Team:LMU-Munich/ApoControl

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Latest revision as of 18:54, 26 October 2010


	Cut'N'Survive System Functional Principle Sequences Schedule Jump-or-Die System Functional Principle Sequences Schedule ProSearch System Schedule

	Functional Principle Sequences Schedule picture gallery

ApoControl

The ApoControl team consists of 12 biology students from the undergraduate school of biology at LMU. We are currently undertaking three subprojects on controllable cell-death. The goal is to develop a system to improve the efficiency and specificity of gene expression in eukaryotic cell-lines and more specifically, to select cells expressing the target gene against cells that do not. Here proapoptotic genes instead of antibiotic resistance are used as selection markers to induce clean cell-death at different stimuli. The project is divided into three parts, each representing different approaches to achieve this goal.

Cut'N'Survive System

In this system, the apoptosis triggering protein Bak is controlled by the tet-on system for inducible apoptosis. Bak can be degraded when the target gene is successfully expressed, therefore ensuring the survival of the wished cells. Bak is combined with a certain interacting protein p14*, TEV-protease recognition site and N-Degron. This construct should be stably integrated into the cell line genome. The target plasmid consists of eGFP as a target gene, another TEV-protease recognition site, the interaction partner of p14* (SF3B) and the TEV-protease. When the target plasmid is expressed, the interaction proteins bring the TEV-protease together with its recognition site; TEV cuts Bak, together with N-Degron, free from the rest of the protein complex, where N-Degron with its free N-terminal acts as a degradation signal of protease in the cells. Thus this system enables selection of cells expressing the target gene while the not transfected cells undergo induced apoptosis.

Jump-or-Die System

This system also uses Bak as apoptosis inducing selection marker together with the tet-on promoter, which is to be stably integrated in the cell-line genome. However, this construct also includes an upstream bacterial attachment site (attB) and a downstream SV40 polyadenylation site (SV40PA). The target gene eGFP is combined with phage attachment site (attP) and another SV40PA but without promoter. The target plasmid is co-transfected with integrase PhiC31o into the cell line. Therefore the target gene can only be expressed if integrated into the cells’ genome, and the stop-codon and SV40PA following the target gene stop the expression of bak, ensuring the survival of the cell while other cells not expressing the target gene undergo induced apoptosis.

ProSearch System

This system is to search for new enhancer-promoter combinations that efficiently trigger gene expression in response to different stimuli. This part of the project is carried out through a bioinformatic modeling process. The end-product is a databank of active genes, their promoters and transcription factors at the input of a wished environmental condition. This system is useful for the construction of new inducible systems trying to utilize apoptosis or other gene expression in the human cell-line 293T.

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-== Apo-Control ==
+[[image:Apologo.png|600px|center|ApoControl logo]]
-Das Team „Apo-Control“ besteht aus 12 Studenten aus dem Fachbereich Biologie. Wir haben uns vorgenommen zwei Unterprojekte zu realisieren, welche mit Apoptose, dem kontrollierten Zelltod arbeiten. Das Ziel ist jeweils die Selektion von Zellen, welche ein Zielgen exprimieren gegenüber Zellen, die dies nicht tun, weil  sie zum Beispiel das entsprechende Plasmid nicht aufgenommen haben. Die Zellen, welche das Zielgen nicht exprimieren sterben daraufhin.
+== ApoControl ==
-Beim „TEV-System“ soll ein Apoptose-auslösendes Protein (Bak) abgebaut werden und gleichzeitig ein Zielgen exprimiert werden. Das funktioniert wie in der Abbildung dargestellt. Bak wird mit interagierendem Protein A, N-Degron und TEV-Schnittstelle verbunden. An eGFP wird ebenfalls eine TEV-Schnittstelle und die TEV-Protease angehängt.  Nun fügen sich die interagierenden Proteine zusammen, dadurch schneidet die TEV-Ptorease an den Schnittstellen und das N-Degron hat einen freien N-Terminus (=Abbausignal in Zellen) und der ganze Proteinkomplex inklusive Bak, exklusive dem Zielgen wird abgebaut. Das Kontrukt mit Bak soll stabil in das Zellgenom eingebaut werden, was durch Antibiotikaresistenz selektiert wird.
+The ApoControl team consists of 12 biology students from the undergraduate school of biology at LMU. We are currently undertaking three subprojects on controllable cell-death. The goal is to develop a system to improve the efficiency and specificity of gene expression in eukaryotic cell-lines and more specifically, to select cells expressing the target gene against cells that do not. Here proapoptotic genes instead of antibiotic resistance are used as selection markers to induce clean cell-death at different stimuli. The project is divided into three parts, each representing different approaches to achieve this goal.
-Für das „Jump-in-System“  wird ein bestimmtes DNA Konstrukt in das eukaryotische Genom eingebaut und mit Antibiotikaresistenz selektiert. Diese Sequenz codiert für einen induzierbaren Promoter, eine „Insert-Sequenz 1“ und ein Apoptose-auslösendes Gen (Bax).  Das Zielgen wird in einen Vektor mit „Insert-Sequenz 2“ und einem Stopcodon aber ohne Promotor eingebracht. Wenn die Zellen nun das Plasmid aufnehmen, bewirkt eine Integrase den Einbau des Zielgens an die Stelle der „Insert-Sequenz 1“. Da im Genom schon ein Promotor eingebaut ist, kann das Zielgen nun abgelesen werden, durch das Stopcodon jedoch nicht das nachfolgende Apoptose-auslösende Gen. In Zellen, wo die Integration des Zielgens ins Genom nicht fuktioniert hat, wird das Pro-Apoptotische Gen abgelesen un die Zelle stirbt.
+[[Image:cnsZelle.png|120px|Logo|right]]
-== Meilensteine ==
+===Cut'N'Survive System===
+In this system, the apoptosis triggering protein Bak is controlled by the tet-on system for inducible apoptosis. Bak can be degraded when the target gene is successfully expressed, therefore ensuring the survival of the wished cells. Bak is combined with a certain interacting protein p14*, TEV-protease recognition site and N-Degron. This construct should be stably integrated into the cell line genome. The target plasmid consists of eGFP as a target gene, another TEV-protease recognition site, the interaction partner of p14* (SF3B) and the TEV-protease. When the target plasmid is expressed, the interaction proteins bring the TEV-protease together with its recognition site; TEV cuts Bak, together with N-Degron, free from the rest of the protein complex, where N-Degron with its free N-terminal acts as a degradation signal of protease in the cells. Thus this system enables selection of cells expressing the target gene while the not transfected cells undergo induced apoptosis.
-Für die Projekte haben wir uns jeweils folgende „Meilensteine“ gesetzt:
+[[Image:Jumplogo.png|120px|Logo|right]]
-=== „Split-Ubiquitin-System“ ===
+===Jump-or-Die System===
+This system also uses Bak as apoptosis inducing selection marker together with the tet-on promoter, which is to be stably integrated in the cell-line genome. However, this construct also includes an upstream bacterial attachment site (attB) and a downstream SV40 polyadenylation site (SV40PA). The target gene eGFP is combined with phage attachment site (attP) and another SV40PA but without promoter. The target plasmid is co-transfected with integrase PhiC31o into the cell line. Therefore the target gene can only be expressed if integrated into the cells’ genome, and the stop-codon and SV40PA following the target gene stop the expression of bak, ensuring the survival of the cell while other cells not expressing the target gene undergo induced apoptosis.
-. Verknüpfen von Bak mit interagierendem Protein A, einem N-Degron und einer Schnittstelle für die TEV-Protesase, sodass Bak noch aktiv bleibt. Davor ist ein induzierbarer Promotor und eine Antibiotikaresistenz geschaltet (Nachweis: induzierbarer Zelltod)
+[[Image:prosearchZelle.jpg|120px|Logo|right]]
-Aufwand: ca. 6-8 Wochen,  2000€
-. Verknüpfen von eGFP als Zielgen mit einer TEV-Schnittstelle, einer  TEV-Protease, und interagierendem Protein B. (Nachweis: eGFP leuchtet, Proteinchromatographie)
+===ProSearch System===
+This system is to search for new enhancer-promoter combinations that efficiently trigger gene expression in response to different stimuli. This part of the project is carried out through a bioinformatic modeling process. The end-product is a databank of active genes, their promoters and transcription factors at the input of a wished environmental condition. This system is useful for the construction of new inducible systems trying to utilize apoptosis or other gene expression in the human cell-line 293T.
-Aufwand: ca. 6-8 Wochen, 2000€, Parallele Arbeit ist möglich und notwendig
-. Einbringen beider Konstrukte in Zellen (Nachweis: eGFP leuchtet, einige Zellen sterben, Proteinchromatographie)
-Aufwand: ca. 2 Wochen, 1000€
-. Parallel wird versucht, das erste Konstrukt stabil ins Zellgenom einzubauen.
-Aufwand: ca. 2 Wochen, 500€
-=== „Jump-in-System“ ===
-. Bau des in die Zelle zu integrierenden Kontrukts: Antibiotikaresistenz mit Promotor, ein weiterer induzierbarer Promotor, Insert-Sequenz 1, proapoptotisches Gen (Bax) (Nachweis:  induzierbare Apoptose)
-Aufwand: 3–4 Wochen, 2000€
-. Integration des Konstruktes in Zellen (Nachweis: Antibiotikaresistenz nach einigen Tagen)
-Aufwand: 2–4 Wochen, 1500€
-. Konstruktion des Zielgenvektors mit Insert-Sequenz 2, eGFP als Zielgen und einem Stopcodon (Nachweis: nicht einzeln möglich)
-Aufwand: 2 Wochen, 500€<br>
-Parallele Arbeit ist möglich und notwendig
-. Transfektion der Zellen mit integriertem Konstrukt mit dem Zielgenvektor. Einbau des Zielgens ins Genom (Nachweis: Induzierter Zelltod bei nicht-Integration, Zielgennahweis bei Integration)
-Aufwand: 2 Wochen, 1000€
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