Difference between revisions of "Team:Freiburg/Demonstrate"

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<h1 align="center">Proof of Concept</h1>
 
<h1 align="center">Proof of Concept</h1>
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<p>The <a href=”https://2017.igem.org/Team:Freiburg/Design”>CARTEL<sup>™</sup>AND gate</a> is a genetic circuit constructed of two promoters interconnected through a protein, to integrate two inputs into one <a href=”https://2017.igem.org/Team:Freiburg/CAR”>output</a>. For input one, two alternatives were evaluated. As a proof of concept we characterized the promoters in human cell lines. </p>
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<p>In order to characterize the promoters we generated stable cell lines, containing the promoters for the inputs driving a reporter protein. During the summer we generated stable cell lines with multiple enhancer elements for the inputs: <a href=”https://2017.igem.org/Team:Freiburg/Design”>pH, <a href=”https://2017.igem.org/Team:Freiburg/Design”>VEGF</a> and <a href=”https://2017.igem.org/Team:Freiburg/Design”>hypoxia</a> in Jurkat and HEK293T cells. Additional transient experiments were performed with HEK293T and CHO-K1 cells. These cells were exposed to <a href=”https://2017.igem.org/Team:Freiburg/Results”>low pH</a>, <a href=”https://2017.igem.org/Team:Freiburg/Results”>high VEGF concentration</a> and <a href=”https://2017.igem.org/Team:Freiburg/Results”>hypoxia</a> and the output was measured via <a href=”https://2017.igem.org/Team:Freiburg/Methods”>flow cytometry</a>.</p>
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Revision as of 12:01, 30 October 2017


Logo
Overview Motivation CAR T Cells Tumor Microenvironment AND Gate Outlook Achievements References
Members Attributions Partners Contact
Main Project Modeling Applied Design Proof of Concept BioBricks Basic Part BioBrick Improvement Interlab Study
Safety Cloning HIF1A Knockdown Cell Culture Lab Book
Human Practice Integrated Human Practice Collaborations

Proof of Concept

The CARTEL™AND gate is a genetic circuit constructed of two promoters interconnected through a protein, to integrate two inputs into one output. For input one, two alternatives were evaluated. As a proof of concept we characterized the promoters in human cell lines.

In order to characterize the promoters we generated stable cell lines, containing the promoters for the inputs driving a reporter protein. During the summer we generated stable cell lines with multiple enhancer elements for the inputs: pH, VEGF and hypoxia in Jurkat and HEK293T cells. Additional transient experiments were performed with HEK293T and CHO-K1 cells. These cells were exposed to low pH, high VEGF concentration and hypoxia and the output was measured via flow cytometry.