to generate a distinct fluorescence signal; while the measurement device recognizes and analyzes the produced signal. On the molecular side, we use the '''[https://2014.igem.org/Team:Aachen/Project/FRET_Reporter REACh construct]''' to transform regular ''E. coli'' cells into ''Cellocks''.<br />
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{{Team:Aachen/Figure|Aachen 17-10-14 The basics of quorum sensing ipo.png||title=The principle of quorum sensing|subtitle=Microorganisms can sense the presence of their own kind based on quorum sensing which is a form of chemical communication. Depending on their cell density, quorum sensing allows these cells to activate or deactivate certain gene expression cascades (Waters and Bassler, 2005) for a specific function.|width=900px}}<br />
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Our '''sensor cells, ''Cellocks'', are immobilized in agar chips'''. To make the chips, we mix the ''Cellocks'' with liquid LB agar. <br />
In the course of our project, we designed a casting mold specifically for the production of our agar chips. When the agar has cooled down, the chips are cut out of the mold and are ready to use. Storage of the readily usable sensor chips is possible for up to two days at 4°C when using LB medium or up to five days if TB medium is used. A detailed description of the sensor chip manufacturing can be found in our [https://2014.igem.org/Team:Aachen/Notebook/Protocols/detection Protocols] section.<br />
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{{Team:Aachen/Figure|Aachen 14-10-14 Flowsheet OD-device part1 ipo.png|title=Assay to detect ''P.&nbsp;aeruginosa'' using ''Cellock Holmes''|subtitle=This flow sheet shows the procedure to sample and detect ''P.&nbsp;aeruginosa'': A sampling chip is briefly put onto the potentially contaminated surface, added onto one of our sensor chips and inserted into ''WatsOn''.|width=900px}}<br />
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Using ''Cellock Holmes'', we developed a simple assay to detect ''P.&nbsp;aeruginosa''. Initially, a so called sampling chip is placed on a solid surface that is potentially contaminated with the pathogen. Subsequently, the sampling chip is removed from the surface and put onto one of our sensor chips. Theorectically, the sensor chips could be directly used for sampling, however, this was avoided in our project to '''match [https://2014.igem.org/Team:Aachen/Safety biosafety regulations]''' and to prevent the spread of genetically modified organisms (GMOs) into the environment. The two layered chip-stack is then put into a petri dish which is inserted into our measurement device [https://2014.igem.org/Team:Aachen/Project/Measurement_Device ''WatsOn''] for evalutation.<br />
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{{Team:Aachen/Figure|Aachen 14-10-14 Flowsheet OD-device part2 ipo.png|title=Mode of action inside ''WatsOn''|subtitle=Chips are incubated at 37°C to stimulate cell growth and then illuminated with blue light to excite fluorescence. A picture is taken and analyzed for fluorescence signals using the software ''Measurarty''.|width=900px}}<br />
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Inside ''WatsOn'', the chips are incubated at 37°C and the sampled populations of microorganisms attached on the sampling chip start to grow and multiply. During incubation the chips can be '''illuminated with blue light''' at any time, and a '''photo of the chips''' is taken. The '''software ''Measurarty''''' then analyzes any fluorescent signal. ''P.&nbsp;aeruginosa'' secrets an increasing number of quorum sensing molecules that are recognized by ''Cellocks'', thereby producing a fluorescence signal. For detection of ''P.&nbsp;aeruginosa'', we focused on a quorum sensing molecule called N-3-oxo-dodecanoyl-L-homoserine lactone (for short: 3-oxo-C<sub>12</sub>-HSL), which is involved in virulence regulation of ''P.&nbsp;aeruginosa'' (Jimenez, Koch, Thompson et al., 2012). The incorporation of the 3-oxo-C<sub>12</sub>-HSL detection system into the ''Cellocks'' is explained in detail in the [https://2014.igem.org/Team:Aachen/Project/FRET_Reporter REACh Construct] section.<br />
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For our biosensor, our team genetically modified ''E. coli'' cells to be able to elecit a '''fluorescent response to autoinducers''' produced by the pathogen ''Pseudomonas aeruginosa'' during quorum sensing. In the case of ''P.&nbsp;aeruginosa'', these autoinducers are N-3-oxo-dodecanoyl-L-homoserine lactone, or 3-oxo-C-12-HSL for short. The quorum sensing system of this pathogen contains the '''LasR activator''' which binds 3-oxo-C-12-HSL, and the '''LasI promoter''', which is activated by the LasR-HSL complex. Both LasR activator and LasI promoter are available as BioBricks [http://parts.igem.org/Part:BBa_C0179 C0179] and [http://parts.igem.org/Part:BBa_J64010 J64010].<br />
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As a reporter gene, we use '''GFP'''. However, expression of GFP is not simply controlled through the LasI promoter activity in our approach. Instead, our sensor cells contain genes for a constitutively expressed fusion protein consisting of GFP and a dark quencher, and an '''HSL-inducible protease'''. We use the REACh protein as dark quencher for GFP and the TEV protease to cleave the complex; [https://2014.igem.org/Team:Aachen/Project/FRET_Reporter here] you can read more about the REACh construct and the TEV protease. <br />
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{{Team:Aachen/Figure|align=center|Aachen_REACh_approach.png|title=Our novel biosensor approach|subtitle=Expression of the TEV protease is induced by HSL. The protease cleaves the GFP-REACh fusion protein to elecit a fluorescence response.|width=500px}}<br />
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When ''P.&nbsp;aeruginosa cells'' are stuck on our agar chip and come close to our sensor cells, the latter take up the HSL molecules secreted by the pathogens. Inside the sensor cells, the autoinducer binds to the LasR gene product and activate the expression of the TEV protease. The protease then cleaves the GFP-REACh construct. When '''illuminated with light of 480&nbsp;nm''', the excitation wavelenght of GFP, our sensor cells in the vicinity of ''P.&nbsp;aeruginosa'' give a '''fluorescence signal'''. On the other hand, sensor cells that were not anywhere close to the pathogens do not express the protease. Therefore, the GFP will still be attached to the dark quencher in these cells, and no fluorescence is produced.<br />
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== Development & Optimization ==<br />
<span class="anchor" id="biosensordevelopment"></span><br />
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{{Team:Aachen/FigureFloat|Aachen_ILOV_GFP_HM_1,5h.png|title=iLOV and GFP in the Gel Doc<sup>TM</sup>|subtitle=Sensor cells producing iLOV (A) and GFP (B) 1.5&nbsp;h after induction.|left|width=500px}}<br />
=== Equipment and medium selection ===<br />
Our first approach (before developing our own device) was to use the Molecular Imager&reg; Gel Doc™ XR+ from BIO-RAD in our lab to detect fluorescence. This device uses UV and white light illuminators. However, only two different filters were available for the excitation light wavelength, which resulted in very limited possibilities for the excitation of fluorescent molecules. For example, it was possible to detect the expression of iLOV in our sensor chips, but not the expression of GFP. Hence, the '''Gel Doc™ was not suitable for our project'''.<br />
{{Team:Aachen/FigureFloat|Aachen_Chip_medium_geldoc.png|title=Differend medium in the Gel Doc™|subtitle=complex media exhibited high background fluorescence while less back- ground fluorescence was observed with the minimal media (HM, M9, NA).|right|width=500px}}<br />
{{Team:Aachen/FigureFloat|Aachen_5days_K131026_neb_tb_1,5h.jpg |title=Testing our chips' shelf-life|subtitle= Chips of [http://parts.igem.org/Part:BBa_K131026 K131026] in NEB were stored five days at 4°C. The right chip was induced with 0.2&nbsp;µL of 500&nbsp;µg/mL HSL and an image was taken after 1.5&nbsp;h.|left|width=500px}}<br />
We tested different media (LB, TB, M9, NA and HM) for the preparation of our sensor chips. The medium compositions can be found in the [https://2014.igem.org/Team:Aachen/Notebook/Protocols Protocols] section. We screened for an optimized medium composition to minimize background fluorescence and to enhance cell growth. The results of the analysis are presented in the table below. Due to the low background fluorescence in ''WatsOn'' and the excellent cell growth, we '''chose LB&nbsp;medium''' over the other tested media for sensor chip manufacturing.<br />
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<center><br />
{| class="wikitable"<br />
! !! LB !! TB !! NA !! M9 !! HM <br />
|-<br />
| Growth of ''Cellock'' || <div style="text-align: center;">'''+'''</div> || <div style="text-align: center;">'''+'''</div> || <div style="text-align: center;">-</div> || <div style="text-align: center;">-</div> || <div style="text-align: center;">-</div><br />
|-<br />
| Background fluorescence in GelDoc || <div style="text-align: center;">'''+'''</div> || <div style="text-align: center;">'''+'''</div> || <div style="text-align: center;">-</div> || <div style="text-align: center;">-</div> || <div style="text-align: center;">-</div><br />
|-<br />
| Background fluorescence in ''WatsOn'' || <div style="text-align: center;">-</div> || <div style="text-align: center;">'''+'''</div> || <div style="text-align: center;">-</div> || <div style="text-align: center;">-</div> || <div style="text-align: center;">-</div><br />
|}<br />
</center><br />
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Another set of experiments were conducted to test the '''long-time storage''' of the sensor chips. We varied the glycerol content of the chips as well as the storage temperature. Storage at -20°C resulted in the loss of our sensor cells. Adding 5-10% (v/v) glycerol ensured survival of the sensor cells, but resulted in the loss of fluorescence ability. Hence, we concluded that long-time storage of the sensor chips at -20°C is not possible under the tested conditions. However, the 'ready-to-use' sensor chips can be kept at at 4°C for two days when using LB medium, and storage at this temperature for 5 days is possible with chips made from TB medium.<br />
<!--Regarding the medium used for our sensor chips, LB medium showed a high background fluorescence when exposed to UV light in the Gel Doc. Surprisingly, the background fluorescence resulting from the LB medium was too high to detect a signal emitted by our sensor cells. Hence, minimal media (NA, M9, Hartman (HM)) was used to minimize background fluorescence, but this approach resulted in less to no growth of our sensor cells. In our device ''WatsOn'', optimized wavelengths of 450&nbsp;nm and 480&nbsp;nm were used for excitation of iLOV and GFP, respectively. When exposed to either excitation wavelength TB medium, which is basically an improved LB medium and highly supports cell growth, showed strong background fluorescence in our own device. High background fluorescence was also observed for TB medum when using the Gel Doc. In contrast to the Gel Doc LB medium showed minimal fluorescence in our device ''WatsOn'' and no difficulties in cultivation of our ''Cellocks'' were observed. Because of the reduced fluorescence compared to TB medium when using ''Watson'' for sensor chip evaluation and because of sufficient cultivation conditions for our 'Cellocks'' LB medium was chosen over TB mediium for sensor chip manufacturing. --><br />
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{{Team:Aachen/FigureFloat|Aachen_2_chipform.jpg|title=Sensor chip manufacturing using the closed mold|subtitle=When injecting the liquid agar into a closed mold we encounter problems due to frequent bubble formation.|left|width=500px}}<br />
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=== Optimal Agarose Concentration for Sensor Chip Manufacturing ===<br />
For the sensor chip manufacturing, agarose was preferred over agar because of the uniform linkage between molecules that results in a better chip homogeneity. In addition, agarose reduced diffusion of the inducer molecules through the chip. A reduction in diffusion is essential for the formation of distinct fluorescent spots on the sensor chips.<br />
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{{Team:Aachen/FigureFloat|Aachen_Final_chipform.jpg|title=The finalized chip mold|subtitle=An open casting mold was found to be optimal for sensor chip manufacturing, because this approach was fast, easy to handle and generated a reproducible chip quality.|left|width=500px}}<br />
=== Optimal Chip Configuration ===<br />
Several approaches were tested for the production of agarose-based sensor chips with reproducible quality. The first approach was to cast every sensor chip individually. To achieve a plain chip surface, a requirement for high quality images, we casted the sensor chips between two microscope slides. However, this approach was not adequate because the agar was too liquid and leaked from the microscope slides. In a second approach, we designed a closed mold into which liquid agar is injected using a pipette, but we encountered a high number of bubbles in the resulting chips. Bubbles in the sensor chips interfered with fluorescence evaluation. Finally, we tried an open casting mold. Once solidified, we cut the agar along precast indentations in the casting mold to form the chips. An advantage of the open mold is the ability to simultaneously produce nine sensor chips while the surface tension of the liquid agar ensures a plane chip surface.<br />
=== Induction of the Sensor Chips ===<br />
To test our molecular constructs, we simulated the presence of ''P.&nbsp;aeruginosa'' by using IPTG or 3-oxo-C<sub>12</sub>-HSL. Initial experiments showed that diffusion of the inducers hinder the formation of distinct fluorescent spots. Through this set of experiments we determined that the best compromise between diffusion and spot intensity is an induction volume of 2.0&nbsp;µL for IPTG and 0.2&nbsp;µL for HSL. Furthermore, detection of growing ''P.&nbsp;aeruginosa'' based on secreted HSLs was possible using the [http://parts.igem.org/Part:BBa_K131026 K131026] construct. The experiments for optimizing the induction of our sensor chips are described in more detail in the [https://2014.igem.org/Team:Aachen/Project/2D_Biosensor#biosensorachievements Achievements] section.<br />
=== Negative Control ===<br />
To ensure that the fluorescence signal resulted from the sensor construct and not from the medium or ''E. coli'' cells themselves, [http://parts.igem.org/Part:BBa_B0015 B0015] in NEB10β cells was used as negative control during sensor chip induction with IPTG, HSL and ''P.&nbsp;aeruginosa''.<br />
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{{Team:Aachen/Figure|Aachen_B0015_IPTG_HSL_Pseudomonas.png|title=B0015 in NEB10β was used as a negativ control|subtitle=Induction with A) 0.2&nbsp;µL of 100&nbsp;mM IPTG, image taken after 2.5&nbsp;h; B) 0.2&nbsp;µL of 500&nbsp;µg/mL 3-oxo-C<sub>12</sub>-HSL, image after 2.5&nbsp;h; C) with five spots of ''Pseudomonas&nbsp;aeruginosa'' liquid culture on the left and one big spot on the right, image taken after 2&nbsp;h.|width=900px}}<br />
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==Achievements==<br />
<span class="anchor" id="biosensorachievements"></span><br />
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We developed and optimized a 2D biosensor, which is able to detect IPTG, 3-oxo-C{{sub|12}}-HSL and living ''Pseudomonas&nbsp;aeruginosa'' cells. <br />
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{{Team:Aachen/FigureFloat|Aachen_K1319042_Platereader.gif|title=Testing K1319042 in our sensor chips|subtitle=Sensor chips based on [http://parts.igem.org/Part:BBa_K1319042 K1319042] were investigated for fluorescence using a plate reader. Blue color indicates the absence of fluorescence, while red color indicates fluorescence. The upper chip was not induced, while the lower chip was induced with IPTG (2.0&nbsp;µL, 100mM).|width=260px}}<br />
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=== Testing our Sensor Chips in a Plate Reader ===<br />
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To establish a prove-of-principle for our sensor chip design, we used our construct [http://parts.igem.org/Part:BBa_K1319042 K1319042], an IPTG inducible iLOV. ''E. coli'' cells carrying the this construct were introduced into sensor chips and fluorescence was measured every 15&nbsp;minutes after induction with 2&nbsp;µL of 100&nbsp;mM IPTG. The results are displayed on the left.<br />
We observed a distinct difference in fluorescence between the non-induced chip (top) and the induced chip (bottom). The middle of the bottom chip started to exhibit a clear and visible fluorescence that increased over time and spread outwards. The top chip, however, also showed an increase in the measured fluorescence over time which was primarily due to the leaky promoter and background fluorescence.<br />
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=== Detecting 3-oxo-C{{sub|12}}-HSL with Sensor Chips ===<br />
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{{Team:Aachen/FigureFloatRight|Aachen_K131026_Platereader.gif|title=Testing K131026 in our sensor chips|subtitle=Sensor chips based on [http://parts.igem.org/Part:BBa_K131026 K131026] were investigated for fluorescence using a plate reader. Blue color indicates the absence of fluorescence, while red color indicates fluorescence. The lower chip was induced with with 3-oxo-C{{sub|12}}-HSL (0.2&nbsp;µL, 500 µg/mL).|width=360px}}<br />
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In an initial attempt to detect 3-oxo-C{{sub|12}}-HSL, we incorporated the [http://parts.igem.org/Part:BBa_K131026 K131026] construct generated by the 2008 iGEM Team Calgary in our sensor chips. This construct generates a fluorescent signal based on GFP in presence of 3-oxo-C{{sub|12}}-HSL molecules produced by ''P.&nbsp;aeruginosa'' during quorum sensing (Jimenez, Koch, Thompson et al., 2012). First, we tested the construct by direct induction with 3-oxo-C{{sub|12}}-HSL (0.2&nbsp;µL, 500&nbsp;µg/mL). The fluorescence measurement was taken every 15&nbsp;minutes with an excitation wavelength of 496&nbsp;nm and an emission wavelength of 516&nbsp;nm. The results of this test are shown on the right.<br />
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A distinct fluorescence signal was observed on the induced chip (bottom) compared to the non-induced chip (top). <br />
Fluorescence started in the middle of the chip, the point of induction, and then extended outwards while growing in intesnity. The basal level of fluorescence was attributed to leakiness of the promoter and general background fluorescence of growing ''E. coli'' cells. In the induced chip (bottom), the background fluorescence was lower than in the non-induced chip (top) because the signal masked the noise. The difference between the induced and non-induced chips indicates a clear response to the HSL and proofed the ability of our 2D sensor chip design to detect HSLs produced by ''Pseudomonas&nbsp;aeruginosa''.<br />
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{{Team:Aachen/FigureFloat|Aachen_I746909_slower_reduced.gif|title=IPTG-inducible superfolder GFP (I746909) in sensor chips|subtitle=Expression of superfolder GFP ([http://parts.igem.org/Part:BBa_I746909 I746909]) was induced by the addition of IPTG (2&nbsp;µL,&nbsp;100mM) on the right chip. The left chip was not induced.|width=480px}}<br />
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=== Detecting IPTG with Sensor Chips ===<br />
The clip displayed on the left side shows the construct [http://parts.igem.org/Part:BBa_I746909 I746909] from the 2007 iGEM Team Cambridge which expresses super folder GFP under the control of a T7 promoter in combination with our 2D sensor chip technology. The [http://parts.igem.org/Part:BBa_I746909 I746909] construct was introduced into BL21(DE3) cells to make the expression IPTG-inducible since the genome of BL21(De3) contains the T7 RNA Polymerase under the control of a lacI promoter.<br />
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While the left chip does not show visible fluorescence, the right chip exhibits a strong fluorescence signal. This proves the ability of our sensor chip technology to detect IPTG. The fluorescence response is also high enough to be detected and analyzed by our measurement device ''WatsOn''.<br />
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{{Team:Aachen/FigureFloatRight|Aachen_K131026_HSLdetection_slow.gif|title=Detection of 3-oxo-C{{sub|12}}-HSL with K131026|subtitle=0.2&nbsp;µL of 3-oxo-C{{sub|12}}-HSL was placed in the middle of a sensor chip based on [http://parts.igem.org/Part:BBa_K131026 K131026] followed by incubation at 37°C in ''WatsOn''.|width=480px}}<br />
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===Detecting the 3-oxo-C{{sub|12}}-HSL with K131026 in our Sensor Chips using ''WatsOn''===<br />
<br />
The next step towards the final goal of detecting living cells of ''Pseudomonas&nbsp;aeruginosa'' was to reproduce the detection of 3-oxo-C{{sub|12}}-HSL, already established in the plate reader, in our own [https://2014.igem.org/Team:Aachen/Project/Measurement_Device ''WatsOn''] device. Therefore, we again used ''E. coli'' BL21(DE3) cells carrying [http://parts.igem.org/Part:BBa_K131026 K131026] and induced with 0.2&nbsp;µL 3-oxo-C{{sub|12}}-HSL of a concentration of 500&nbsp;µg/mL. In the clip displayed on the left, the right chip was induced and - as a negative control - the left chip was not induced. Pictures were taken every four&nbsp;minutes.<br />
<br />
The result was a clear replication of the success of the plate reader experiment. The induced chip showed a clear fluorescence response eminating from the center, where the induction with HSL took place. This demonstrated the ability of not only our sensor chip technology but also our measurement device ''WatsOn to successfully'' detect 3-oxo-C{{sub|12}}-HSL.<br />
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{{Team:Aachen/FigureFloat|Aachen_K131026_Pseudomonas_aeruginosa_detection.gif|title=Detection of ''Pseudomonas&nbsp;aeruginosa'' with K131026|subtitle=Direct detection of ''Pseudomonas&nbsp;aeruginosa'' on sensor chips based on [http://parts.igem.org/Part:BBa_K131026 K131026].|width=480px}}<br />
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===Detecting ''Pseudomonas&nbsp;aeruginosa'' with K131026 in our Sensor Chip with ''WatsOn''===<br />
<br />
After establishing the successful detection of 3-oxo-C{{sub|12}}-HSLs with our sensor chips the next step was to detect living cells of ''Pseudomonas aeruginosa'' with our measurement device ''WatsOn''. Therefore sensor chips based on [http://parts.igem.org/Part:BBa_K131026 K131026] were prepared and the right chip was induced with 0.2&nbsp;µL of a ''Pseudomonas aeruginosa'' culture while the left chip was not induced (Detection of 3-oxo-C12 HSL with K131026, displayed below). On the induced chip, a clear fluorescence signal was visible in response to ''P. aeruginosa''. The fluorescence signal emerged outward from the induction point and showed a significant difference to the non-induced chip. The results clearly demonstrate the ability of our sensor chip technology and our measurement device ''WatsOn'' to detect ''P. aeruginosa''!<br />
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== Outlook ==<br />
<span class="anchor" id="biosensoroutlook"></span><br />
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We are '''committed to improve''' our sensor chip platform. The current technique of using a simple agarose chip is not sufficient to collect all microorganisms from the sampled surface. Therefore, the aim is to improve the sampling chip by using a different, more adhesive material. <br />
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Furthermore, diffusion in the sensor chips will be reduced to '''limit the spread of the fluorescence signal'''. Currently, the fluorescence spot grows beyond the point of induction and makes it difficult to differentitate between multiple points of induction. By introducing diffusion barriers into our chips, the growth of the fluorescence spots might be reduced, thus enabling the detection of multiple sources of fluorescence lying close together. <br />
<br />
Additionally, the application of our sensor chips in combination with our ''WatsOn'' device is currently being evaluated for the detection of signals other than fluorescence. '''Detecting bio- and chemiluminescence''' has been identified and will be investigated as an area of potential future application. <br />
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==References==<br />
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* Waters, C. M., & Bassler, B. L. (2005). QUORUM SENSING: Cell-to-Cell Communication In Bacteria. Annual Review of Cell and Developmental Biology, 21(1), 319-346. Available online at http://www.annualreviews.org/doi/full/10.1146/annurev.cellbio.21.012704.131001.<br />
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* Jimenez, P. N., Koch, G., Thompson, J. A., Xavier, K. B., Cool, R. H., & Quax, W. J. (2012). The Multiple Signaling Systems Regulating Virulence in ''Pseudomonas aeruginosa''. Microbiology and Molecular Biology Reviews, 76(1), 46-65. Available online at http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3294424/#B63.<br />
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{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/Project/2D_BiosensorTeam:Aachen/Project/2D Biosensor2014-10-18T03:19:06Z<p>Nbailly: /* Achievements */</p>
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= 2D Biosensor =<br />
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With our 2D biosensor technology we are able to detect the pathogen ''Pseudomonas aeruginosa'' on solid surfaces. The sensor system is comprised of '''two distinct but inseparable modules''', a biological and a technical part:<br />
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* Sensor chips containing '''''Cellocks''''', our '''engineered detective cells''' that fluoresce in the presence of the pathogen, make up the biological part of ''Cellock Holmes''.<br />
* Our '''measurement device [https://2014.igem.org/Team:Aachen/Project/Measurement_Device ''WatsOn'']''' and the complementary '''software [https://2014.igem.org/Team:Aachen/Notebook/Software/Measurarty ''Measurarty'']''' complete our sensing technology on the technical side. <br />
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== Principle of Operation ==<br />
<span class="anchor" id="biosensorpoo"></span><br />
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''Cellock Holmes'' is designed upon a SynBio approach comprising a '''two-dimensional biosensor and a measurement unit'''. The two-dimensional biosensor is devised to recognize quorum sensing molecules secreted by the pathogen cells and to generate a distinct fluorescence signal; while the measurement device recognizes and analyzes the produced signal. On the molecular side, we use the '''[https://2014.igem.org/Team:Aachen/Project/FRET_Reporter REACh construct]''' to transform regular ''E. coli'' cells into ''Cellocks''.<br />
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{{Team:Aachen/Figure|Aachen 17-10-14 The basics of quorum sensing ipo.png||title=The principle of quorum sensing|subtitle=Microorganisms can sense the presence of their own kind based on quorum sensing which is a form of chemical communication. Depending on their cell density, quorum sensing allows these cells to activate or deactivate certain gene expression cascades (Waters and Bassler, 2005) for a specific function.|width=900px}}<br />
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Our '''sensor cells, ''Cellocks'', are immobilized in agar chips'''. To make the chips, we mix the ''Cellocks'' with liquid LB agar. <br />
In the course of our project, we designed a casting mold specifically for the production of our agar chips. When the agar has cooled down, the chips are cut out of the mold and are ready to use. Storage of the readily usable sensor chips is possible for up to two days at 4°C when using LB medium or up to five days if TB medium is used. A detailed description of the sensor chip manufacturing can be found in our [https://2014.igem.org/Team:Aachen/Notebook/Protocols/detection Protocols] section.<br />
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{{Team:Aachen/Figure|Aachen 14-10-14 Flowsheet OD-device part1 ipo.png|title=Assay to detect ''P.&nbsp;aeruginosa'' using ''Cellock Holmes''|subtitle=This flow sheet shows the procedure to sample and detect ''P.&nbsp;aeruginosa'': A sampling chip is briefly put onto the potentially contaminated surface, added onto one of our sensor chips and inserted into ''WatsOn''.|width=900px}}<br />
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Using ''Cellock Holmes'', we developed a simple assay to detect ''P.&nbsp;aeruginosa''. Initially, a so called sampling chip is placed on a solid surface that is potentially contaminated with the pathogen. Subsequently, the sampling chip is removed from the surface and put onto one of our sensor chips. Theorectically, the sensor chips could be directly used for sampling, however, this was avoided in our project to '''match [https://2014.igem.org/Team:Aachen/Safety biosafety regulations]''' and to prevent the spread of genetically modified organisms (GMOs) into the environment. The two layered chip-stack is then put into a petri dish which is inserted into our measurement device [https://2014.igem.org/Team:Aachen/Project/Measurement_Device ''WatsOn''] for evalutation.<br />
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{{Team:Aachen/Figure|Aachen 14-10-14 Flowsheet OD-device part2 ipo.png|title=Mode of action inside ''WatsOn''|subtitle=Chips are incubated at 37°C to stimulate cell growth and then illuminated with blue light to excite fluorescence. A picture is taken and analyzed for fluorescence signals using the software ''Measurarty''.|width=900px}}<br />
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Inside ''WatsOn'', the chips are incubated at 37°C and the sampled populations of microorganisms attached on the sampling chip start to grow and multiply. During incubation the chips can be '''illuminated with blue light''' at any time, and a '''photo of the chips''' is taken. The '''software ''Measurarty''''' then analyzes any fluorescent signal. ''P.&nbsp;aeruginosa'' secrets an increasing number of quorum sensing molecules that are recognized by ''Cellocks'', thereby producing a fluorescence signal. For detection of ''P.&nbsp;aeruginosa'', we focused on a quorum sensing molecule called N-3-oxo-dodecanoyl-L-homoserine lactone (for short: 3-oxo-C<sub>12</sub>-HSL), which is involved in virulence regulation of ''P.&nbsp;aeruginosa'' (Jimenez, Koch, Thompson et al., 2012). The incorporation of the 3-oxo-C<sub>12</sub>-HSL detection system into the ''Cellocks'' is explained in detail in the [https://2014.igem.org/Team:Aachen/Project/FRET_Reporter REACh Construct] section.<br />
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<!-- ==A Novel Molecular Approach==<br />
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For our biosensor, our team genetically modified ''E. coli'' cells to be able to elecit a '''fluorescent response to autoinducers''' produced by the pathogen ''Pseudomonas aeruginosa'' during quorum sensing. In the case of ''P.&nbsp;aeruginosa'', these autoinducers are N-3-oxo-dodecanoyl-L-homoserine lactone, or 3-oxo-C-12-HSL for short. The quorum sensing system of this pathogen contains the '''LasR activator''' which binds 3-oxo-C-12-HSL, and the '''LasI promoter''', which is activated by the LasR-HSL complex. Both LasR activator and LasI promoter are available as BioBricks [http://parts.igem.org/Part:BBa_C0179 C0179] and [http://parts.igem.org/Part:BBa_J64010 J64010].<br />
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As a reporter gene, we use '''GFP'''. However, expression of GFP is not simply controlled through the LasI promoter activity in our approach. Instead, our sensor cells contain genes for a constitutively expressed fusion protein consisting of GFP and a dark quencher, and an '''HSL-inducible protease'''. We use the REACh protein as dark quencher for GFP and the TEV protease to cleave the complex; [https://2014.igem.org/Team:Aachen/Project/FRET_Reporter here] you can read more about the REACh construct and the TEV protease. <br />
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{{Team:Aachen/Figure|align=center|Aachen_REACh_approach.png|title=Our novel biosensor approach|subtitle=Expression of the TEV protease is induced by HSL. The protease cleaves the GFP-REACh fusion protein to elecit a fluorescence response.|width=500px}}<br />
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When ''P.&nbsp;aeruginosa cells'' are stuck on our agar chip and come close to our sensor cells, the latter take up the HSL molecules secreted by the pathogens. Inside the sensor cells, the autoinducer binds to the LasR gene product and activate the expression of the TEV protease. The protease then cleaves the GFP-REACh construct. When '''illuminated with light of 480&nbsp;nm''', the excitation wavelenght of GFP, our sensor cells in the vicinity of ''P.&nbsp;aeruginosa'' give a '''fluorescence signal'''. On the other hand, sensor cells that were not anywhere close to the pathogens do not express the protease. Therefore, the GFP will still be attached to the dark quencher in these cells, and no fluorescence is produced.<br />
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== Development & Optimization ==<br />
<span class="anchor" id="biosensordevelopment"></span><br />
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{{Team:Aachen/FigureFloat|Aachen_ILOV_GFP_HM_1,5h.png|title=iLOV and GFP in the Gel Doc<sup>TM</sup>|subtitle=Sensor cells producing iLOV (A) and GFP (B) 1.5&nbsp;h after induction.|left|width=500px}}<br />
=== Equipment and medium selection ===<br />
Our first approach (before developing our own device) was to use the Molecular Imager&reg; Gel Doc™ XR+ from BIO-RAD in our lab to detect fluorescence. This device uses UV and white light illuminators. However, only two different filters were available for the excitation light wavelength, which resulted in very limited possibilities for the excitation of fluorescent molecules. For example, it was possible to detect the expression of iLOV in our sensor chips, but not the expression of GFP. Hence, the '''Gel Doc™ was not suitable for our project'''.<br />
{{Team:Aachen/FigureFloat|Aachen_Chip_medium_geldoc.png|title=Differend medium in the Gel Doc™|subtitle=complex media exhibited high background fluorescence while less back- ground fluorescence was observed with the minimal media (HM, M9, NA).|right|width=500px}}<br />
{{Team:Aachen/FigureFloat|Aachen_5days_K131026_neb_tb_1,5h.jpg |title=Testing our chips' shelf-life|subtitle= Chips of [http://parts.igem.org/Part:BBa_K131026 K131026] in NEB were stored five days at 4°C. The right chip was induced with 0.2&nbsp;µL of 500&nbsp;µg/mL HSL and an image was taken after 1.5&nbsp;h.|left|width=500px}}<br />
We tested different media (LB, TB, M9, NA and HM) for the preparation of our sensor chips. The medium compositions can be found in the [https://2014.igem.org/Team:Aachen/Notebook/Protocols Protocols] section. We screened for an optimized medium composition to minimize background fluorescence and to enhance cell growth. The results of the analysis are presented in the table below. Due to the low background fluorescence in ''WatsOn'' and the excellent cell growth, we '''chose LB&nbsp;medium''' over the other tested media for sensor chip manufacturing.<br />
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<center><br />
{| class="wikitable"<br />
! !! LB !! TB !! NA !! M9 !! HM <br />
|-<br />
| Growth of ''Cellock'' || <div style="text-align: center;">'''+'''</div> || <div style="text-align: center;">'''+'''</div> || <div style="text-align: center;">-</div> || <div style="text-align: center;">-</div> || <div style="text-align: center;">-</div><br />
|-<br />
| Background fluorescence in GelDoc || <div style="text-align: center;">'''+'''</div> || <div style="text-align: center;">'''+'''</div> || <div style="text-align: center;">-</div> || <div style="text-align: center;">-</div> || <div style="text-align: center;">-</div><br />
|-<br />
| Background fluorescence in ''WatsOn'' || <div style="text-align: center;">-</div> || <div style="text-align: center;">'''+'''</div> || <div style="text-align: center;">-</div> || <div style="text-align: center;">-</div> || <div style="text-align: center;">-</div><br />
|}<br />
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Another set of experiments were conducted to test the '''long-time storage''' of the sensor chips. We varied the glycerol content of the chips as well as the storage temperature. Storage at -20°C resulted in the loss of our sensor cells. Adding 5-10% (v/v) glycerol ensured survival of the sensor cells, but resulted in the loss of fluorescence ability. Hence, we concluded that long-time storage of the sensor chips at -20°C is not possible under the tested conditions. However, the 'ready-to-use' sensor chips can be kept at at 4°C for two days when using LB medium, and storage at this temperature for 5 days is possible with chips made from TB medium.<br />
<!--Regarding the medium used for our sensor chips, LB medium showed a high background fluorescence when exposed to UV light in the Gel Doc. Surprisingly, the background fluorescence resulting from the LB medium was too high to detect a signal emitted by our sensor cells. Hence, minimal media (NA, M9, Hartman (HM)) was used to minimize background fluorescence, but this approach resulted in less to no growth of our sensor cells. In our device ''WatsOn'', optimized wavelengths of 450&nbsp;nm and 480&nbsp;nm were used for excitation of iLOV and GFP, respectively. When exposed to either excitation wavelength TB medium, which is basically an improved LB medium and highly supports cell growth, showed strong background fluorescence in our own device. High background fluorescence was also observed for TB medum when using the Gel Doc. In contrast to the Gel Doc LB medium showed minimal fluorescence in our device ''WatsOn'' and no difficulties in cultivation of our ''Cellocks'' were observed. Because of the reduced fluorescence compared to TB medium when using ''Watson'' for sensor chip evaluation and because of sufficient cultivation conditions for our 'Cellocks'' LB medium was chosen over TB mediium for sensor chip manufacturing. --><br />
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{{Team:Aachen/FigureFloat|Aachen_2_chipform.jpg|title=Sensor chip manufacturing using the closed mold|subtitle=When injecting the liquid agar into a closed mold we encounter problems due to frequent bubble formation.|left|width=500px}}<br />
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=== Optimal Agarose Concentration for Sensor Chip Manufacturing ===<br />
For the sensor chip manufacturing, agarose was preferred over agar because of the uniform linkage between molecules that results in a better chip homogeneity. In addition, agarose reduced diffusion of the inducer molecules through the chip. A reduction in diffusion is essential for the formation of distinct fluorescent spots on the sensor chips.<br />
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{{Team:Aachen/FigureFloat|Aachen_Final_chipform.jpg|title=The finalized chip mold|subtitle=An open casting mold was found to be optimal for sensor chip manufacturing, because this approach was fast, easy to handle and generated a reproducible chip quality.|left|width=500px}}<br />
=== Optimal Chip Configuration ===<br />
Several approaches were tested for the production of agarose-based sensor chips with reproducible quality. The first approach was to cast every sensor chip individually. To achieve a plain chip surface, a requirement for high quality images, we casted the sensor chips between two microscope slides. However, this approach was not adequate because the agar was too liquid and leaked from the microscope slides. In a second approach, we designed a closed mold into which liquid agar is injected using a pipette, but we encountered a high number of bubbles in the resulting chips. Bubbles in the sensor chips interfered with fluorescence evaluation. Finally, we tried an open casting mold. Once solidified, we cut the agar along precast indentations in the casting mold to form the chips. An advantage of the open mold is the ability to simultaneously produce nine sensor chips while the surface tension of the liquid agar ensures a plane chip surface.<br />
=== Induction of the Sensor Chips ===<br />
To test our molecular constructs, we simulated the presence of ''P.&nbsp;aeruginosa'' by using IPTG or 3-oxo-C<sub>12</sub>-HSL. Initial experiments showed that diffusion of the inducers hinder the formation of distinct fluorescent spots. Through this set of experiments we determined that the best compromise between diffusion and spot intensity is an induction volume of 2.0&nbsp;µL for IPTG and 0.2&nbsp;µL for HSL. Furthermore, detection of growing ''P.&nbsp;aeruginosa'' based on secreted HSLs was possible using the [http://parts.igem.org/Part:BBa_K131026 K131026] construct. The experiments for optimizing the induction of our sensor chips are described in more detail in the [https://2014.igem.org/Team:Aachen/Project/2D_Biosensor#biosensorachievements Achievements] section.<br />
=== Negative Control ===<br />
To ensure that the fluorescence signal resulted from the sensor construct and not from the medium or ''E. coli'' cells themselves, [http://parts.igem.org/Part:BBa_B0015 B0015] in NEB10β cells was used as negative control during sensor chip induction with IPTG, HSL and ''P.&nbsp;aeruginosa''.<br />
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{{Team:Aachen/Figure|Aachen_B0015_IPTG_HSL_Pseudomonas.png|title=B0015 in NEB10β was used as a negativ control|subtitle=Induction with A) 0.2&nbsp;µL of 100&nbsp;mM IPTG, image taken after 2.5&nbsp;h; B) 0.2&nbsp;µL of 500&nbsp;µg/mL 3-oxo-C<sub>12</sub>-HSL, image after 2.5&nbsp;h; C) with five spots of ''Pseudomonas&nbsp;aeruginosa'' liquid culture on the left and one big spot on the right, image taken after 2&nbsp;h.|width=900px}}<br />
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==Achievements==<br />
<span class="anchor" id="biosensorachievements"></span><br />
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We developed and optimized a 2D biosensor, which is able to detect IPTG, 3-oxo-C{{sub|12}}-HSL and living ''Pseudomonas&nbsp;aeruginosa'' cells. <br />
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{{Team:Aachen/FigureFloat|Aachen_K1319042_Platereader.gif|title=Testing K1319042 in our sensor chips|subtitle=Sensor chips based on [http://parts.igem.org/Part:BBa_K1319042 K1319042] were investigated for fluorescence using a plate reader. Blue color indicates the absence of fluorescence, while red color indicates fluorescence. The upper chip was not induced, while the lower chip was induced with IPTG (2.0&nbsp;µL, 100mM).|width=260px}}<br />
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=== Testing our Sensor Chips in a Plate Reader ===<br />
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To establish a prove-of-principle for our sensor chip design, we used our construct [http://parts.igem.org/Part:BBa_K1319042 K1319042], an IPTG inducible iLOV. ''E. coli'' cells carrying the this construct were introduced into sensor chips and fluorescence was measured every 15&nbsp;minutes after induction with 2&nbsp;µL of 100&nbsp;mM IPTG. The results are displayed on the left.<br />
We observed a distinct difference in fluorescence between the non-induced chip (top) and the induced chip (bottom). The middle of the bottom chip started to exhibit a clear and visible fluorescence that increased over time and spread outwards. The top chip, however, also showed an increase in the measured fluorescence over time which was primarily due to the leaky promoter and background fluorescence.<br />
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=== Detecting 3-oxo-C{{sub|12}}-HSL with Sensor Chips ===<br />
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{{Team:Aachen/FigureFloatRight|Aachen_K131026_Platereader.gif|title=Testing K131026 in our sensor chips|subtitle=Sensor chips based on [http://parts.igem.org/Part:BBa_K131026 K131026] were investigated for fluorescence using a plate reader. Blue color indicates the absence of fluorescence, while red color indicates fluorescence. The lower chip was induced with with 3-oxo-C{{sub|12}}-HSL (0.2&nbsp;µL, 500 µg/mL).|width=360px}}<br />
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In an initial attempt to detect 3-oxo-C{{sub|12}}-HSL, we incorporated the [http://parts.igem.org/Part:BBa_K131026 K131026] construct generated by the 2008 iGEM Team Calgary in our sensor chips. This construct generates a fluorescent signal based on GFP in presence of 3-oxo-C{{sub|12}}-HSL molecules produced by ''P.&nbsp;aeruginosa'' during quorum sensing (Jimenez, Koch, Thompson et al., 2012). First, we tested the construct by direct induction with 3-oxo-C{{sub|12}}-HSL (0.2&nbsp;µL, 500&nbsp;µg/mL). The fluorescence measurement was taken every 15&nbsp;minutes with an excitation wavelength of 496&nbsp;nm and an emission wavelength of 516&nbsp;nm. The results of this test are shown on the right.<br />
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A distinct fluorescence signal was observed on the induced chip (bottom) compared to the non-induced chip (top). <br />
Fluorescence started in the middle of the chip, the point of induction, and then extended outwards while growing in intesnity. The basal level of fluorescence was attributed to leakiness of the promoter and general background fluorescence of growing ''E. coli'' cells. In the induced chip (bottom), the background fluorescence was lower than in the non-induced chip (top) because the signal masked the noise. The difference between the induced and non-induced chips indicates a clear response to the HSL and proofed the ability of our 2D sensor chip design to detect HSLs produced by ''Pseudomonas&nbsp;aeruginosa''.<br />
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{{Team:Aachen/FigureFloat|Aachen_I746909_slower_reduced.gif|title=IPTG-inducible superfolder GFP (I746909) in sensor chips|subtitle=Expression of superfolder GFP ([http://parts.igem.org/Part:BBa_I746909 I746909]) was induced by the addition of IPTG (2&nbsp;µL,&nbsp;100mM) on the right chip. The left chip was not induced.|width=480px}}<br />
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=== Detecting IPTG with Sensor Chips ===<br />
The clip displayed on the left side shows the construct [http://parts.igem.org/Part:BBa_I746909 I746909] from the 2007 iGEM Team Cambridge which expresses super folder GFP under the control of a T7 promoter in combination with our 2D sensor chip technology. The [http://parts.igem.org/Part:BBa_I746909 I746909] construct was introduced into BL21(DE3) cells to make the expression IPTG-inducible since the genome of BL21(De3) contains the T7 RNA Polymerase under the control of a lacI promoter.<br />
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While the left chip does not show visible fluorescence, the right chip exhibits a strong fluorescence signal. This proves the ability of our sensor chip technology to detect IPTG. The fluorescence response is also high enough to be detected and analyzed by our measurement device ''WatsOn''.<br />
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{{Team:Aachen/FigureFloatRight|Aachen_K131026_HSLdetection_slow.gif|title=Detection of 3-oxo-C{{sub|12}}-HSL with K131026|subtitle=0.2&nbsp;µL of 3-oxo-C{{sub|12}}-HSL was placed in the middle of a sensor chip based on [http://parts.igem.org/Part:BBa_K131026 K131026] followed by incubation at 37°C in ''WatsOn''.|width=480px}}<br />
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===Detecting the 3-oxo-C{{sub|12}}-HSL with K131026 in our Sensor Chips using ''WatsOn''===<br />
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The next step towards the final goal of detecting living cells of ''Pseudomonas&nbsp;aeruginosa'' was to reproduce the detection of 3-oxo-C{{sub|12}}-HSL, already established in the plate reader, in our own [https://2014.igem.org/Team:Aachen/Project/Measurement_Device ''WatsOn''] device. Therefore, we again used ''E. coli'' BL21(DE3) cells carrying [http://parts.igem.org/Part:BBa_K131026 K131026] and induced with 0.2&nbsp;µL 3-oxo-C{{sub|12}}-HSL of a concentration of 500&nbsp;µg/mL. In the clip displayed on the left, the right chip was induced and - as a negative control - the left chip was not induced. Pictures were taken every four&nbsp;minutes.<br />
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The result was a clear replication of the success of the plate reader experiment. The induced chip showed a clear fluorescence response eminating from the center, where the induction with HSL took place. This demonstrated the ability of not only our sensor chip technology but also our measurement device ''WatsOn to successfully'' detect 3-oxo-C{{sub|12}}-HSL.<br />
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{{Team:Aachen/FigureFloat|Aachen_K131026_Pseudomonas_aeruginosa_detection.gif|title=Detection of ''Pseudomonas&nbsp;aeruginosa'' with K131026|subtitle=Direct detection of ''Pseudomonas&nbsp;aeruginosa'' on sensor chips based on [http://parts.igem.org/Part:BBa_K131026 K131026].|width=480px}}<br />
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===Detecting ''Pseudomonas&nbsp;aeruginosa'' with K131026 in our Sensor Chip with ''WatsOn''===<br />
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After establishing the successful detection of 3-oxo-C{{sub|12}}-HSLs with our sensor chips the next step was to detect living cells of ''Pseudomonas aeruginosa'' with our measurement device ''WatsOn''. Therefore sensor chips based on [http://parts.igem.org/Part:BBa_K131026 K131026] were prepared and the right chip was induced with 0.2&nbsp;µL of a ''Pseudomonas aeruginosa'' culture while the left chip was not induced (Detection of 3-oxo-C12 HSL with K131026, displayed below). On the induced chip, a clear fluorescence signal was visible in response to ''P. aeruginosa''. The fluorescence signal emerged outward from the induction point and showed a significant difference to the non-induced chip. The results clearly demonstrate the ability of our sensor chip technology and our measurement device ''WatsOn'' to detect ''P. aeruginosa''!<br />
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== Outlook ==<br />
<span class="anchor" id="biosensoroutlook"></span><br />
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We are '''committed to improve''' our sensor chip platform. The current technique of using a simple agarose chip is not sufficient to collect all microorganisms from the sampled surface. Therefore, the aim is to improve the sampling chip by using a different, more adhesive material. <br />
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Furthermore, diffusion in the sensor chips will be reduced to '''limit the spread of the fluorescence signal'''. Currently, the fluorescence spot grows beyond the point of induction and makes it difficult to differentitate between multiple points of induction. By introducing diffusion barriers into our chips, the growth of the fluorescence spots might be reduced, thus enabling the detection of multiple sources of fluorescence lying close together. <br />
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Additionally, the application of our sensor chips in combination with our ''WatsOn'' device is currently being evaluated for the detection of signals other than fluorescence. '''Detecting bio- and chemiluminescence''' has been identified and will be investigated as an area of potential future application. <br />
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==References==<br />
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* Waters, C. M., & Bassler, B. L. (2005). QUORUM SENSING: Cell-to-Cell Communication In Bacteria. Annual Review of Cell and Developmental Biology, 21(1), 319-346. Available online at http://www.annualreviews.org/doi/full/10.1146/annurev.cellbio.21.012704.131001.<br />
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* Jimenez, P. N., Koch, G., Thompson, J. A., Xavier, K. B., Cool, R. H., & Quax, W. J. (2012). The Multiple Signaling Systems Regulating Virulence in ''Pseudomonas aeruginosa''. Microbiology and Molecular Biology Reviews, 76(1), 46-65. Available online at http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3294424/#B63.<br />
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{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/Project/2D_BiosensorTeam:Aachen/Project/2D Biosensor2014-10-18T03:18:50Z<p>Nbailly: /* Achievements */</p>
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= 2D Biosensor =<br />
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With our 2D biosensor technology we are able to detect the pathogen ''Pseudomonas aeruginosa'' on solid surfaces. The sensor system is comprised of '''two distinct but inseparable modules''', a biological and a technical part:<br />
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* Sensor chips containing '''''Cellocks''''', our '''engineered detective cells''' that fluoresce in the presence of the pathogen, make up the biological part of ''Cellock Holmes''.<br />
* Our '''measurement device [https://2014.igem.org/Team:Aachen/Project/Measurement_Device ''WatsOn'']''' and the complementary '''software [https://2014.igem.org/Team:Aachen/Notebook/Software/Measurarty ''Measurarty'']''' complete our sensing technology on the technical side. <br />
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== Principle of Operation ==<br />
<span class="anchor" id="biosensorpoo"></span><br />
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''Cellock Holmes'' is designed upon a SynBio approach comprising a '''two-dimensional biosensor and a measurement unit'''. The two-dimensional biosensor is devised to recognize quorum sensing molecules secreted by the pathogen cells and to generate a distinct fluorescence signal; while the measurement device recognizes and analyzes the produced signal. On the molecular side, we use the '''[https://2014.igem.org/Team:Aachen/Project/FRET_Reporter REACh construct]''' to transform regular ''E. coli'' cells into ''Cellocks''.<br />
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{{Team:Aachen/Figure|Aachen 17-10-14 The basics of quorum sensing ipo.png||title=The principle of quorum sensing|subtitle=Microorganisms can sense the presence of their own kind based on quorum sensing which is a form of chemical communication. Depending on their cell density, quorum sensing allows these cells to activate or deactivate certain gene expression cascades (Waters and Bassler, 2005) for a specific function.|width=900px}}<br />
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Our '''sensor cells, ''Cellocks'', are immobilized in agar chips'''. To make the chips, we mix the ''Cellocks'' with liquid LB agar. <br />
In the course of our project, we designed a casting mold specifically for the production of our agar chips. When the agar has cooled down, the chips are cut out of the mold and are ready to use. Storage of the readily usable sensor chips is possible for up to two days at 4°C when using LB medium or up to five days if TB medium is used. A detailed description of the sensor chip manufacturing can be found in our [https://2014.igem.org/Team:Aachen/Notebook/Protocols/detection Protocols] section.<br />
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{{Team:Aachen/Figure|Aachen 14-10-14 Flowsheet OD-device part1 ipo.png|title=Assay to detect ''P.&nbsp;aeruginosa'' using ''Cellock Holmes''|subtitle=This flow sheet shows the procedure to sample and detect ''P.&nbsp;aeruginosa'': A sampling chip is briefly put onto the potentially contaminated surface, added onto one of our sensor chips and inserted into ''WatsOn''.|width=900px}}<br />
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Using ''Cellock Holmes'', we developed a simple assay to detect ''P.&nbsp;aeruginosa''. Initially, a so called sampling chip is placed on a solid surface that is potentially contaminated with the pathogen. Subsequently, the sampling chip is removed from the surface and put onto one of our sensor chips. Theorectically, the sensor chips could be directly used for sampling, however, this was avoided in our project to '''match [https://2014.igem.org/Team:Aachen/Safety biosafety regulations]''' and to prevent the spread of genetically modified organisms (GMOs) into the environment. The two layered chip-stack is then put into a petri dish which is inserted into our measurement device [https://2014.igem.org/Team:Aachen/Project/Measurement_Device ''WatsOn''] for evalutation.<br />
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{{Team:Aachen/Figure|Aachen 14-10-14 Flowsheet OD-device part2 ipo.png|title=Mode of action inside ''WatsOn''|subtitle=Chips are incubated at 37°C to stimulate cell growth and then illuminated with blue light to excite fluorescence. A picture is taken and analyzed for fluorescence signals using the software ''Measurarty''.|width=900px}}<br />
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Inside ''WatsOn'', the chips are incubated at 37°C and the sampled populations of microorganisms attached on the sampling chip start to grow and multiply. During incubation the chips can be '''illuminated with blue light''' at any time, and a '''photo of the chips''' is taken. The '''software ''Measurarty''''' then analyzes any fluorescent signal. ''P.&nbsp;aeruginosa'' secrets an increasing number of quorum sensing molecules that are recognized by ''Cellocks'', thereby producing a fluorescence signal. For detection of ''P.&nbsp;aeruginosa'', we focused on a quorum sensing molecule called N-3-oxo-dodecanoyl-L-homoserine lactone (for short: 3-oxo-C<sub>12</sub>-HSL), which is involved in virulence regulation of ''P.&nbsp;aeruginosa'' (Jimenez, Koch, Thompson et al., 2012). The incorporation of the 3-oxo-C<sub>12</sub>-HSL detection system into the ''Cellocks'' is explained in detail in the [https://2014.igem.org/Team:Aachen/Project/FRET_Reporter REACh Construct] section.<br />
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For our biosensor, our team genetically modified ''E. coli'' cells to be able to elecit a '''fluorescent response to autoinducers''' produced by the pathogen ''Pseudomonas aeruginosa'' during quorum sensing. In the case of ''P.&nbsp;aeruginosa'', these autoinducers are N-3-oxo-dodecanoyl-L-homoserine lactone, or 3-oxo-C-12-HSL for short. The quorum sensing system of this pathogen contains the '''LasR activator''' which binds 3-oxo-C-12-HSL, and the '''LasI promoter''', which is activated by the LasR-HSL complex. Both LasR activator and LasI promoter are available as BioBricks [http://parts.igem.org/Part:BBa_C0179 C0179] and [http://parts.igem.org/Part:BBa_J64010 J64010].<br />
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As a reporter gene, we use '''GFP'''. However, expression of GFP is not simply controlled through the LasI promoter activity in our approach. Instead, our sensor cells contain genes for a constitutively expressed fusion protein consisting of GFP and a dark quencher, and an '''HSL-inducible protease'''. We use the REACh protein as dark quencher for GFP and the TEV protease to cleave the complex; [https://2014.igem.org/Team:Aachen/Project/FRET_Reporter here] you can read more about the REACh construct and the TEV protease. <br />
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{{Team:Aachen/Figure|align=center|Aachen_REACh_approach.png|title=Our novel biosensor approach|subtitle=Expression of the TEV protease is induced by HSL. The protease cleaves the GFP-REACh fusion protein to elecit a fluorescence response.|width=500px}}<br />
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When ''P.&nbsp;aeruginosa cells'' are stuck on our agar chip and come close to our sensor cells, the latter take up the HSL molecules secreted by the pathogens. Inside the sensor cells, the autoinducer binds to the LasR gene product and activate the expression of the TEV protease. The protease then cleaves the GFP-REACh construct. When '''illuminated with light of 480&nbsp;nm''', the excitation wavelenght of GFP, our sensor cells in the vicinity of ''P.&nbsp;aeruginosa'' give a '''fluorescence signal'''. On the other hand, sensor cells that were not anywhere close to the pathogens do not express the protease. Therefore, the GFP will still be attached to the dark quencher in these cells, and no fluorescence is produced.<br />
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== Development & Optimization ==<br />
<span class="anchor" id="biosensordevelopment"></span><br />
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{{Team:Aachen/FigureFloat|Aachen_ILOV_GFP_HM_1,5h.png|title=iLOV and GFP in the Gel Doc<sup>TM</sup>|subtitle=Sensor cells producing iLOV (A) and GFP (B) 1.5&nbsp;h after induction.|left|width=500px}}<br />
=== Equipment and medium selection ===<br />
Our first approach (before developing our own device) was to use the Molecular Imager&reg; Gel Doc™ XR+ from BIO-RAD in our lab to detect fluorescence. This device uses UV and white light illuminators. However, only two different filters were available for the excitation light wavelength, which resulted in very limited possibilities for the excitation of fluorescent molecules. For example, it was possible to detect the expression of iLOV in our sensor chips, but not the expression of GFP. Hence, the '''Gel Doc™ was not suitable for our project'''.<br />
{{Team:Aachen/FigureFloat|Aachen_Chip_medium_geldoc.png|title=Differend medium in the Gel Doc™|subtitle=complex media exhibited high background fluorescence while less back- ground fluorescence was observed with the minimal media (HM, M9, NA).|right|width=500px}}<br />
{{Team:Aachen/FigureFloat|Aachen_5days_K131026_neb_tb_1,5h.jpg |title=Testing our chips' shelf-life|subtitle= Chips of [http://parts.igem.org/Part:BBa_K131026 K131026] in NEB were stored five days at 4°C. The right chip was induced with 0.2&nbsp;µL of 500&nbsp;µg/mL HSL and an image was taken after 1.5&nbsp;h.|left|width=500px}}<br />
We tested different media (LB, TB, M9, NA and HM) for the preparation of our sensor chips. The medium compositions can be found in the [https://2014.igem.org/Team:Aachen/Notebook/Protocols Protocols] section. We screened for an optimized medium composition to minimize background fluorescence and to enhance cell growth. The results of the analysis are presented in the table below. Due to the low background fluorescence in ''WatsOn'' and the excellent cell growth, we '''chose LB&nbsp;medium''' over the other tested media for sensor chip manufacturing.<br />
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{| class="wikitable"<br />
! !! LB !! TB !! NA !! M9 !! HM <br />
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| Growth of ''Cellock'' || <div style="text-align: center;">'''+'''</div> || <div style="text-align: center;">'''+'''</div> || <div style="text-align: center;">-</div> || <div style="text-align: center;">-</div> || <div style="text-align: center;">-</div><br />
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| Background fluorescence in GelDoc || <div style="text-align: center;">'''+'''</div> || <div style="text-align: center;">'''+'''</div> || <div style="text-align: center;">-</div> || <div style="text-align: center;">-</div> || <div style="text-align: center;">-</div><br />
|-<br />
| Background fluorescence in ''WatsOn'' || <div style="text-align: center;">-</div> || <div style="text-align: center;">'''+'''</div> || <div style="text-align: center;">-</div> || <div style="text-align: center;">-</div> || <div style="text-align: center;">-</div><br />
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Another set of experiments were conducted to test the '''long-time storage''' of the sensor chips. We varied the glycerol content of the chips as well as the storage temperature. Storage at -20°C resulted in the loss of our sensor cells. Adding 5-10% (v/v) glycerol ensured survival of the sensor cells, but resulted in the loss of fluorescence ability. Hence, we concluded that long-time storage of the sensor chips at -20°C is not possible under the tested conditions. However, the 'ready-to-use' sensor chips can be kept at at 4°C for two days when using LB medium, and storage at this temperature for 5 days is possible with chips made from TB medium.<br />
<!--Regarding the medium used for our sensor chips, LB medium showed a high background fluorescence when exposed to UV light in the Gel Doc. Surprisingly, the background fluorescence resulting from the LB medium was too high to detect a signal emitted by our sensor cells. Hence, minimal media (NA, M9, Hartman (HM)) was used to minimize background fluorescence, but this approach resulted in less to no growth of our sensor cells. In our device ''WatsOn'', optimized wavelengths of 450&nbsp;nm and 480&nbsp;nm were used for excitation of iLOV and GFP, respectively. When exposed to either excitation wavelength TB medium, which is basically an improved LB medium and highly supports cell growth, showed strong background fluorescence in our own device. High background fluorescence was also observed for TB medum when using the Gel Doc. In contrast to the Gel Doc LB medium showed minimal fluorescence in our device ''WatsOn'' and no difficulties in cultivation of our ''Cellocks'' were observed. Because of the reduced fluorescence compared to TB medium when using ''Watson'' for sensor chip evaluation and because of sufficient cultivation conditions for our 'Cellocks'' LB medium was chosen over TB mediium for sensor chip manufacturing. --><br />
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{{Team:Aachen/FigureFloat|Aachen_2_chipform.jpg|title=Sensor chip manufacturing using the closed mold|subtitle=When injecting the liquid agar into a closed mold we encounter problems due to frequent bubble formation.|left|width=500px}}<br />
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=== Optimal Agarose Concentration for Sensor Chip Manufacturing ===<br />
For the sensor chip manufacturing, agarose was preferred over agar because of the uniform linkage between molecules that results in a better chip homogeneity. In addition, agarose reduced diffusion of the inducer molecules through the chip. A reduction in diffusion is essential for the formation of distinct fluorescent spots on the sensor chips.<br />
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{{Team:Aachen/FigureFloat|Aachen_Final_chipform.jpg|title=The finalized chip mold|subtitle=An open casting mold was found to be optimal for sensor chip manufacturing, because this approach was fast, easy to handle and generated a reproducible chip quality.|left|width=500px}}<br />
=== Optimal Chip Configuration ===<br />
Several approaches were tested for the production of agarose-based sensor chips with reproducible quality. The first approach was to cast every sensor chip individually. To achieve a plain chip surface, a requirement for high quality images, we casted the sensor chips between two microscope slides. However, this approach was not adequate because the agar was too liquid and leaked from the microscope slides. In a second approach, we designed a closed mold into which liquid agar is injected using a pipette, but we encountered a high number of bubbles in the resulting chips. Bubbles in the sensor chips interfered with fluorescence evaluation. Finally, we tried an open casting mold. Once solidified, we cut the agar along precast indentations in the casting mold to form the chips. An advantage of the open mold is the ability to simultaneously produce nine sensor chips while the surface tension of the liquid agar ensures a plane chip surface.<br />
=== Induction of the Sensor Chips ===<br />
To test our molecular constructs, we simulated the presence of ''P.&nbsp;aeruginosa'' by using IPTG or 3-oxo-C<sub>12</sub>-HSL. Initial experiments showed that diffusion of the inducers hinder the formation of distinct fluorescent spots. Through this set of experiments we determined that the best compromise between diffusion and spot intensity is an induction volume of 2.0&nbsp;µL for IPTG and 0.2&nbsp;µL for HSL. Furthermore, detection of growing ''P.&nbsp;aeruginosa'' based on secreted HSLs was possible using the [http://parts.igem.org/Part:BBa_K131026 K131026] construct. The experiments for optimizing the induction of our sensor chips are described in more detail in the [https://2014.igem.org/Team:Aachen/Project/2D_Biosensor#biosensorachievements Achievements] section.<br />
=== Negative Control ===<br />
To ensure that the fluorescence signal resulted from the sensor construct and not from the medium or ''E. coli'' cells themselves, [http://parts.igem.org/Part:BBa_B0015 B0015] in NEB10β cells was used as negative control during sensor chip induction with IPTG, HSL and ''P.&nbsp;aeruginosa''.<br />
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{{Team:Aachen/Figure|Aachen_B0015_IPTG_HSL_Pseudomonas.png|title=B0015 in NEB10β was used as a negativ control|subtitle=Induction with A) 0.2&nbsp;µL of 100&nbsp;mM IPTG, image taken after 2.5&nbsp;h; B) 0.2&nbsp;µL of 500&nbsp;µg/mL 3-oxo-C<sub>12</sub>-HSL, image after 2.5&nbsp;h; C) with five spots of ''Pseudomonas&nbsp;aeruginosa'' liquid culture on the left and one big spot on the right, image taken after 2&nbsp;h.|width=900px}}<br />
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==Achievements==<br />
<span class="anchor" id="biosensorachievements"></span><br />
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We developed and optimized a 2D biosensor, which is able to detect IPTG, 3-oxo-C{{sub|12}}-HSL and living ''Pseudomonas&nbsp;aeruginosa'' cells. <br />
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{{Team:Aachen/FigureFloat|Aachen_K1319042_Platereader.gif|title=Testing K1319042 in our sensor chips|subtitle=Sensor chips based on [http://parts.igem.org/Part:BBa_K1319042 K1319042] were investigated for fluorescence using a plate reader. Blue color indicates the absence of fluorescence, while red color indicates fluorescence. The upper chip was not induced, while the lower chip was induced with IPTG (2.0&nbsp;µL, 100mM).|width=260px}}<br />
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=== Testing our Sensor Chips in a Plate Reader ===<br />
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To establish a prove-of-principle for our sensor chip design, we used our construct [http://parts.igem.org/Part:BBa_K1319042 K1319042], an IPTG inducible iLOV. ''E. coli'' cells carrying the this construct were introduced into sensor chips and fluorescence was measured every 15&nbsp;minutes after induction with 2&nbsp;µL of 100&nbsp;mM IPTG. The results are displayed on the left.<br />
We observed a distinct difference in fluorescence between the non-induced chip (top) and the induced chip (bottom). The middle of the bottom chip started to exhibit a clear and visible fluorescence that increased over time and spread outwards. The top chip, however, also showed an increase in the measured fluorescence over time which was primarily due to the leaky promoter and background fluorescence.<br />
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=== Detecting 3-oxo-C{{sub|12}}-HSL with Sensor Chips ===<br />
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{{Team:Aachen/FigureFloatRight|Aachen_K131026_Platereader.gif|title=Testing K131026 in our sensor chips|subtitle=Sensor chips based on [http://parts.igem.org/Part:BBa_K131026 K131026] were investigated for fluorescence using a plate reader. Blue color indicates the absence of fluorescence, while red color indicates fluorescence. The lower chip was induced with with 3-oxo-C{{sub|12}}-HSL (0.2&nbsp;µL, 500 µg/mL).|width=360px}}<br />
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In an initial attempt to detect 3-oxo-C{{sub|12}}-HSL, we incorporated the [http://parts.igem.org/Part:BBa_K131026 K131026] construct generated by the 2008 iGEM Team Calgary in our sensor chips. This construct generates a fluorescent signal based on GFP in presence of 3-oxo-C{{sub|12}}-HSL molecules produced by ''P.&nbsp;aeruginosa'' during quorum sensing (Jimenez, Koch, Thompson et al., 2012). First, we tested the construct by direct induction with 3-oxo-C{{sub|12}}-HSL (0.2&nbsp;µL, 500&nbsp;µg/mL). The fluorescence measurement was taken every 15&nbsp;minutes with an excitation wavelength of 496&nbsp;nm and an emission wavelength of 516&nbsp;nm. The results of this test are shown on the right.<br />
<br />
A distinct fluorescence signal was observed on the induced chip (bottom) compared to the non-induced chip (top). <br />
Fluorescence started in the middle of the chip, the point of induction, and then extended outwards while growing in intesnity. The basal level of fluorescence was attributed to leakiness of the promoter and general background fluorescence of growing ''E. coli'' cells. In the induced chip (bottom), the background fluorescence was lower than in the non-induced chip (top) because the signal masked the noise. The difference between the induced and non-induced chips indicates a clear response to the HSL and proofed the ability of our 2D sensor chip design to detect HSLs produced by ''Pseudomonas&nbsp;aeruginosa''.<br />
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{{Team:Aachen/FigureFloat|Aachen_I746909_slower_reduced.gif|title=IPTG-inducible superfolder GFP (I746909) in sensor chips|subtitle=Expression of superfolder GFP ([http://parts.igem.org/Part:BBa_I746909 I746909]) was induced by the addition of IPTG (2&nbsp;µL,&nbsp;100mM) on the right chip. The left chip was not induced.|width=480px}}<br />
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=== Detecting IPTG with Sensor Chips ===<br />
The clip displayed on the left side shows the construct [http://parts.igem.org/Part:BBa_I746909 I746909] from the 2007 iGEM Team Cambridge which expresses super folder GFP under the control of a T7 promoter in combination with our 2D sensor chip technology. The [http://parts.igem.org/Part:BBa_I746909 I746909] construct was introduced into BL21(DE3) cells to make the expression IPTG-inducible since the genome of BL21(De3) contains the T7 RNA Polymerase under the control of a lacI promoter.<br />
<br />
While the left chip does not show visible fluorescence, the right chip exhibits a strong fluorescence signal. This proves the ability of our sensor chip technology to detect IPTG. The fluorescence response is also high enough to be detected and analyzed by our measurement device ''WatsOn''.<br />
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{{Team:Aachen/FigureFloatRight|Aachen_K131026_HSLdetection_slow.gif|title=Detection of 3-oxo-C{{sub|12}}-HSL with K131026|subtitle=0.2&nbsp;µL of 3-oxo-C{{sub|12}}-HSL was placed in the middle of a sensor chip based on [http://parts.igem.org/Part:BBa_K131026 K131026] followed by incubation at 37°C in ''WatsOn''.|width=480px}}<br />
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===Detecting the 3-oxo-C{{sub|12}}-HSL with K131026 in our Sensor Chips using ''WatsOn''===<br />
<br />
The next step towards the final goal of detecting living cells of ''Pseudomonas&nbsp;aeruginosa'' was to reproduce the detection of 3-oxo-C{{sub|12}}-HSL, already established in the plate reader, in our own [https://2014.igem.org/Team:Aachen/Project/Measurement_Device ''WatsOn''] device. Therefore, we again used ''E. coli'' BL21(DE3) cells carrying [http://parts.igem.org/Part:BBa_K131026 K131026] and induced with 0.2&nbsp;µL 3-oxo-C{{sub|12}}-HSL of a concentration of 500&nbsp;µg/mL. In the clip displayed on the left, the right chip was induced and - as a negative control - the left chip was not induced. Pictures were taken every four&nbsp;minutes.<br />
<br />
The result was a clear replication of the success of the plate reader experiment. The induced chip showed a clear fluorescence response eminating from the center, where the induction with HSL took place. This demonstrated the ability of not only our sensor chip technology but also our measurement device ''WatsOn to successfully'' detect 3-oxo-C{{sub|12}}-HSL.<br />
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{{Team:Aachen/FigureFloat|Aachen_K131026_Pseudomonas_aeruginosa_detection.gif|title=Detection of ''Pseudomonas&nbsp;aeruginosa'' with K131026|subtitle=Direct detection of ''Pseudomonas&nbsp;aeruginosa'' on sensor chips based on [http://parts.igem.org/Part:BBa_K131026 K131026].|width=480px}}<br />
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===Detecting ''Pseudomonas&nbsp;aeruginosa'' with K131026 in our Sensor Chip with ''WatsOn''===<br />
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After establishing the successful detection of 3-oxo-C{{sub|12}}-HSLs with our sensor chips the next step was to detect living cells of ''Pseudomonas aeruginosa'' with our measurement device ''WatsOn''. Therefore sensor chips based on [http://parts.igem.org/Part:BBa_K131026 K131026] were prepared and the right chip was induced with 0.2&nbsp;µL of a ''Pseudomonas aeruginosa'' culture while the left chip was not induced (Detection of 3-oxo-C12 HSL with K131026, displayed below). On the induced chip, a clear fluorescence signal was visible in response to ''P. aeruginosa''. The fluorescence signal emerged outward from the induction point and showed a significant difference to the non-induced chip. The results clearly demonstrate the ability of our sensor chip technology and our measurement device ''WatsOn'' to detect ''P. aeruginosa''!<br />
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== Outlook ==<br />
<span class="anchor" id="biosensoroutlook"></span><br />
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We are '''committed to improve''' our sensor chip platform. The current technique of using a simple agarose chip is not sufficient to collect all microorganisms from the sampled surface. Therefore, the aim is to improve the sampling chip by using a different, more adhesive material. <br />
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Furthermore, diffusion in the sensor chips will be reduced to '''limit the spread of the fluorescence signal'''. Currently, the fluorescence spot grows beyond the point of induction and makes it difficult to differentitate between multiple points of induction. By introducing diffusion barriers into our chips, the growth of the fluorescence spots might be reduced, thus enabling the detection of multiple sources of fluorescence lying close together. <br />
<br />
Additionally, the application of our sensor chips in combination with our ''WatsOn'' device is currently being evaluated for the detection of signals other than fluorescence. '''Detecting bio- and chemiluminescence''' has been identified and will be investigated as an area of potential future application. <br />
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==References==<br />
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* Waters, C. M., & Bassler, B. L. (2005). QUORUM SENSING: Cell-to-Cell Communication In Bacteria. Annual Review of Cell and Developmental Biology, 21(1), 319-346. Available online at http://www.annualreviews.org/doi/full/10.1146/annurev.cellbio.21.012704.131001.<br />
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* Jimenez, P. N., Koch, G., Thompson, J. A., Xavier, K. B., Cool, R. H., & Quax, W. J. (2012). The Multiple Signaling Systems Regulating Virulence in ''Pseudomonas aeruginosa''. Microbiology and Molecular Biology Reviews, 76(1), 46-65. Available online at http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3294424/#B63.<br />
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{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/Project/2D_BiosensorTeam:Aachen/Project/2D Biosensor2014-10-18T03:18:30Z<p>Nbailly: /* Achievements */</p>
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= 2D Biosensor =<br />
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With our 2D biosensor technology we are able to detect the pathogen ''Pseudomonas aeruginosa'' on solid surfaces. The sensor system is comprised of '''two distinct but inseparable modules''', a biological and a technical part:<br />
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* Sensor chips containing '''''Cellocks''''', our '''engineered detective cells''' that fluoresce in the presence of the pathogen, make up the biological part of ''Cellock Holmes''.<br />
* Our '''measurement device [https://2014.igem.org/Team:Aachen/Project/Measurement_Device ''WatsOn'']''' and the complementary '''software [https://2014.igem.org/Team:Aachen/Notebook/Software/Measurarty ''Measurarty'']''' complete our sensing technology on the technical side. <br />
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== Principle of Operation ==<br />
<span class="anchor" id="biosensorpoo"></span><br />
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''Cellock Holmes'' is designed upon a SynBio approach comprising a '''two-dimensional biosensor and a measurement unit'''. The two-dimensional biosensor is devised to recognize quorum sensing molecules secreted by the pathogen cells and to generate a distinct fluorescence signal; while the measurement device recognizes and analyzes the produced signal. On the molecular side, we use the '''[https://2014.igem.org/Team:Aachen/Project/FRET_Reporter REACh construct]''' to transform regular ''E. coli'' cells into ''Cellocks''.<br />
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{{Team:Aachen/Figure|Aachen 17-10-14 The basics of quorum sensing ipo.png||title=The principle of quorum sensing|subtitle=Microorganisms can sense the presence of their own kind based on quorum sensing which is a form of chemical communication. Depending on their cell density, quorum sensing allows these cells to activate or deactivate certain gene expression cascades (Waters and Bassler, 2005) for a specific function.|width=900px}}<br />
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Our '''sensor cells, ''Cellocks'', are immobilized in agar chips'''. To make the chips, we mix the ''Cellocks'' with liquid LB agar. <br />
In the course of our project, we designed a casting mold specifically for the production of our agar chips. When the agar has cooled down, the chips are cut out of the mold and are ready to use. Storage of the readily usable sensor chips is possible for up to two days at 4°C when using LB medium or up to five days if TB medium is used. A detailed description of the sensor chip manufacturing can be found in our [https://2014.igem.org/Team:Aachen/Notebook/Protocols/detection Protocols] section.<br />
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{{Team:Aachen/Figure|Aachen 14-10-14 Flowsheet OD-device part1 ipo.png|title=Assay to detect ''P.&nbsp;aeruginosa'' using ''Cellock Holmes''|subtitle=This flow sheet shows the procedure to sample and detect ''P.&nbsp;aeruginosa'': A sampling chip is briefly put onto the potentially contaminated surface, added onto one of our sensor chips and inserted into ''WatsOn''.|width=900px}}<br />
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Using ''Cellock Holmes'', we developed a simple assay to detect ''P.&nbsp;aeruginosa''. Initially, a so called sampling chip is placed on a solid surface that is potentially contaminated with the pathogen. Subsequently, the sampling chip is removed from the surface and put onto one of our sensor chips. Theorectically, the sensor chips could be directly used for sampling, however, this was avoided in our project to '''match [https://2014.igem.org/Team:Aachen/Safety biosafety regulations]''' and to prevent the spread of genetically modified organisms (GMOs) into the environment. The two layered chip-stack is then put into a petri dish which is inserted into our measurement device [https://2014.igem.org/Team:Aachen/Project/Measurement_Device ''WatsOn''] for evalutation.<br />
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{{Team:Aachen/Figure|Aachen 14-10-14 Flowsheet OD-device part2 ipo.png|title=Mode of action inside ''WatsOn''|subtitle=Chips are incubated at 37°C to stimulate cell growth and then illuminated with blue light to excite fluorescence. A picture is taken and analyzed for fluorescence signals using the software ''Measurarty''.|width=900px}}<br />
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Inside ''WatsOn'', the chips are incubated at 37°C and the sampled populations of microorganisms attached on the sampling chip start to grow and multiply. During incubation the chips can be '''illuminated with blue light''' at any time, and a '''photo of the chips''' is taken. The '''software ''Measurarty''''' then analyzes any fluorescent signal. ''P.&nbsp;aeruginosa'' secrets an increasing number of quorum sensing molecules that are recognized by ''Cellocks'', thereby producing a fluorescence signal. For detection of ''P.&nbsp;aeruginosa'', we focused on a quorum sensing molecule called N-3-oxo-dodecanoyl-L-homoserine lactone (for short: 3-oxo-C<sub>12</sub>-HSL), which is involved in virulence regulation of ''P.&nbsp;aeruginosa'' (Jimenez, Koch, Thompson et al., 2012). The incorporation of the 3-oxo-C<sub>12</sub>-HSL detection system into the ''Cellocks'' is explained in detail in the [https://2014.igem.org/Team:Aachen/Project/FRET_Reporter REACh Construct] section.<br />
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For our biosensor, our team genetically modified ''E. coli'' cells to be able to elecit a '''fluorescent response to autoinducers''' produced by the pathogen ''Pseudomonas aeruginosa'' during quorum sensing. In the case of ''P.&nbsp;aeruginosa'', these autoinducers are N-3-oxo-dodecanoyl-L-homoserine lactone, or 3-oxo-C-12-HSL for short. The quorum sensing system of this pathogen contains the '''LasR activator''' which binds 3-oxo-C-12-HSL, and the '''LasI promoter''', which is activated by the LasR-HSL complex. Both LasR activator and LasI promoter are available as BioBricks [http://parts.igem.org/Part:BBa_C0179 C0179] and [http://parts.igem.org/Part:BBa_J64010 J64010].<br />
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As a reporter gene, we use '''GFP'''. However, expression of GFP is not simply controlled through the LasI promoter activity in our approach. Instead, our sensor cells contain genes for a constitutively expressed fusion protein consisting of GFP and a dark quencher, and an '''HSL-inducible protease'''. We use the REACh protein as dark quencher for GFP and the TEV protease to cleave the complex; [https://2014.igem.org/Team:Aachen/Project/FRET_Reporter here] you can read more about the REACh construct and the TEV protease. <br />
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{{Team:Aachen/Figure|align=center|Aachen_REACh_approach.png|title=Our novel biosensor approach|subtitle=Expression of the TEV protease is induced by HSL. The protease cleaves the GFP-REACh fusion protein to elecit a fluorescence response.|width=500px}}<br />
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When ''P.&nbsp;aeruginosa cells'' are stuck on our agar chip and come close to our sensor cells, the latter take up the HSL molecules secreted by the pathogens. Inside the sensor cells, the autoinducer binds to the LasR gene product and activate the expression of the TEV protease. The protease then cleaves the GFP-REACh construct. When '''illuminated with light of 480&nbsp;nm''', the excitation wavelenght of GFP, our sensor cells in the vicinity of ''P.&nbsp;aeruginosa'' give a '''fluorescence signal'''. On the other hand, sensor cells that were not anywhere close to the pathogens do not express the protease. Therefore, the GFP will still be attached to the dark quencher in these cells, and no fluorescence is produced.<br />
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== Development & Optimization ==<br />
<span class="anchor" id="biosensordevelopment"></span><br />
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{{Team:Aachen/FigureFloat|Aachen_ILOV_GFP_HM_1,5h.png|title=iLOV and GFP in the Gel Doc<sup>TM</sup>|subtitle=Sensor cells producing iLOV (A) and GFP (B) 1.5&nbsp;h after induction.|left|width=500px}}<br />
=== Equipment and medium selection ===<br />
Our first approach (before developing our own device) was to use the Molecular Imager&reg; Gel Doc™ XR+ from BIO-RAD in our lab to detect fluorescence. This device uses UV and white light illuminators. However, only two different filters were available for the excitation light wavelength, which resulted in very limited possibilities for the excitation of fluorescent molecules. For example, it was possible to detect the expression of iLOV in our sensor chips, but not the expression of GFP. Hence, the '''Gel Doc™ was not suitable for our project'''.<br />
{{Team:Aachen/FigureFloat|Aachen_Chip_medium_geldoc.png|title=Differend medium in the Gel Doc™|subtitle=complex media exhibited high background fluorescence while less back- ground fluorescence was observed with the minimal media (HM, M9, NA).|right|width=500px}}<br />
{{Team:Aachen/FigureFloat|Aachen_5days_K131026_neb_tb_1,5h.jpg |title=Testing our chips' shelf-life|subtitle= Chips of [http://parts.igem.org/Part:BBa_K131026 K131026] in NEB were stored five days at 4°C. The right chip was induced with 0.2&nbsp;µL of 500&nbsp;µg/mL HSL and an image was taken after 1.5&nbsp;h.|left|width=500px}}<br />
We tested different media (LB, TB, M9, NA and HM) for the preparation of our sensor chips. The medium compositions can be found in the [https://2014.igem.org/Team:Aachen/Notebook/Protocols Protocols] section. We screened for an optimized medium composition to minimize background fluorescence and to enhance cell growth. The results of the analysis are presented in the table below. Due to the low background fluorescence in ''WatsOn'' and the excellent cell growth, we '''chose LB&nbsp;medium''' over the other tested media for sensor chip manufacturing.<br />
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{| class="wikitable"<br />
! !! LB !! TB !! NA !! M9 !! HM <br />
|-<br />
| Growth of ''Cellock'' || <div style="text-align: center;">'''+'''</div> || <div style="text-align: center;">'''+'''</div> || <div style="text-align: center;">-</div> || <div style="text-align: center;">-</div> || <div style="text-align: center;">-</div><br />
|-<br />
| Background fluorescence in GelDoc || <div style="text-align: center;">'''+'''</div> || <div style="text-align: center;">'''+'''</div> || <div style="text-align: center;">-</div> || <div style="text-align: center;">-</div> || <div style="text-align: center;">-</div><br />
|-<br />
| Background fluorescence in ''WatsOn'' || <div style="text-align: center;">-</div> || <div style="text-align: center;">'''+'''</div> || <div style="text-align: center;">-</div> || <div style="text-align: center;">-</div> || <div style="text-align: center;">-</div><br />
|}<br />
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Another set of experiments were conducted to test the '''long-time storage''' of the sensor chips. We varied the glycerol content of the chips as well as the storage temperature. Storage at -20°C resulted in the loss of our sensor cells. Adding 5-10% (v/v) glycerol ensured survival of the sensor cells, but resulted in the loss of fluorescence ability. Hence, we concluded that long-time storage of the sensor chips at -20°C is not possible under the tested conditions. However, the 'ready-to-use' sensor chips can be kept at at 4°C for two days when using LB medium, and storage at this temperature for 5 days is possible with chips made from TB medium.<br />
<!--Regarding the medium used for our sensor chips, LB medium showed a high background fluorescence when exposed to UV light in the Gel Doc. Surprisingly, the background fluorescence resulting from the LB medium was too high to detect a signal emitted by our sensor cells. Hence, minimal media (NA, M9, Hartman (HM)) was used to minimize background fluorescence, but this approach resulted in less to no growth of our sensor cells. In our device ''WatsOn'', optimized wavelengths of 450&nbsp;nm and 480&nbsp;nm were used for excitation of iLOV and GFP, respectively. When exposed to either excitation wavelength TB medium, which is basically an improved LB medium and highly supports cell growth, showed strong background fluorescence in our own device. High background fluorescence was also observed for TB medum when using the Gel Doc. In contrast to the Gel Doc LB medium showed minimal fluorescence in our device ''WatsOn'' and no difficulties in cultivation of our ''Cellocks'' were observed. Because of the reduced fluorescence compared to TB medium when using ''Watson'' for sensor chip evaluation and because of sufficient cultivation conditions for our 'Cellocks'' LB medium was chosen over TB mediium for sensor chip manufacturing. --><br />
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{{Team:Aachen/FigureFloat|Aachen_2_chipform.jpg|title=Sensor chip manufacturing using the closed mold|subtitle=When injecting the liquid agar into a closed mold we encounter problems due to frequent bubble formation.|left|width=500px}}<br />
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=== Optimal Agarose Concentration for Sensor Chip Manufacturing ===<br />
For the sensor chip manufacturing, agarose was preferred over agar because of the uniform linkage between molecules that results in a better chip homogeneity. In addition, agarose reduced diffusion of the inducer molecules through the chip. A reduction in diffusion is essential for the formation of distinct fluorescent spots on the sensor chips.<br />
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{{Team:Aachen/FigureFloat|Aachen_Final_chipform.jpg|title=The finalized chip mold|subtitle=An open casting mold was found to be optimal for sensor chip manufacturing, because this approach was fast, easy to handle and generated a reproducible chip quality.|left|width=500px}}<br />
=== Optimal Chip Configuration ===<br />
Several approaches were tested for the production of agarose-based sensor chips with reproducible quality. The first approach was to cast every sensor chip individually. To achieve a plain chip surface, a requirement for high quality images, we casted the sensor chips between two microscope slides. However, this approach was not adequate because the agar was too liquid and leaked from the microscope slides. In a second approach, we designed a closed mold into which liquid agar is injected using a pipette, but we encountered a high number of bubbles in the resulting chips. Bubbles in the sensor chips interfered with fluorescence evaluation. Finally, we tried an open casting mold. Once solidified, we cut the agar along precast indentations in the casting mold to form the chips. An advantage of the open mold is the ability to simultaneously produce nine sensor chips while the surface tension of the liquid agar ensures a plane chip surface.<br />
=== Induction of the Sensor Chips ===<br />
To test our molecular constructs, we simulated the presence of ''P.&nbsp;aeruginosa'' by using IPTG or 3-oxo-C<sub>12</sub>-HSL. Initial experiments showed that diffusion of the inducers hinder the formation of distinct fluorescent spots. Through this set of experiments we determined that the best compromise between diffusion and spot intensity is an induction volume of 2.0&nbsp;µL for IPTG and 0.2&nbsp;µL for HSL. Furthermore, detection of growing ''P.&nbsp;aeruginosa'' based on secreted HSLs was possible using the [http://parts.igem.org/Part:BBa_K131026 K131026] construct. The experiments for optimizing the induction of our sensor chips are described in more detail in the [https://2014.igem.org/Team:Aachen/Project/2D_Biosensor#biosensorachievements Achievements] section.<br />
=== Negative Control ===<br />
To ensure that the fluorescence signal resulted from the sensor construct and not from the medium or ''E. coli'' cells themselves, [http://parts.igem.org/Part:BBa_B0015 B0015] in NEB10β cells was used as negative control during sensor chip induction with IPTG, HSL and ''P.&nbsp;aeruginosa''.<br />
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{{Team:Aachen/Figure|Aachen_B0015_IPTG_HSL_Pseudomonas.png|title=B0015 in NEB10β was used as a negativ control|subtitle=Induction with A) 0.2&nbsp;µL of 100&nbsp;mM IPTG, image taken after 2.5&nbsp;h; B) 0.2&nbsp;µL of 500&nbsp;µg/mL 3-oxo-C<sub>12</sub>-HSL, image after 2.5&nbsp;h; C) with five spots of ''Pseudomonas&nbsp;aeruginosa'' liquid culture on the left and one big spot on the right, image taken after 2&nbsp;h.|width=900px}}<br />
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==Achievements==<br />
<span class="anchor" id="biosensorachievements"></span><br />
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We developed and optimized a 2D biosensor, which is able to detect IPTG, 3-oxo-C{{sub|12}}-HSL and living ''Pseudomonas&nbsp;aeruginosa'' cells. <br />
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{{Team:Aachen/FigureFloat|Aachen_K1319042_Platereader.gif|title=Testing K1319042 in our sensor chips|subtitle=Sensor chips based on [http://parts.igem.org/Part:BBa_K1319042 K1319042] were investigated for fluorescence using a plate reader. Blue color indicates the absence of fluorescence, while red color indicates fluorescence. The upper chip was not induced, while the lower chip was induced with IPTG (2.0&nbsp;µL, 100mM).|width=260px}}<br />
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=== Testing our Sensor Chips in a Plate Reader ===<br />
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To establish a prove-of-principle for our sensor chip design, we used our construct [http://parts.igem.org/Part:BBa_K1319042 K1319042], an IPTG inducible iLOV. ''E. coli'' cells carrying the this construct were introduced into sensor chips and fluorescence was measured every 15&nbsp;minutes after induction with 2&nbsp;µL of 100&nbsp;mM IPTG. The results are displayed on the left.<br />
We observed a distinct difference in fluorescence between the non-induced chip (top) and the induced chip (bottom). The middle of the bottom chip started to exhibit a clear and visible fluorescence that increased over time and spread outwards. The top chip, however, also showed an increase in the measured fluorescence over time which was primarily due to the leaky promoter and background fluorescence.<br />
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=== Detecting 3-oxo-C{{sub|12}}-HSL with Sensor Chips ===<br />
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{{Team:Aachen/FigureFloatRight|Aachen_K131026_Platereader.gif|title=Testing K131026 in our sensor chips|subtitle=Sensor chips based on [http://parts.igem.org/Part:BBa_K131026 K131026] were investigated for fluorescence using a plate reader. Blue color indicates the absence of fluorescence, while red color indicates fluorescence. The lower chip was induced with with 3-oxo-C{{sub|12}}-HSL (0.2&nbsp;µL, 500 µg/mL).|width=360px}}<br />
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In an initial attempt to detect 3-oxo-C{{sub|12}}-HSL, we incorporated the [http://parts.igem.org/Part:BBa_K131026 K131026] construct generated by the 2008 iGEM Team Calgary in our sensor chips. This construct generates a fluorescent signal based on GFP in presence of 3-oxo-C{{sub|12}}-HSL molecules produced by ''P.&nbsp;aeruginosa'' during quorum sensing (Jimenez, Koch, Thompson et al., 2012). First, we tested the construct by direct induction with 3-oxo-C{{sub|12}}-HSL (0.2&nbsp;µL, 500&nbsp;µg/mL). The fluorescence measurement was taken every 15&nbsp;minutes with an excitation wavelength of 496&nbsp;nm and an emission wavelength of 516&nbsp;nm. The results of this test are shown on the right.<br />
<br />
A distinct fluorescence signal was observed on the induced chip (bottom) compared to the non-induced chip (top). <br />
Fluorescence started in the middle of the chip, the point of induction, and then extended outwards while growing in intesnity. The basal level of fluorescence was attributed to leakiness of the promoter and general background fluorescence of growing ''E. coli'' cells. In the induced chip (bottom), the background fluorescence was lower than in the non-induced chip (top) because the signal masked the noise. The difference between the induced and non-induced chips indicates a clear response to the HSL and proofed the ability of our 2D sensor chip design to detect HSLs produced by ''Pseudomonas&nbsp;aeruginosa''.<br />
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{{Team:Aachen/FigureFloat|Aachen_I746909_slower_reduced.gif|title=IPTG-inducible superfolder GFP (I746909) in sensor chips|subtitle=Expression of superfolder GFP ([http://parts.igem.org/Part:BBa_I746909 I746909]) was induced by the addition of IPTG (2&nbsp;µL,&nbsp;100mM) on the right chip. The left chip was not induced.|width=480px}}<br />
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=== Detecting IPTG with Sensor Chips ===<br />
The clip displayed on the left side shows the construct [http://parts.igem.org/Part:BBa_I746909 I746909] from the 2007 iGEM Team Cambridge which expresses super folder GFP under the control of a T7 promoter in combination with our 2D sensor chip technology. The [http://parts.igem.org/Part:BBa_I746909 I746909] construct was introduced into BL21(DE3) cells to make the expression IPTG-inducible since the genome of BL21(De3) contains the T7 RNA Polymerase under the control of a lacI promoter.<br />
<br />
While the left chip does not show visible fluorescence, the right chip exhibits a strong fluorescence signal. This proves the ability of our sensor chip technology to detect IPTG. The fluorescence response is also high enough to be detected and analyzed by our measurement device ''WatsOn''.<br />
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{{Team:Aachen/FigureFloatRight|Aachen_K131026_HSLdetection_slow.gif|title=Detection of 3-oxo-C{{sub|12}}-HSL with K131026|subtitle=0.2&nbsp;µL of 3-oxo-C{{sub|12}}-HSL was placed in the middle of a sensor chip based on [http://parts.igem.org/Part:BBa_K131026 K131026] followed by incubation at 37°C in ''WatsOn''.|width=480px}}<br />
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===Detecting the 3-oxo-C{{sub|12}}-HSL with K131026 in our Sensor Chips using ''WatsOn''===<br />
<br />
The next step towards the final goal of detecting living cells of ''Pseudomonas&nbsp;aeruginosa'' was to reproduce the detection of 3-oxo-C{{sub|12}}-HSL, already established in the plate reader, in our own [https://2014.igem.org/Team:Aachen/Project/Measurement_Device ''WatsOn''] device. Therefore, we again used ''E. coli'' BL21(DE3) cells carrying [http://parts.igem.org/Part:BBa_K131026 K131026] and induced with 0.2&nbsp;µL 3-oxo-C{{sub|12}}-HSL of a concentration of 500&nbsp;µg/mL. In the clip displayed on the left, the right chip was induced and - as a negative control - the left chip was not induced. Pictures were taken every four&nbsp;minutes.<br />
<br />
The result was a clear replication of the success of the plate reader experiment. The induced chip showed a clear fluorescence response eminating from the center, where the induction with HSL took place. This demonstrated the ability of not only our sensor chip technology but also our measurement device ''WatsOn to successfully'' detect 3-oxo-C{{sub|12}}-HSL.<br />
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{{Team:Aachen/FigureFloat|Aachen_K131026_Pseudomonas_aeruginosa_detection.gif|title=Detection of ''Pseudomonas&nbsp;aeruginosa'' with K131026|subtitle=Direct detection of ''Pseudomonas&nbsp;aeruginosa'' on sensor chips based on [http://parts.igem.org/Part:BBa_K131026 K131026].|width=480px}}<br />
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===Detecting ''Pseudomonas&nbsp;aeruginosa'' with K131026 in our Sensor Chip with ''WatsOn''===<br />
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After establishing the successful detection of 3-oxo-C{{sub|12}}-HSLs with our sensor chips the next step was to detect living cells of ''Pseudomonas aeruginosa'' with our measurement device ''WatsOn''. Therefore sensor chips based on [http://parts.igem.org/Part:BBa_K131026 K131026] were prepared and the right chip was induced with 0.2&nbsp;µL of a ''Pseudomonas aeruginosa'' culture while the left chip was not induced (Detection of 3-oxo-C12 HSL with K131026, displayed below). On the induced chip, a clear fluorescence signal was visible in response to ''P. aeruginosa''. The fluorescence signal emerged outward from the induction point and showed a significant difference to the non-induced chip. The results clearly demonstrate the ability of our sensor chip technology and our measurement device ''WatsOn'' to detect ''P. aeruginosa''!<br />
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== Outlook ==<br />
<span class="anchor" id="biosensoroutlook"></span><br />
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We are '''committed to improve''' our sensor chip platform. The current technique of using a simple agarose chip is not sufficient to collect all microorganisms from the sampled surface. Therefore, the aim is to improve the sampling chip by using a different, more adhesive material. <br />
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Furthermore, diffusion in the sensor chips will be reduced to '''limit the spread of the fluorescence signal'''. Currently, the fluorescence spot grows beyond the point of induction and makes it difficult to differentitate between multiple points of induction. By introducing diffusion barriers into our chips, the growth of the fluorescence spots might be reduced, thus enabling the detection of multiple sources of fluorescence lying close together. <br />
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Additionally, the application of our sensor chips in combination with our ''WatsOn'' device is currently being evaluated for the detection of signals other than fluorescence. '''Detecting bio- and chemiluminescence''' has been identified and will be investigated as an area of potential future application. <br />
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==References==<br />
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* Waters, C. M., & Bassler, B. L. (2005). QUORUM SENSING: Cell-to-Cell Communication In Bacteria. Annual Review of Cell and Developmental Biology, 21(1), 319-346. Available online at http://www.annualreviews.org/doi/full/10.1146/annurev.cellbio.21.012704.131001.<br />
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* Jimenez, P. N., Koch, G., Thompson, J. A., Xavier, K. B., Cool, R. H., & Quax, W. J. (2012). The Multiple Signaling Systems Regulating Virulence in ''Pseudomonas aeruginosa''. Microbiology and Molecular Biology Reviews, 76(1), 46-65. Available online at http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3294424/#B63.<br />
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{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/Team:Aachen/Project/2D_BiosensorTeam:Aachen/Project/2D Biosensor2014-10-18T03:18:06Z<p>Nbailly: /* Achievements */</p>
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= 2D Biosensor =<br />
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With our 2D biosensor technology we are able to detect the pathogen ''Pseudomonas aeruginosa'' on solid surfaces. The sensor system is comprised of '''two distinct but inseparable modules''', a biological and a technical part:<br />
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* Sensor chips containing '''''Cellocks''''', our '''engineered detective cells''' that fluoresce in the presence of the pathogen, make up the biological part of ''Cellock Holmes''.<br />
* Our '''measurement device [https://2014.igem.org/Team:Aachen/Project/Measurement_Device ''WatsOn'']''' and the complementary '''software [https://2014.igem.org/Team:Aachen/Notebook/Software/Measurarty ''Measurarty'']''' complete our sensing technology on the technical side. <br />
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== Principle of Operation ==<br />
<span class="anchor" id="biosensorpoo"></span><br />
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''Cellock Holmes'' is designed upon a SynBio approach comprising a '''two-dimensional biosensor and a measurement unit'''. The two-dimensional biosensor is devised to recognize quorum sensing molecules secreted by the pathogen cells and to generate a distinct fluorescence signal; while the measurement device recognizes and analyzes the produced signal. On the molecular side, we use the '''[https://2014.igem.org/Team:Aachen/Project/FRET_Reporter REACh construct]''' to transform regular ''E. coli'' cells into ''Cellocks''.<br />
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{{Team:Aachen/Figure|Aachen 17-10-14 The basics of quorum sensing ipo.png||title=The principle of quorum sensing|subtitle=Microorganisms can sense the presence of their own kind based on quorum sensing which is a form of chemical communication. Depending on their cell density, quorum sensing allows these cells to activate or deactivate certain gene expression cascades (Waters and Bassler, 2005) for a specific function.|width=900px}}<br />
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Our '''sensor cells, ''Cellocks'', are immobilized in agar chips'''. To make the chips, we mix the ''Cellocks'' with liquid LB agar. <br />
In the course of our project, we designed a casting mold specifically for the production of our agar chips. When the agar has cooled down, the chips are cut out of the mold and are ready to use. Storage of the readily usable sensor chips is possible for up to two days at 4°C when using LB medium or up to five days if TB medium is used. A detailed description of the sensor chip manufacturing can be found in our [https://2014.igem.org/Team:Aachen/Notebook/Protocols/detection Protocols] section.<br />
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{{Team:Aachen/Figure|Aachen 14-10-14 Flowsheet OD-device part1 ipo.png|title=Assay to detect ''P.&nbsp;aeruginosa'' using ''Cellock Holmes''|subtitle=This flow sheet shows the procedure to sample and detect ''P.&nbsp;aeruginosa'': A sampling chip is briefly put onto the potentially contaminated surface, added onto one of our sensor chips and inserted into ''WatsOn''.|width=900px}}<br />
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Using ''Cellock Holmes'', we developed a simple assay to detect ''P.&nbsp;aeruginosa''. Initially, a so called sampling chip is placed on a solid surface that is potentially contaminated with the pathogen. Subsequently, the sampling chip is removed from the surface and put onto one of our sensor chips. Theorectically, the sensor chips could be directly used for sampling, however, this was avoided in our project to '''match [https://2014.igem.org/Team:Aachen/Safety biosafety regulations]''' and to prevent the spread of genetically modified organisms (GMOs) into the environment. The two layered chip-stack is then put into a petri dish which is inserted into our measurement device [https://2014.igem.org/Team:Aachen/Project/Measurement_Device ''WatsOn''] for evalutation.<br />
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{{Team:Aachen/Figure|Aachen 14-10-14 Flowsheet OD-device part2 ipo.png|title=Mode of action inside ''WatsOn''|subtitle=Chips are incubated at 37°C to stimulate cell growth and then illuminated with blue light to excite fluorescence. A picture is taken and analyzed for fluorescence signals using the software ''Measurarty''.|width=900px}}<br />
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Inside ''WatsOn'', the chips are incubated at 37°C and the sampled populations of microorganisms attached on the sampling chip start to grow and multiply. During incubation the chips can be '''illuminated with blue light''' at any time, and a '''photo of the chips''' is taken. The '''software ''Measurarty''''' then analyzes any fluorescent signal. ''P.&nbsp;aeruginosa'' secrets an increasing number of quorum sensing molecules that are recognized by ''Cellocks'', thereby producing a fluorescence signal. For detection of ''P.&nbsp;aeruginosa'', we focused on a quorum sensing molecule called N-3-oxo-dodecanoyl-L-homoserine lactone (for short: 3-oxo-C<sub>12</sub>-HSL), which is involved in virulence regulation of ''P.&nbsp;aeruginosa'' (Jimenez, Koch, Thompson et al., 2012). The incorporation of the 3-oxo-C<sub>12</sub>-HSL detection system into the ''Cellocks'' is explained in detail in the [https://2014.igem.org/Team:Aachen/Project/FRET_Reporter REACh Construct] section.<br />
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For our biosensor, our team genetically modified ''E. coli'' cells to be able to elecit a '''fluorescent response to autoinducers''' produced by the pathogen ''Pseudomonas aeruginosa'' during quorum sensing. In the case of ''P.&nbsp;aeruginosa'', these autoinducers are N-3-oxo-dodecanoyl-L-homoserine lactone, or 3-oxo-C-12-HSL for short. The quorum sensing system of this pathogen contains the '''LasR activator''' which binds 3-oxo-C-12-HSL, and the '''LasI promoter''', which is activated by the LasR-HSL complex. Both LasR activator and LasI promoter are available as BioBricks [http://parts.igem.org/Part:BBa_C0179 C0179] and [http://parts.igem.org/Part:BBa_J64010 J64010].<br />
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As a reporter gene, we use '''GFP'''. However, expression of GFP is not simply controlled through the LasI promoter activity in our approach. Instead, our sensor cells contain genes for a constitutively expressed fusion protein consisting of GFP and a dark quencher, and an '''HSL-inducible protease'''. We use the REACh protein as dark quencher for GFP and the TEV protease to cleave the complex; [https://2014.igem.org/Team:Aachen/Project/FRET_Reporter here] you can read more about the REACh construct and the TEV protease. <br />
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{{Team:Aachen/Figure|align=center|Aachen_REACh_approach.png|title=Our novel biosensor approach|subtitle=Expression of the TEV protease is induced by HSL. The protease cleaves the GFP-REACh fusion protein to elecit a fluorescence response.|width=500px}}<br />
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When ''P.&nbsp;aeruginosa cells'' are stuck on our agar chip and come close to our sensor cells, the latter take up the HSL molecules secreted by the pathogens. Inside the sensor cells, the autoinducer binds to the LasR gene product and activate the expression of the TEV protease. The protease then cleaves the GFP-REACh construct. When '''illuminated with light of 480&nbsp;nm''', the excitation wavelenght of GFP, our sensor cells in the vicinity of ''P.&nbsp;aeruginosa'' give a '''fluorescence signal'''. On the other hand, sensor cells that were not anywhere close to the pathogens do not express the protease. Therefore, the GFP will still be attached to the dark quencher in these cells, and no fluorescence is produced.<br />
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== Development & Optimization ==<br />
<span class="anchor" id="biosensordevelopment"></span><br />
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{{Team:Aachen/FigureFloat|Aachen_ILOV_GFP_HM_1,5h.png|title=iLOV and GFP in the Gel Doc<sup>TM</sup>|subtitle=Sensor cells producing iLOV (A) and GFP (B) 1.5&nbsp;h after induction.|left|width=500px}}<br />
=== Equipment and medium selection ===<br />
Our first approach (before developing our own device) was to use the Molecular Imager&reg; Gel Doc™ XR+ from BIO-RAD in our lab to detect fluorescence. This device uses UV and white light illuminators. However, only two different filters were available for the excitation light wavelength, which resulted in very limited possibilities for the excitation of fluorescent molecules. For example, it was possible to detect the expression of iLOV in our sensor chips, but not the expression of GFP. Hence, the '''Gel Doc™ was not suitable for our project'''.<br />
{{Team:Aachen/FigureFloat|Aachen_Chip_medium_geldoc.png|title=Differend medium in the Gel Doc™|subtitle=complex media exhibited high background fluorescence while less back- ground fluorescence was observed with the minimal media (HM, M9, NA).|right|width=500px}}<br />
{{Team:Aachen/FigureFloat|Aachen_5days_K131026_neb_tb_1,5h.jpg |title=Testing our chips' shelf-life|subtitle= Chips of [http://parts.igem.org/Part:BBa_K131026 K131026] in NEB were stored five days at 4°C. The right chip was induced with 0.2&nbsp;µL of 500&nbsp;µg/mL HSL and an image was taken after 1.5&nbsp;h.|left|width=500px}}<br />
We tested different media (LB, TB, M9, NA and HM) for the preparation of our sensor chips. The medium compositions can be found in the [https://2014.igem.org/Team:Aachen/Notebook/Protocols Protocols] section. We screened for an optimized medium composition to minimize background fluorescence and to enhance cell growth. The results of the analysis are presented in the table below. Due to the low background fluorescence in ''WatsOn'' and the excellent cell growth, we '''chose LB&nbsp;medium''' over the other tested media for sensor chip manufacturing.<br />
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{| class="wikitable"<br />
! !! LB !! TB !! NA !! M9 !! HM <br />
|-<br />
| Growth of ''Cellock'' || <div style="text-align: center;">'''+'''</div> || <div style="text-align: center;">'''+'''</div> || <div style="text-align: center;">-</div> || <div style="text-align: center;">-</div> || <div style="text-align: center;">-</div><br />
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| Background fluorescence in GelDoc || <div style="text-align: center;">'''+'''</div> || <div style="text-align: center;">'''+'''</div> || <div style="text-align: center;">-</div> || <div style="text-align: center;">-</div> || <div style="text-align: center;">-</div><br />
|-<br />
| Background fluorescence in ''WatsOn'' || <div style="text-align: center;">-</div> || <div style="text-align: center;">'''+'''</div> || <div style="text-align: center;">-</div> || <div style="text-align: center;">-</div> || <div style="text-align: center;">-</div><br />
|}<br />
</center><br />
<br />
Another set of experiments were conducted to test the '''long-time storage''' of the sensor chips. We varied the glycerol content of the chips as well as the storage temperature. Storage at -20°C resulted in the loss of our sensor cells. Adding 5-10% (v/v) glycerol ensured survival of the sensor cells, but resulted in the loss of fluorescence ability. Hence, we concluded that long-time storage of the sensor chips at -20°C is not possible under the tested conditions. However, the 'ready-to-use' sensor chips can be kept at at 4°C for two days when using LB medium, and storage at this temperature for 5 days is possible with chips made from TB medium.<br />
<!--Regarding the medium used for our sensor chips, LB medium showed a high background fluorescence when exposed to UV light in the Gel Doc. Surprisingly, the background fluorescence resulting from the LB medium was too high to detect a signal emitted by our sensor cells. Hence, minimal media (NA, M9, Hartman (HM)) was used to minimize background fluorescence, but this approach resulted in less to no growth of our sensor cells. In our device ''WatsOn'', optimized wavelengths of 450&nbsp;nm and 480&nbsp;nm were used for excitation of iLOV and GFP, respectively. When exposed to either excitation wavelength TB medium, which is basically an improved LB medium and highly supports cell growth, showed strong background fluorescence in our own device. High background fluorescence was also observed for TB medum when using the Gel Doc. In contrast to the Gel Doc LB medium showed minimal fluorescence in our device ''WatsOn'' and no difficulties in cultivation of our ''Cellocks'' were observed. Because of the reduced fluorescence compared to TB medium when using ''Watson'' for sensor chip evaluation and because of sufficient cultivation conditions for our 'Cellocks'' LB medium was chosen over TB mediium for sensor chip manufacturing. --><br />
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{{Team:Aachen/FigureFloat|Aachen_2_chipform.jpg|title=Sensor chip manufacturing using the closed mold|subtitle=When injecting the liquid agar into a closed mold we encounter problems due to frequent bubble formation.|left|width=500px}}<br />
<br />
=== Optimal Agarose Concentration for Sensor Chip Manufacturing ===<br />
For the sensor chip manufacturing, agarose was preferred over agar because of the uniform linkage between molecules that results in a better chip homogeneity. In addition, agarose reduced diffusion of the inducer molecules through the chip. A reduction in diffusion is essential for the formation of distinct fluorescent spots on the sensor chips.<br />
<br />
{{Team:Aachen/FigureFloat|Aachen_Final_chipform.jpg|title=The finalized chip mold|subtitle=An open casting mold was found to be optimal for sensor chip manufacturing, because this approach was fast, easy to handle and generated a reproducible chip quality.|left|width=500px}}<br />
=== Optimal Chip Configuration ===<br />
Several approaches were tested for the production of agarose-based sensor chips with reproducible quality. The first approach was to cast every sensor chip individually. To achieve a plain chip surface, a requirement for high quality images, we casted the sensor chips between two microscope slides. However, this approach was not adequate because the agar was too liquid and leaked from the microscope slides. In a second approach, we designed a closed mold into which liquid agar is injected using a pipette, but we encountered a high number of bubbles in the resulting chips. Bubbles in the sensor chips interfered with fluorescence evaluation. Finally, we tried an open casting mold. Once solidified, we cut the agar along precast indentations in the casting mold to form the chips. An advantage of the open mold is the ability to simultaneously produce nine sensor chips while the surface tension of the liquid agar ensures a plane chip surface.<br />
=== Induction of the Sensor Chips ===<br />
To test our molecular constructs, we simulated the presence of ''P.&nbsp;aeruginosa'' by using IPTG or 3-oxo-C<sub>12</sub>-HSL. Initial experiments showed that diffusion of the inducers hinder the formation of distinct fluorescent spots. Through this set of experiments we determined that the best compromise between diffusion and spot intensity is an induction volume of 2.0&nbsp;µL for IPTG and 0.2&nbsp;µL for HSL. Furthermore, detection of growing ''P.&nbsp;aeruginosa'' based on secreted HSLs was possible using the [http://parts.igem.org/Part:BBa_K131026 K131026] construct. The experiments for optimizing the induction of our sensor chips are described in more detail in the [https://2014.igem.org/Team:Aachen/Project/2D_Biosensor#biosensorachievements Achievements] section.<br />
=== Negative Control ===<br />
To ensure that the fluorescence signal resulted from the sensor construct and not from the medium or ''E. coli'' cells themselves, [http://parts.igem.org/Part:BBa_B0015 B0015] in NEB10β cells was used as negative control during sensor chip induction with IPTG, HSL and ''P.&nbsp;aeruginosa''.<br />
<br />
{{Team:Aachen/Figure|Aachen_B0015_IPTG_HSL_Pseudomonas.png|title=B0015 in NEB10β was used as a negativ control|subtitle=Induction with A) 0.2&nbsp;µL of 100&nbsp;mM IPTG, image taken after 2.5&nbsp;h; B) 0.2&nbsp;µL of 500&nbsp;µg/mL 3-oxo-C<sub>12</sub>-HSL, image after 2.5&nbsp;h; C) with five spots of ''Pseudomonas&nbsp;aeruginosa'' liquid culture on the left and one big spot on the right, image taken after 2&nbsp;h.|width=900px}}<br />
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[[File:Aachen_14-10-15_Medal_Cellocks_iNB.png|right|150px]]<br />
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==Achievements==<br />
<span class="anchor" id="biosensorachievements"></span><br />
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We developed and optimized a 2D biosensor, which is able to detect IPTG, 3-oxo-C{{sub|12}}-HSL and living ''Pseudomonas&nbsp;aeruginosa'' cells. <br />
<br />
{{Team:Aachen/FigureFloat|Aachen_K1319042_Platereader.gif|title=Testing K1319042 in our sensor chips|subtitle=Sensor chips based on [http://parts.igem.org/Part:BBa_K1319042 K1319042] were investigated for fluorescence using a plate reader. Blue color indicates the absence of fluorescence, while red color indicates fluorescence. The upper chip was not induced, while the lower chip was induced with IPTG (2.0&nbsp;µL, 100mM).|width=260px}}<br />
<br />
=== Testing our Sensor Chips in a Plate Reader ===<br />
<br />
To establish a prove-of-principle for our sensor chip design, we used our construct [http://parts.igem.org/Part:BBa_K1319042 K1319042], an IPTG inducible iLOV. ''E. coli'' cells carrying the this construct were introduced into sensor chips and fluorescence was measured every 15&nbsp;minutes after induction with 2&nbsp;µL of 100&nbsp;mM IPTG. The results are displayed on the left.<br />
We observed a distinct difference in fluorescence between the non-induced chip (top) and the induced chip (bottom). The middle of the bottom chip started to exhibit a clear and visible fluorescence that increased over time and spread outwards. The top chip, however, also showed an increase in the measured fluorescence over time which was primarily due to the leaky promoter and background fluorescence.<br />
<br />
=== Detecting 3-oxo-C{{sub|12}}-HSL with Sensor Chips ===<br />
<br />
{{Team:Aachen/FigureFloatRight|Aachen_K131026_Platereader.gif|title=Testing K131026 in our sensor chips|subtitle=Sensor chips based on [http://parts.igem.org/Part:BBa_K131026 K131026] were investigated for fluorescence using a plate reader. Blue color indicates the absence of fluorescence, while red color indicates fluorescence. The lower chip was induced with with 3-oxo-C{{sub|12}}-HSL (0.2&nbsp;µL, 500 µg/mL).|width=360px}}<br />
<br />
In an initial attempt to detect 3-oxo-C{{sub|12}}-HSL, we incorporated the [http://parts.igem.org/Part:BBa_K131026 K131026] construct generated by the 2008 iGEM Team Calgary in our sensor chips. This construct generates a fluorescent signal based on GFP in presence of 3-oxo-C{{sub|12}}-HSL molecules produced by ''P.&nbsp;aeruginosa'' during quorum sensing (Jimenez, Koch, Thompson et al., 2012). First, we tested the construct by direct induction with 3-oxo-C{{sub|12}}-HSL (0.2&nbsp;µL, 500&nbsp;µg/mL). The fluorescence measurement was taken every 15&nbsp;minutes with an excitation wavelength of 496&nbsp;nm and an emission wavelength of 516&nbsp;nm. The results of this test are shown on the right.<br />
<br />
A distinct fluorescence signal was observed on the induced chip (bottom) compared to the non-induced chip (top). <br />
Fluorescence started in the middle of the chip, the point of induction, and then extended outwards while growing in intesnity. The basal level of fluorescence was attributed to leakiness of the promoter and general background fluorescence of growing ''E. coli'' cells. In the induced chip (bottom), the background fluorescence was lower than in the non-induced chip (top) because the signal masked the noise. The difference between the induced and non-induced chips indicates a clear response to the HSL and proofed the ability of our 2D sensor chip design to detect HSLs produced by ''Pseudomonas&nbsp;aeruginosa''.<br />
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{{Team:Aachen/FigureFloat|Aachen_I746909_slower_reduced.gif|title=IPTG-inducible superfolder GFP (I746909) in sensor chips|subtitle=Expression of superfolder GFP ([http://parts.igem.org/Part:BBa_I746909 I746909]) was induced by the addition of IPTG (2&nbsp;µL,&nbsp;100mM) on the right chip. The left chip was not induced.|width=480px}}<br />
<br />
=== Detecting IPTG with Sensor Chips ===<br />
The clip displayed on the left side shows the construct [http://parts.igem.org/Part:BBa_I746909 I746909] from the 2007 iGEM Team Cambridge which expresses super folder GFP under the control of a T7 promoter in combination with our 2D sensor chip technology. The [http://parts.igem.org/Part:BBa_I746909 I746909] construct was introduced into BL21(DE3) cells to make the expression IPTG-inducible since the genome of BL21(De3) contains the T7 RNA Polymerase under the control of a lacI promoter.<br />
<br />
While the left chip does not show visible fluorescence, the right chip exhibits a strong fluorescence signal. This proves the ability of our sensor chip technology to detect IPTG. The fluorescence response is also high enough to be detected and analyzed by our measurement device ''WatsOn''.<br />
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<br />
{{Team:Aachen/FigureFloatRight|Aachen_K131026_HSLdetection_slow.gif|title=Detection of 3-oxo-C{{sub|12}}-HSL with K131026|subtitle=0.2&nbsp;µL of 3-oxo-C{{sub|12}}-HSL was placed in the middle of a sensor chip based on [http://parts.igem.org/Part:BBa_K131026 K131026] followed by incubation at 37°C in ''WatsOn''.|width=480px}}<br />
<br />
===Detecting the 3-oxo-C{{sub|12}}-HSL with K131026 in our Sensor Chips using ''WatsOn''===<br />
<br />
The next step towards the final goal of detecting living cells of ''Pseudomonas&nbsp;aeruginosa'' was to reproduce the detection of 3-oxo-C{{sub|12}}-HSL, already established in the plate reader, in our own [https://2014.igem.org/Team:Aachen/Project/Measurement_Device ''WatsOn''] device. Therefore, we again used ''E. coli'' BL21(DE3) cells carrying [http://parts.igem.org/Part:BBa_K131026 K131026] and induced with 0.2&nbsp;µL 3-oxo-C{{sub|12}}-HSL of a concentration of 500&nbsp;µg/mL. In the clip displayed on the left, the right chip was induced and - as a negative control - the left chip was not induced. Pictures were taken every four&nbsp;minutes.<br />
<br />
The result was a clear replication of the success of the plate reader experiment. The induced chip showed a clear fluorescence response eminating from the center, where the induction with HSL took place. This demonstrated the ability of not only our sensor chip technology but also our measurement device ''WatsOn to successfully'' detect 3-oxo-C{{sub|12}}-HSL.<br />
<br />
{{Team:Aachen/FigureFloat|Aachen_K131026_Pseudomonas_aeruginosa_detection.gif|title=Detection of ''Pseudomonas&nbsp;aeruginosa'' with K131026|subtitle=Direct detection of ''Pseudomonas&nbsp;aeruginosa'' on sensor chips based on [http://parts.igem.org/Part:BBa_K131026 K131026].|width=480px}}<br />
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<br />
===Detecting ''Pseudomonas&nbsp;aeruginosa'' with K131026 in our Sensor Chip with ''WatsOn''===<br />
<br />
After establishing the successful detection of 3-oxo-C{{sub|12}}-HSLs with our sensor chips the next step was to detect living cells of ''Pseudomonas aeruginosa'' with our measurement device ''WatsOn''. Therefore sensor chips based on [http://parts.igem.org/Part:BBa_K131026 K131026] were prepared and the right chip was induced with 0.2&nbsp;µL of a ''Pseudomonas aeruginosa'' culture while the left chip was not induced (Detection of 3-oxo-C12 HSL with K131026, displayed below). On the induced chip, a clear fluorescence signal was visible in response to ''P. aeruginosa''. The fluorescence signal emerged outward from the induction point and showed a significant difference to the non-induced chip. The results clearly demonstrate the ability of our sensor chip technology and our measurement device ''WatsOn'' to detect ''P. aeruginosa''!<br />
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[[File:Aachen_14-10-16_Outlook_Cellocks_iNB.png|right|150px]]<br />
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== Outlook ==<br />
<span class="anchor" id="biosensoroutlook"></span><br />
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We are '''committed to improve''' our sensor chip platform. The current technique of using a simple agarose chip is not sufficient to collect all microorganisms from the sampled surface. Therefore, the aim is to improve the sampling chip by using a different, more adhesive material. <br />
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Furthermore, diffusion in the sensor chips will be reduced to '''limit the spread of the fluorescence signal'''. Currently, the fluorescence spot grows beyond the point of induction and makes it difficult to differentitate between multiple points of induction. By introducing diffusion barriers into our chips, the growth of the fluorescence spots might be reduced, thus enabling the detection of multiple sources of fluorescence lying close together. <br />
<br />
Additionally, the application of our sensor chips in combination with our ''WatsOn'' device is currently being evaluated for the detection of signals other than fluorescence. '''Detecting bio- and chemiluminescence''' has been identified and will be investigated as an area of potential future application. <br />
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==References==<br />
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* Waters, C. M., & Bassler, B. L. (2005). QUORUM SENSING: Cell-to-Cell Communication In Bacteria. Annual Review of Cell and Developmental Biology, 21(1), 319-346. Available online at http://www.annualreviews.org/doi/full/10.1146/annurev.cellbio.21.012704.131001.<br />
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* Jimenez, P. N., Koch, G., Thompson, J. A., Xavier, K. B., Cool, R. H., & Quax, W. J. (2012). The Multiple Signaling Systems Regulating Virulence in ''Pseudomonas aeruginosa''. Microbiology and Molecular Biology Reviews, 76(1), 46-65. Available online at http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3294424/#B63.<br />
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{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/User:VeraAUser:VeraA2014-10-18T03:17:15Z<p>Nbailly: /* */</p>
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'''Vera Alexandrova - Lab Division'''<br />
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''Biology (B.Sc. student)''<br />
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I was born and grew up in Russia and decided to move to Germany in 2010. One year later I began my studies in biology at RWTH. Last year I completed a research internship at the Institute of Biochemistry at the RWTH Aachen Teaching Hospital, where I also wrote my bachelor thesis. For me the most exciting part of the iGEM competition is about working in the lab, because I already have experience in this field, and I'm glad to share it with other members of the team. I like modern and classical ballet, and would like to try other dance arts and participate in some dance festivals.<br />
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'''Mainly involved in...'''<br />
* [https://2014.igem.org/Team:Aachen/Notebook/Wetlab/August#5th QuikChange] at the Schwaneberg Group<br />
* all of our molecular approaches<br />
* graphics design for our wiki<br />
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{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/User:StefanReinholdUser:StefanReinhold2014-10-18T03:16:53Z<p>Nbailly: /* */</p>
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'''Stefan Reinhold - Lab Division & Treasurer'''<br />
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''Molecular and Applied Biotechnology (M.Sc. student)''<br />
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I wrote my bachelor thesis about “Determination of factors that influence Growth and Productivity of Chinese Hamster Ovary Cells” at the Cell Culture Laboratory of University of Applied Science Aachen. Additionally, I'm working as a student assistant in the cell culture laboratory at RWTH Aachen Teaching Hospital.<br />
I'm also a member of the Aachen Entrepreneurship Club (AC.E) and work with other students on different projects like “3 Day Startup”. Besides these tasks, I keep different kinds of bird spiders, like to read (there is always time for a good book!) and to meet friends.<br />
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<br />
'''Mainly involved in...'''<br />
* lab work<br />
* [https://2014.igem.org/Team:Aachen/PolicyPractices/Economics economical considerations]<br />
* finances<br />
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{{Team:Aachen/Footer}}</div>Nbaillyhttp://2014.igem.org/User:R.hankeUser:R.hanke2014-10-18T03:16:07Z<p>Nbailly: /* */</p>
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== ==<br />
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'''René Hanke - Lab Division & Public Relations'''<br />
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''Molecular and Applied Biotechnology (B.Sc. student)''<br />
<br />
What facinates me about the iGEM is the chance of designing and realising such an enormous project, autonomously. As Co-founder of our team, I am really glad that we have established such a diverse team, together exploring synthetic biology as a promising, novel field of reasearch.<br />
In my opinion, the iGEM-competition bears a unique potential not only to learn new biotechnological techniques and methods, but to gain experience in a lot of other areas such as engineering, public relations and fundraising.<br />
Moreover, the prospect to face and solve the challenges of participating in an international competition tempts me. I particularly await visiting Boston and taking part in the final Jamboree!!<br />
<br />
If there i some time apart from working for the iGEM I love to go dancing or enjoy reading some thrilling books.<br />
<br />
'''Mainly involved in...'''<br />
* [https://2014.igem.org/Team:Aachen/Collaborations/Kaiser-Karls-Gymnasium Kaiser-Karls-Gymnasium] chool project<br />
* presenting our devices at the [https://2014.igem.org/Team:Aachen/Collaborations/MakerFaire MakerFaire]<br />
* organisation, lab work, fundraising, presentation, embellishing our wiki<br />
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{{Team:Aachen/Footer}}</div>Nbailly