Meetings

From 2008.igem.org

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<font face="Arial Rounded MT Bold" style="color:#010369">_meetings</font></div>
<font face="Arial Rounded MT Bold" style="color:#010369">_meetings</font></div>
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;April 24th 2008 <br>
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;Apr. 24th 2008
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Recruitment of team<br>
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;Apr. 30th 2008
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;May 8th 2008 <br>
;May 8th 2008 <br>
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'''Teilnehmer'''<br>
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'''attendees'''<br>
Philipp Mappes, Wolfgang Schamel, Kilian Bartholomé, Katja Arndt, Kristian Müller, Dario Hermida Aponte, Normann Kilb, Robert Gawlik, Simone Weber, Veronika Götz, Kristina Brückner, Kathrin Pieper, Michael Kneib, Moritz Busacker, Sabine Jägle
Philipp Mappes, Wolfgang Schamel, Kilian Bartholomé, Katja Arndt, Kristian Müller, Dario Hermida Aponte, Normann Kilb, Robert Gawlik, Simone Weber, Veronika Götz, Kristina Brückner, Kathrin Pieper, Michael Kneib, Moritz Busacker, Sabine Jägle
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'''Wie soll das Projekt durchgeführt werden?''' <br>
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'''How to organize the project?''' <br>
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Zuerst soll die DNA Struktur auf dem etablierten T-Zellsystem getestet werden und dann werden wir versuchen ein künstliches System herzustellen. Als cytoplasmatische Domäne kommen beispielsweise der EGFR oder Fas in Frage. Da Fas bei der Apoptose beteiligt ist könnte man hier dann die erfolgte Signalweiterleitung anhand des Zellsterbens beurteilen. Weitere Systeme die für die Signalweiterleitung in Frage kommen wären z.B. NF-kappaB, das MAP-Kinase-Jun-Fos-System oder durch Split-Enzyme, die bei Stimulation der Zelle zusammenkommen und dann aktiv werden.  
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The DNA origamis shall be tested on the established T-cell system. Then we will try to establish an artificial system. Extracellular fragments could be the EGFR or Fas for example. Since the Fas receptor is involved in apoptosis, signaltransduction could be detectable by cell death. Further systems for detecting signaltransduction could be NF-kappaB, the MAP-Kinase-pathway, the Jun-Fos-system or splitenzymes.<br>
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'''DNA-origami structure'''<br>
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How expensive will the synthesis of the NIP coupled Oligos be? Phillip and Michael will contact the company and ask for an calculation of costs. NIP can be ordered by the company Biosearch technology.<br>
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'''Aufbau der Nanostruktur'''<br>
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'''Controls for the DNA origamis'''<br>
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Wie teuer wird es, wenn wir uns die mit Nitrojodophenol gekoppelten Oligos synthetisieren lassen? Philipp und Michael werden sich mit der Firma in Verbindung setzen und ein Preisangebot einholen. NIP kann von der Firma Biosearch Technologie gekauft werden.
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* We should produce a DNA origami without NIP-coupled Oligos to check if there also a cell binding occurs (negative control)<br>     
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* To check the calculated distances between the NIP's on the origami, DNA-origami with fluorophores could be made. By using FRET the distance between the oligo's can be estimated.<br>
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'''Kontrolle der Nanostruktur'''<br>
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<br>
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* Als eine Negativkontrolle sollten wir eine DNA Nanostruktur ohne gekoppelte Oligos herstellen und prüfen, ob hier auch eine Bindung zustande kommt
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* Um zu prüfen ob die Oligos bzw die NIP wirklich die geplanten Abstände haben könnte eine Nanostruktur mit einem Fluorophoren FRET Paar gemacht werden. Dadurch könnten die Abstände der gekoppelten Oligos genau bestimmt werden
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;May 23th 2008  
;May 23th 2008  
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;Jun 2th 2008 <br>
;Jun 2th 2008 <br>
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'''Teilnehmer'''<br>
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'''attendees'''<br>
Sabine Jägle, Normann Kilb, Robert Gawlik, Kristian Müller, Philipp Mappes
Sabine Jägle, Normann Kilb, Robert Gawlik, Kristian Müller, Philipp Mappes
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'''Besprochenes'''<br>
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'''We have talked about...'''<br>
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* Anbringen längerer NIP-Linker um optimale Bindung an den AK zu gewährleisten.
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* To ensure an optimal NIP-scFV-Anti-NIP binding a longer NIP-Linker should be used
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* NIP-Abstände betragen (rechnerisch) 6 nm - 7 nm.  
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* Calculated distances between the NIP's are 6-7 nm.  
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* Einbau des TCR in die Grafik um einen Größenvergleich zu haben.
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* Integration of the TCR in the model to achieve a comparison of the sizes
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* Suche nach einer geeigneten Transmembranhelix für den künstlichen Rezeptor.
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* Searching a suitable transmembraneregion for the artificial receptor
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* Kostenvoranschlag von BIOSEARCH für 10 Oligos mit NIP am 5' bzw. 3'-Ende: 3000,-US$ bei unbekannter Kopplungseffizienz
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* Calculation of costs for 10 Oligos wirh NIP coupled to the 5' or 3'-end: 3000,-US$ at uncertain coupling efficiency
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* => Bestellung voraussichtlich über PURIMEX, Anfrage/Planung läuft...
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* => Order presumably from PURIMEX
;Jun 13th 2008  
;Jun 13th 2008  
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'''Teilnehmer'''<br>
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'''attendees'''<br>
Michael Kneib, Robert Gawlik, Philipp Mappes, Simone Weber, Kristina Brückner, Sabine Jägle, Kathrin Pieper
Michael Kneib, Robert Gawlik, Philipp Mappes, Simone Weber, Kristina Brückner, Sabine Jägle, Kathrin Pieper
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'''Bestellung der NIPs'''<br>
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'''Ordering of NIPs'''<br>
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Aufbau der DNA-Nanostruktur mit den gekoppelten NIPs wurde besprochen und entschieden:
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Assembly of the DNA origami with coupled NIPs was discussed and decided:
[[image:Freiburg08_NIP_13_06_08.jpg]]
[[image:Freiburg08_NIP_13_06_08.jpg]]
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Frage: Wie sind die genauen Abstände zwischen den DNA-Helices? --> Rothemund <br>
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Question: What are the exact distances between the DNA-helices/nod-points? --> Rothemund <br>
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'''Die nächsten Schritte'''<br>
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'''The next steps'''<br>
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- Literatursuche für TCR-Clustering<br>
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- searching for literature on TCR-Clustering<br>
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- Literatursuche Transmembranhelices, Signalsequenzen<br>
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- searching for literature on transmembrane-helices, signal sequences<br>
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- Planung des künstlichen Systems mit Antikörperfragment, Transmembranregion, Split-Enzym, Enzymtest<br>
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- planning of the artificial system with antibodyfragment, transmembraneregion, splitenzymes, enzyme assay<br>
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- EGFR als weiteres Testsystem<br>
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- EGFR as further test-system<br>
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;16. Juni 2008 10.00h
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;Jun 16th 2008 10.00h
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Vorbeireitung der Präsentation für Dienstag 12.00h
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Preparation of intern presentation on Tuesday
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;17. Juni 2008 12.00Uhr
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;Jun 17th 2008 12.00h
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'''Besprochenes'''<br>
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'''We have talked about...'''<br>
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- Bis Freitag werden nach Rücksprache mit Schamels Doktorandin die NIP-gekoppelten-Oligos bestellt --> Phillip<br>
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- Until Friday we will order the NIP-coupled-Oligos. Consultation of Mahima: Philipp<br>
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- Die Vektoren für die Klonierungen zusammentragen (Dina/Christina: Vektor mit CMV-Promotor) --> Kathrin <br>
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- We should organize to get the vectors for cloning (Dinah/Christina: Vector with CMV-Promoter) --> Kathrin <br>
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- Schamels Versuche zum TCR "nachkochen" --> Terminabsprache mit der Doktorandin<br>
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- Reproduce Schamels experiment with the TCR --> appointment with Mahima<br>
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- Klonierungsstrategien entwerfen (dabei IGEM-Schnittstellen-Vorgaben beachten) <br>
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- Think about a cloning strategy using the BioBrick pre- and suffix <br>
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- Literatur-Recherche Transmembranhelices WF DeGrado<br>
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- Literature transmembranhelices WF DeGrado<br>
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- Programm für Klonierungen: Emboss (emboss.org)<br>
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- Programme for cloning: Emboss (emboss.org)<br>
<br>
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;23. Juni 2008 12.00Uhr
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;Jun 23rd 2008 12.00h
<div class=Section1>
<div class=Section1>
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<p class=MsoNormal style='margin-left:18.0pt'><b>Anwesend</b></p>
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<p class=MsoNormal style='margin-left:18.0pt'><b>attendees</b></p>
<p class=MsoNormal style='margin-left:18.0pt'>Robert Gawlik, Philipp Mappes,
<p class=MsoNormal style='margin-left:18.0pt'>Robert Gawlik, Philipp Mappes,
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<p class=MsoNormal style='margin-left:18.0pt'>&nbsp;</p>
<p class=MsoNormal style='margin-left:18.0pt'>&nbsp;</p>
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<p class=MsoNormal style='margin-left:18.0pt'><b>Besprochenes</b></p>
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<p class=MsoNormal style='margin-left:18.0pt'><b>We have talked about...</b></p>
<p class=MsoNormal style='margin-left:29.35pt;text-indent:-11.35pt'><span
<p class=MsoNormal style='margin-left:29.35pt;text-indent:-11.35pt'><span
style='font-family:Symbol'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
style='font-family:Symbol'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
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</span></span>Oligos sind bestellt und sollten in der Woche vom 10.Juli
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</span></span>Oligos are ordered. They should be delivered until the 10th of July.</p>
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geschickt werden.</p>
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<p class=MsoNormal style='margin-left:29.35pt;text-indent:-11.35pt'><span
<p class=MsoNormal style='margin-left:29.35pt;text-indent:-11.35pt'><span
style='font-family:Symbol'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
style='font-family:Symbol'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
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</span></span>Transmembran-Region: </p>
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</span></span>Transmembraneregion: </p>
<p class=MsoNormal style='margin-left:54.0pt;text-indent:-18.0pt'><span
<p class=MsoNormal style='margin-left:54.0pt;text-indent:-18.0pt'><span
style='font-family:"Courier New"'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
style='font-family:"Courier New"'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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</span></span>Literaturrecherche zur Transmembranregion</p>
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</span></span>Literature about the transmembraneregion</p>
<p class=MsoNormal style='margin-left:54.0pt;text-indent:-18.0pt'><span
<p class=MsoNormal style='margin-left:54.0pt;text-indent:-18.0pt'><span
style='font-family:"Courier New"'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
style='font-family:"Courier New"'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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</span></span>Welche Transmembranregionen können benutzt werden z.B. EGF-R</p>
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</span></span>Which transmembraneregion can be used? For example EGF-R</p>
<p class=MsoNormal style='margin-left:54.0pt;text-indent:-18.0pt'><span
<p class=MsoNormal style='margin-left:54.0pt;text-indent:-18.0pt'><span
style='font-family:"Courier New"'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
style='font-family:"Courier New"'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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</span></span>Signalpeptid?!</p>
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</span></span>Signalpeptide?!</p>
<p class=MsoNormal style='margin-left:54.0pt;text-indent:-18.0pt'><span
<p class=MsoNormal style='margin-left:54.0pt;text-indent:-18.0pt'><span
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<p class=MsoNormal style='margin-left:27.0pt;text-indent:-11.35pt'><span
lang=EN-GB style='font-family:Symbol'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
lang=EN-GB style='font-family:Symbol'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
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</span></span><span lang=EN-GB>synthetische Transmembranregion: </span></p>
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</span></span><span lang=EN-GB>synthetic transmembraneregion: </span></p>
<p class=MsoNormal style='margin-left:90.0pt;text-indent:-18.0pt'><span
<p class=MsoNormal style='margin-left:90.0pt;text-indent:-18.0pt'><span
style='font-family:"Courier New"'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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</span></span>Aus welchen Teilen soll der synthetische Rezeptor zusammen gebaut
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</span></span>Which parts shall build the synthetic receptor?</p>
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sein?</p>
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<p class=MsoNormal style='margin-left:72.0pt'>&nbsp;</p>
<p class=MsoNormal style='margin-left:72.0pt'>&nbsp;</p>
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<p class=MsoNormal style='margin-left:27.0pt;text-indent:-11.35pt'><span
lang=EN-GB style='font-family:Symbol'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
lang=EN-GB style='font-family:Symbol'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
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</span></span><span lang=EN-GB>Vektoren: </span></p>
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</span></span><span lang=EN-GB>Vectors: </span></p>
<p class=MsoNormal style='margin-left:54.0pt;text-indent:-18.0pt'><span
<p class=MsoNormal style='margin-left:54.0pt;text-indent:-18.0pt'><span
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style='font-family:"Courier New"'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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</span></span>Genkarten für Vektoren müssen gesammelt werden</p>
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</span></span>Genemaps for the Vectors</p>
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<p class=MsoNormal style='margin-left:54.0pt;text-indent:-18.0pt'><span
style='font-family:"Courier New"'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
style='font-family:"Courier New"'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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</span></span>Vorhandene Vektoren müssen mit den IGEM-System(Freiburg2007-WIKI
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</span></span>Available Vectors have to be compared to the iGEM-Systeme(Freiburg2007-WIKI)</p>
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Seite) verglichen werden</p>
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<p class=MsoNormal style='margin-left:54.0pt;text-indent:-18.0pt'><span
<p class=MsoNormal style='margin-left:54.0pt;text-indent:-18.0pt'><span
style='font-family:"Courier New"'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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</span></span>evtl. suche nach geeigneten Vektoren</p>
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</span></span>possibly searching suitable vectors</p>
<p class=MsoNormal><span lang=EN-GB>&nbsp;</span></p>
<p class=MsoNormal><span lang=EN-GB>&nbsp;</span></p>
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<p class=MsoNormal style='margin-left:15.65pt'><b><span lang=EN-GB>Aufgaben</span></b></p>
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<p class=MsoNormal style='margin-left:15.65pt'><b><span lang=EN-GB>jobs</span></b></p>
<p class=MsoNormal style='margin-left:29.35pt;text-indent:-11.35pt'><span
<p class=MsoNormal style='margin-left:29.35pt;text-indent:-11.35pt'><span
lang=IT style='font-family:Symbol'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
lang=IT style='font-family:Symbol'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
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</span></span><span lang=IT>Literaturrecherche Transmembranregion/Signalpeptid </span><span
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</span></span><span lang=IT>Literature transmembraneregion/signalepeptide </span><span
lang=EN-GB style='font-family:Wingdings'>:</span><span lang=IT> Simone, Kristina,
lang=EN-GB style='font-family:Wingdings'>:</span><span lang=IT> Simone, Kristina,
Sabine</span></p>
Sabine</span></p>
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<p class=MsoNormal style='margin-left:29.35pt;text-indent:-11.35pt'><span
lang=IT style='font-family:Symbol'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
lang=IT style='font-family:Symbol'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
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</span></span><span lang=EN-GB>Vektorsequenzen </span><span lang=EN-GB
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</span></span><span lang=EN-GB>Vectorsequences </span><span lang=EN-GB
style='font-family:Wingdings'>:</span><span lang=EN-GB> Philipp, Kathrin,
style='font-family:Wingdings'>:</span><span lang=EN-GB> Philipp, Kathrin,
Michael</span></p>
Michael</span></p>
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style='font-family:Symbol'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;
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</span></span>Einrichten unserer Gruppe  auf offizielle Wikiseite <span
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</span></span>Registry on the official WIKI<span
lang=EN-GB style='font-family:Wingdings'>:</span>Robert</p>
lang=EN-GB style='font-family:Wingdings'>:</span>Robert</p>
</div>
</div>
<br />
<br />
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;3. Juli 12.00 Uhr
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;July 3rd 12.00h
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'''Teilnehmer''' <br>
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'''attendees''' <br>
Robert Gawlik, Michael Kneib, Kathrin Pieper, Simone Weber, Daniel Hautzinger, Sabine Jägle, Philipp Mappes, Kristian Müller
Robert Gawlik, Michael Kneib, Kathrin Pieper, Simone Weber, Daniel Hautzinger, Sabine Jägle, Philipp Mappes, Kristian Müller
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'''1. Vektoren'''<br>
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'''1. Vectors'''<br>
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Philipp und Kathrin haben sich in GCG eingearbeitet. Dabei hat sich gezeigt, dass in der Fab Fragment Sequenz eine IGEM Standard Schnittstelle befindet. In dem Vektor von Christina befinden sich 7 Schnittstellen.<br>
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Philipp and Kathrin have worked with GCG. In doing so they saw that the fab fragment has iGEM restriction side. The vector from Christina, which we wanted to use, has also 7 iGEM restrictions sides.<br>
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Es bieten sich die Möglichkeiten der klassischen Mutagenese oder der Gensynthese. Von Geneart gibt es das Gensyntheseangebot 20 Cent pro Base. Dies gilt allerdings nur für 2500 Euro, anschließend gilt der Preis von 50 Cent pro Base. Falls wir die Gensynthese machen wollen, sollte man bei Geneart anfragen, ob die Vektoren, die sie verwenden auch keine IGEM (EcoRI, XbaI, SpeI, PstI) und keine von unseren Schnittstellen (NgoMIV, AgeI) haben.<br>
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We could mutagenesis or gensynthesis to get the iGEM restrictions sides. Genart is offering a special price for gen synthesis. If we want to use this special offer, we have to check which vector they use. It is important that their vector has not any of the iGEM restrictions sides (EcoRI, XbaI, SpeI, PstI) and non of ours restriction sides(NgoMIV, AgeI).<br>
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Die restlichen Sequenzen, die noch gefehlt haben, hat Mahima inzwischen im richtigen Format geschickt, sodass diese Sequenzen nun auch noch auf die Schnittstellen untersucht werden können (Kathrin und Philipp?).<br>
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All of the sequences from Mahima arrived by now and they can be checked. A vector wiith CMV-promotor could be also saerched on the wiki 2007.<br>
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Man könnte auch noch mal auf der bei den IGEM parts nach Vektoren mit CMV Promotor schauen.<br>
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'''2. Konzentration der DNA Struktur'''<br>
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'''2. concentration of DNA structure'''<br>
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Daniel hat eine Konzentration von 20 nM berechnet, die eventuell auch auf 200 nM erhöht werden könnte . Laut Mahima ist aber mindestens eine Konzentration von 2 µM nötig. Eventuell können wir eine geringere Konzentration einsetzen, allerdings können wir dann wahrscheinlich keine Unterschiede mehr detektieren, ob 2, 3 oder 4 NIP's gebunden haben.<br>
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theoretically possible: 20 nM, maybe even 200 nM; according we need at least 2 µM to distinct between 2, 3 or 4 NIPs<br>
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'''3. DNA Strukturen'''<br>
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'''3. DNA-Origami'''<br>
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* Kontrolle ob DNA mit NIP's an die Zellen bindet. Dafür DNA Struktur mit 4 NIP's und 2 Fluorophoren. Den Nachweis der Fluoreszenz als Beweis für die Bindung der DNA Struktur an die Zellen. Siehe auch Konfokale Mikroskopie.
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* binding measurement: Origami with 4 NIP molecules and 2 fluorophores<br>
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* Negativkontrolle. DNA Struktur ohne NIP's, mit 2 Fluorophoren. Nachweis dass diese Struktur alleine nicht zu einer Stimulation des T-Zellrezeptors führt.
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* negative control: DNA-Origami without NIP but with fluorophores<br>
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* Michael, Simone und Daniel machen M13 Phagen Prep und den Oligopool.<br>
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* Michael, Simone and Daniel: preparation of M13 phage DNA and assessment of oligo-pool<br>
    
    
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'''4. Nachweis der T-Zellrezeptor-Stimulation'''<br>
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'''4. proof of principle: T-cell-receptor activation'''<br>
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* Ca-Messung: Bei 530 nm. Gerät im MPI ist allerdings kaputt?
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* Ca-measurement at 530 nm<br>
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* GFP Marker: Genexpression ist nach 12-18 h nachweisbar. GFP positive Zellen werden mit dem FACS bestimmt. Eventuell wäre es möglich ein FACS in der Uniklinik zu verwenden. Kathrin erkundigt sich bei Dr. Eibel.<br>
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* GFP-marker: detection of product after 12 to 18 hours; maybe we can measure FACS at the universities hospital?<br>
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'''5. Konfokale Mikroskopie'''
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'''5. confocal microscopy'''
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* Im MPI muss man zuerst ein Trainingsprogramm absolvieren, worauf man allerdings auch 3 Wochen warten muss
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*the MPI requires the absolvation of a training, we don´t have time for that;<br>
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* Wir könnten das konfokale Mikroskop im Fischbach Labor nutzen. Da stehen die Wellenlängen 488nm und 568nm zur Verfügung.Ansprechpartner ist Gerit.<br>
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*wavelength 488nm and 568nm might be measured at another lab<br>
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'''6. Allgemeines'''<br>
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'''6. "in general"'''<br>
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* Rezepte von Puffern usw auf's Wiki schreiben
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* upload more data onto the intern wiki<br>
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* Wenn man etwas Neues in Erfahrung gebracht hat Rundmail schreiben, später dann aber auch ins Wiki
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* meet more frequently
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* Laborjournal wird chronologisch geführt
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* protocol/write labjournal on wiki
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* Anlegen eines Registers z.B. auf der ersten Seite des Laborbuchs. Hier kann man dann noch mal ausführlicher beschreiben was genau in den Eppi's drin ist<br>
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* precise documentation of every generated sample<br>
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'''10. Juli 2008 12:00 Uhr'''
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'''July 10th, 2008'''
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Teilnehmer:
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Attendees:
<p><font face="Arial">Simone Weber, Sabine Jägle, Kathrin Pieper, Phillip Mappes,  
<p><font face="Arial">Simone Weber, Sabine Jägle, Kathrin Pieper, Phillip Mappes,  
Norman Kilb, Robert Gawlik, Daniel Hautzinger, Michael Kneib, Kristian Müller,  
Norman Kilb, Robert Gawlik, Daniel Hautzinger, Michael Kneib, Kristian Müller,  
Kristina Brückner</font></p>
Kristina Brückner</font></p>
<p>&nbsp;</p>
<p>&nbsp;</p>
-
<p><font face="Arial"><b>1. Sicherheitseinführung</b></font></p>
+
<p><font face="Arial"><b>1. Security instruction</b></font></p>
-
<p><font face="Arial"><b>&gt; </b>interne Notrufnummer: 2000</font></p>
+
<p><font face="Arial"><b>&gt; </b>emergency call: 2000</font></p>
-
<p><font face="Arial"><b>2. Stand der Dinge</b></font></p>
+
<p><font face="Arial"><b>2.actual state of affairs</b></font></p>
<ul>
<ul>
-
<li><font face="Arial">M13-Phagen DNA wurde geprept</font></li>
+
<li><font face="Arial">M13mp18 phage DNA was preped</font></li>
-
<li><font face="Arial">inzwischen sind alle Oligos eingetroffen --&gt; Pool
+
<li><font face="Arial">The Oligios we ordered arrived --&gt; A poll was made, the pool contains all the Oligos except of the 10 which are to be marked with NIP or fluorophor </font></li>
-
wurde hergestellt indem alle drin sind, außer denen, die Fluoreszenzmarkiert
+
<li><font face="Arial">Ca<sup>2+</sup> - Daniels experiment (making DNA-Origami 1:20 has been reproduced, but we have to check on the AFM if the Origami formed right -> AFM measurement is tomorrow.</font></li>
-
werden oder NIPs tragen</font></li>
+
<li><font face="Arial">We have to reorder some of the Oligo</font></li>
-
<li><font face="Arial">mit DNA-Origamie wurde begonnen --&gt; AFM-Betrachtung
+
-
11.07.08</font></li>
+
-
<li><font face="Arial">ersten Oligos müssen nachbestellt werden (~ 50€)</font></li>
+
</ul>
</ul>
-
<p><font face="Arial"><b>3. Lösung für Konzentrationsproblem</b></font></p>
+
<p><font face="Arial"><b>3. We need to solve the Origami-concentration problem</b></font></p>
<ul>
<ul>
-
<li><font face="Arial">Ca<sup>2+</sup> - Einstrom direkt unter dem
+
<li><font face="Arial">Ca<sup>2+</sup>We could measure the calcium influx with a flourescence microscope. Therefore we have to find out which dye to use</font></li>
-
Fluoreszenzmikroskop anschauen</font></li>
+
-
<li><font face="Arial">welcher Farbstoff hier verwendet werden soll --&gt; wird
+
-
am Freitag, 11.07. besprochen</font></li>
+
</ul>
</ul>
-
<p><font face="Arial"><b>4. Überlegung für einfaches synthetisches System</b></font></p>
+
<p><font face="Arial"><b>4. Think about the synthetic receptor system</b></font></p>
<ul>
<ul>
-
<li><font face="Arial">könnten bereits vorhandenen Vektor nutzen, der
+
<li><font face="Arial">we could use a vector which already has the beta-chain of the TCR coupled to the NIP fab fragment and fuse half of &#946;-Lactamase to each of it</font></li>
-
&#946;-Kette des TCR + das Fab-Fragment enthält</font></li>
+
<li><font face="Arial">if the receptors get clustered by the NIP the &#946;-Lactamase would be functional </font></li>
-
<li><font face="Arial">auf cytoplasmatische Seite einfach noch &#946;-Lactamase  
+
-
dran synthetisieren</font></li>
+
-
<li><font face="Arial">zeigt nur, das die NIPs die &#946;-Ketten zusammen und
+
-
dadurch die die &#946;-Lactamase aktiviert wird</font></li>
+
</ul>
</ul>
-
<p><b><font face="Arial">5. Substrat für &#946;-Lactamase</font></b></p>
+
<p><b><font face="Arial">5. substrates for the &#946;-Lactamase</font></b></p>
<ul>
<ul>
-
<li><font face="Arial">muss demnächst bestellt werden</font></li>
+
<li><font face="Arial">have to be ordered</font></li>
-
<li><font face="Arial">welches --&gt; Norman informiert sich</font></li>
+
<li><font face="Arial">Norman will get some information about diffrent &#946;-Lactamase and order it </font></li>
</ul>
</ul>
-
<p><b><font face="Arial">6. Protokolle für Transformationen</font></b></p>
+
<p><b><font face="Arial">6. Transfection Protocol</font></b></p>
<ul>
<ul>
-
<li><font face="Arial">in Kristians Labor vorhanden</font></li>
+
<li><font face="Arial">available in Kristians lab</font></li>
</ul>
</ul>
-
<p><b><font face="Arial">7. Positiv Kontrolle für Transfektionen</font></b></p>
+
<p><b><font face="Arial">7. Positive control for the transfections</font></b></p>
<ul>
<ul>
-
<li><font face="Arial">Norman kümmert sich darum </font></li>
+
<li><font face="Arial">Norman will read about it</font></li>
</ul>
</ul>
-
<p><b><font face="Arial">8. Suche nach weiteren Vektoren</font></b></p>
+
<p><b><font face="Arial">8. Searching other vectores</font></b></p>
<ul>
<ul>
-
<li><font face="Arial">bei Toolbox Freiburg nachfragen</font></li>
+
<li><font face="Arial">Call Tollbox Freiburg</font></li>
</ul>
</ul>
-
<p><b><font face="Arial">9. zweites System für Multimerisierung mit anderen
+
<p><b><font face="Arial">9. second system for multimerisation with different antibodieszweitesn</font></b></p>
-
Antikörpern</font></b></p>
+
<ul>
<ul>
<li><font face="Arial">Antifluorescin-Antikalin (Arne Skerra) --&gt;  
<li><font face="Arial">Antifluorescin-Antikalin (Arne Skerra) --&gt;  
-
Publikation in Literatur!</font></li>
+
Publication in literature!</font></li>
-
<li><font face="Arial">Andreas Blukthuhn (?) --&gt; Dapin (&gt; Kristian kümmert
+
<li><font face="Arial">Andreas Blukthuhn (?) --&gt; Dapin (&gt; Kristian will ask him)</font></li>
-
sich darum)</font></li>
+
</ul>
</ul>
-
<p><font face="Arial"><b>10. intrazellzläre Domäne des EGF-R</b></font></p>
+
<p><font face="Arial"><b>10. intracellular domain of EGF-R</b></font></p>
<ul>
<ul>
-
<li><font face="Arial"> <a href="http://www.ebi.ac.uk">www.ebi.ac.uk</a> &nbsp;&nbsp;&nbsp;&nbsp;  
+
<li><font face="Arial"> http://www.ebi.ac.uk &nbsp;&nbsp;&nbsp;&nbsp;  
--&gt; SRS&nbsp;&nbsp;&nbsp; --&gt; Uniprot</font></li>
--&gt; SRS&nbsp;&nbsp;&nbsp; --&gt; Uniprot</font></li>
-
<li><font face="Arial"><a href="http://www.expasy.ch">www.expasy.ch</a>&nbsp;&nbsp;&nbsp;  
+
<li><font face="Arial"> http://www.expasy.ch &nbsp;&nbsp;&nbsp;  
--&gt; swissprot</font></li>
--&gt; swissprot</font></li>
-
<li><font face="Arial">Michael kümmert sich darum</font></li>
+
<li><font face="Arial">Michael will read about it</font></li>
</ul>
</ul>
<p>&nbsp;</p>
<p>&nbsp;</p>
<br>
<br>
-
<p><font face="Arial"><b>16 Juli 2008 12.00h</b></font></p>
+
<p><font face="Arial"><b> July 16th 2008 12.00h</b></font></p>
-
'''Teilnehmer:''' <br>
+
'''Attendees:''' <br>
Robert Gawlik, Michael Kneib, Kathrin Pieper, Simone Weber, Moritz Busacker, Sabine Jägle, Philipp Mappes, Kristian Müller<br>
Robert Gawlik, Michael Kneib, Kathrin Pieper, Simone Weber, Moritz Busacker, Sabine Jägle, Philipp Mappes, Kristian Müller<br>
'''1. DNA-Origami:''' <br>
'''1. DNA-Origami:''' <br>
-
*nach geglücktem 1. Ansatz jetzt der Versuch mit DNA:Oligos = 1:5 (anstelle von 1:20 wie bisher)<br>
+
We reproduced the experiment of Daniel(Origami 1:20). Now we want to test, if we able to form also Origami when we use less Oligos (ratio 1:10, ratio 1:5) <br>
-
*RPMI-Medium oder Ringer-Solution imaging-geeigneter?<br>
+
We have to test, if it is possible to use Krebs-Ringer-Hepes buffer for the calcium imaging<br>
-
*Herstellung von Fluorophor-Origami für erste Imaging-Versuche; Fluoreszenz-Mikroskopie im MPI oder Fischbach-Labor?<br>
+
We will also produce some Origami coupled with Alexa 488 for first binding measurements. therefore we have also to check were we could measure Alexa 488<br>
-
*Stabilität des Produktes (Transport etc.)?<br>
+
 
-
'''Vektoren / Synthetisches Rezeptorsystem: '''<br>
+
'''Vectors and synthetic receptor system: '''<br>
-
*nach wie vor noch kein uneingeschränkt nutzbarer Vektor verfügbar; evtl. vom Ljubljana-Projekt 2007 (CMV-Promoter als Modul)?<br>
+
still we have not any vector which we can use for the clonings -> we have to check the vector of the Ljubljana-Projekt 2007<br>
-
*deshalb evtl. Umweg über "Zwischenlagerungs-Vektor"<br>
+
Those parts should be planed and ordered:<br>
-
*Planung und Bestellung folgender Parts:<br>
+
-Fab-Singlechain<br>
-Fab-Singlechain<br>
-
-Signalsequenz für die Membranintegration?<br>
+
-Which signal sequence coats for the transmembrane domain<br>
-
-Transmembranregion?<br>
+
-Which transmembran region will we use?<br>
-
-evtl. Split-Fluorophor?<br>
+
-The transmembrane region could be attached to split-fluorophors, so the receptor clustering could be detected in a      fluorescence microscope<br>
-
*Integration von Kurzlinkern in die einzelnen Parts?<br>
+
- Which linker will we use?<br>
-
*für eventuelle EGFR-Parts Rücksprache mit Tillmann Brunner, ZBSA (downstream-signaling)<br>  
+
- We should talk to Tillmann Brunner, ZBSA about using the EGFR<br>
 +
<br>
 +
<br>
<br>
<br>
<br>
<br>
-
'''23 Juli 2008 12.00h'''<br>
+
'''July 23rd 2008 12.00h'''<br>
<br>
<br>
-
<span style="font-weight: bold;">Teilnehmer:</span> <br>
+
<span style="font-weight: bold;">Attendees</span> <br>
Robert Gawlik, Philipp Mappes, Kathrin Pieper,Sabine J&auml;gle,
Robert Gawlik, Philipp Mappes, Kathrin Pieper,Sabine J&auml;gle,
Simone Weber.<br>
Simone Weber.<br>
<br>
<br>
-
<span style="font-weight: bold;">EGF-Rezeptor (EGFR):</span><br>
+
<span style="font-weight: bold;">EGF-Receptor (EGFR):</span><br>
<ul>
<ul>
-
   <li>EGFR als Transmembrandom&auml;ne sinnvoll da er sehr
+
   <li>The T-cell surface has a lot of big molecules on the surface. Those molecules are much bigger than the receptor itself, therefore they could disturb the interaction between the ligand (e.g. Origami) and the receptor.
-
gro&szlig; ist.Auf der T-Zell-Oberfl&auml;che befinden sich
+
-> We should think about using the EGFR as receptor for our clonings, because this one has a relatively big extra cellular domain.</li>
-
viele gro&szlig;e Molek&uuml;le, diese sind viel
+
-
Gr&ouml;&szlig;er als die Rezeptoren. Bei einem kleineren
+
-
Rezeptor w&auml;re es m&ouml;glich, dass die gro&szlig;en
+
-
Molek&uuml;le den Rezeptor &uuml;berlagern und die Origamis
+
-
nicht binden k&ouml;nnen.</li>
+
</ul>
</ul>
<ul>
<ul>
-
   <li>Der EGFR k&ouml;nnte als ganzes System genutzt werden,
+
   <li>Not only a part of the EGFR could be used but the whole receptor, which is additionally linked to the NIP fab fragment by a long linker. So the ligand binding domain should be as big as the surface molecules.</li>
-
an dem die single chain &uuml;ber einen langen Linker gebunden
+
-
wird. So werden Molek&uuml;le weit genug heraus gebracht um
+
-
&uuml;ber andere Molek&uuml;le herauszuragen.</li>
+
</ul>
</ul>
<ul>
<ul>
-
   <li>Drei m&ouml;gliche Klonierungsvorschl&auml;ge:</li>
+
   <li> This 3 constructs could be cloned:</li>
</ul>
</ul>
-
<div style="margin-left: 40px;">1)EGFR + Single chain + 1.
+
<div style="margin-left: 40px;">1)EGFR + Single chain + one half of beta-Lactamase</div>
-
H&auml;lfte der beta-Lactamase</div>
+
<div style="margin-left: 40px;">2)EGFR + Single chain + second half of beta-Lactamase</div>
-
<div style="margin-left: 40px;">2)EGFR + Single chain + 2.
+
<div style="margin-left: 40px;">3)The whole EGFR fused to the NIP fab fragment. With this construct it might be also possible to activate the receptor by adding EGF. Hence we would have a additionally positive control. But first we would have to find a readout for the activation with EGF.<br>
-
H&auml;lfte der beta-Lactamase</div>
+
-
<div style="margin-left: 40px;">3)vollst&auml;ndige EGFR (mit single chain): k&ouml;nnte man mit EGF die Aktivierungdes EGFR messen (welches read out?)<br>
+
-
<br>
+
</div>
</div>
<span style="font-weight: bold;">Fura 2/ Indo-1:</span>
<span style="font-weight: bold;">Fura 2/ Indo-1:</span>
<br>
<br>
<ul>
<ul>
-
   <li>k&ouml;nnen als Farbstoff f&uuml;r Ca2+ Farbstoffe
+
   <li>Both could be used for the Ca2+ signalling. Indo is often used for measurements on the FACS.</li>
-
verwendet werden.</li>
+
</ul>
</ul>
<ul>
<ul>
-
   <li>wir benutzen Fura 2AM -&gt; wird auf die Zelle gegeben,
+
   <li>We will use Fura-2-AM for the measurements on the microscope.</li>
-
aufgenommen, in den Zellen spalten Proteasen den AM- teil ab. Der
+
-
Farbstoff kann dann als gespaltenen Form die Zelle nicht mehr verlassen.</li>
+
</ul>
</ul>
<ul>
<ul>
-
  <li>gemessen werden Emissionswerte in einem so genannten
 
-
Ratiomeasurement, d.h. es wird bei 2 Wellenl&auml;ngen gemessen und
 
-
das Verh&auml;ltnis daraus berechnet.</li>
 
-
</ul>
 
<br>
<br>
'''Aug. 7th 2008 '''
'''Aug. 7th 2008 '''
-
<span style="font-weight: bold;">Teilnehmer:</span> <br>
+
<span style="font-weight: bold;">Attendees:</span> <br>
Robert Gawlik, Philipp Mappes, Normann Kilb, Sabine J&auml;gle,
Robert Gawlik, Philipp Mappes, Normann Kilb, Sabine J&auml;gle,
-
Kristian Müller, Moritz Busaker.<br>
+
Kristian Müller, Moritz Busacker.<br>
<ul> <li> '''problem:''' Where is the next Ca2+ measurement? </ul>  
<ul> <li> '''problem:''' Where is the next Ca2+ measurement? </ul>  
<ul> <li> Take data of the Ca2+-measurement from Prof. Schamel. Titration to get the right concentration?</ul>  
<ul> <li> Take data of the Ca2+-measurement from Prof. Schamel. Titration to get the right concentration?</ul>  
Line 378: Line 337:
<body>
<body>
<span style="font-weight: bold;">Attendees:</span>
<span style="font-weight: bold;">Attendees:</span>
-
Sabine J&auml;gle, Phillip, Norman Kilb, Christian M&uuml;ller,
+
Sabine J&auml;gle, Philipp Mappes, Norman Kilb, Christian M&uuml;ller,
Wolfgang Schamel, Simone Weber<br>
Wolfgang Schamel, Simone Weber<br>
<br>
<br>
Line 392: Line 351:
to amplificate it.<br>
to amplificate it.<br>
   </li>
   </li>
-
   <li>Prove, if we used the right primer.</li>
+
   <li>Prove if we used the right primer.</li>
   <li>Turn the temperature down a bit.</li>
   <li>Turn the temperature down a bit.</li>
   <li>Use a positive control to be sure the polymerase and buffer
   <li>Use a positive control to be sure the polymerase and buffer
Line 484: Line 443:
'''Sept. 24th. 2008'''<br>
'''Sept. 24th. 2008'''<br>
<br>
<br>
-
 
+
-Modeling<br>
 +
-measurement of fura-stained T-and B-cells (ZBSA)<br>
 +
-AFM-measurement at IMTEK?<br>
 +
-consult MPI concerning antibody B1-8<br>
 +
-cloning: CMV+YFP transfection control positive<br>
 +
-order new oligos, this time modified with FluA aswell<br>
'''Sept. 29th 2008'''<br>
'''Sept. 29th 2008'''<br>
<br>
<br>
 +
-forms for traveling on "company" (university) business have to be filled in<br>
 +
-deadlines for short description of the project and standard variance request are close<br>
 +
-can we "digest" (papain/pepsin) the B1-8 IgM pentameres to visualize the antigen pattern on the origami under the AFM?<br>
 +
-received synthesis from GeneArt and ATG:biosynthetics<br>
 +
-FACS still doesn´t show positive results, amount of origami seems still too low<br<
 +
-organization of hotel rooms at special rate<br>
Line 500: Line 470:
'''Final report'''
'''Final report'''
<ul>
<ul>
-
<li> structure equal to a scientific paper containing the parts Abstract, Introduction, Materials and Methods, Results</li>
+
<li> structure maybe equal to scientific paper, containing Abstract, Introduction, Materials and Methods, Results</li>
<li> fusion of all sections we worked in: Origami, AFM, FACS, CA<sup>2+</sup>/Fura measuremt, cell cultures, modeling, ethics</li>
<li> fusion of all sections we worked in: Origami, AFM, FACS, CA<sup>2+</sup>/Fura measuremt, cell cultures, modeling, ethics</li>
<li> quotation of literature through autor and year of publication in the text (Max Muster, 2007), full quotation in references in the end</li>
<li> quotation of literature through autor and year of publication in the text (Max Muster, 2007), full quotation in references in the end</li>
Line 521: Line 491:
'''attendees:<br>'''
'''attendees:<br>'''
-
Moritz Busaker, Philipp Mappes, Kristian Müller, Normann Kilb, Robert Gawlik, Michael Kneib, Kathrin Pieper, Simone Weber<br>
+
Moritz Busacker, Philipp Mappes, Kristian Müller, Normann Kilb, Robert Gawlik, Michael Kneib, Kathrin Pieper, Simone Weber<br>
<br>
<br>
Line 529: Line 499:
construcs: <br>
construcs: <br>
<br>
<br>
-
pMA - singalpeptide – scFv-anti-NIP – gggs-Linker – transmembran region - '''X''' –  pMA<br><br>
+
pMA - signal peptide – scFv-anti-NIP – gggs-Linker – transmembrane region(BCR/EGFR) - '''X''' –  pMA<br><br>
'''X :'''
'''X :'''
* bla1(1/2 beta-Lactamase)-<br>
* bla1(1/2 beta-Lactamase)-<br>
Line 542: Line 512:
<br>
<br>
'''Next steps:''' <br>
'''Next steps:''' <br>
-
1) We want to test, if the transmembran region really is located in the membrane<br>
+
1) We want to test wheather the transmembrane region really is located in the membrane<br>
-
Therefore we want to clone a constructe which expresses a YFP on the cytoplasmic side of the receptor.<br>
+
Therefore we want to clone a construct expressing YFP on the cytoplasmic side of the receptor.<br>
Construct:<br>
Construct:<br>
-
singalpeptide –  scFv-anti-NIP  –  gggs-Linker  –  transmembran region  - split-linker  -  bla1  - YFP -<br>
+
singal peptide –  scFv-anti-NIP  –  gggs-Linker  –  transmembrane region  - split-linker  -  bla1  - YFP -<br>
<br>
<br>
-
2) We also want to test, if we able to bringe the receptors together by using a NIP coupled peptide.<br>
+
2) We also want to test if we able to bring the receptors together using a NIP coupled peptide.<br>
Therefore we need the peptides from Schamels group -> Norman will ask them.<br>
Therefore we need the peptides from Schamels group -> Norman will ask them.<br>
<br>
<br>
Line 553: Line 523:
Have to produce new Origami -> Michael and Norman<br>
Have to produce new Origami -> Michael and Norman<br>
<br>
<br>
-
Next meeting: Oct. 23th 2008<br>
+
<!--Next meeting: Oct. 23th 2008<br>-->
<br>
<br>
}}
}}

Latest revision as of 01:30, 20 November 2008


Freiburg2008 small header.gif



Home

The Team

Project Report

Parts

Modeling

Notebook

Safety

CoLABoration

_meetings



April 24th 2008

Recruitment of team

May 8th 2008

attendees
Philipp Mappes, Wolfgang Schamel, Kilian Bartholomé, Katja Arndt, Kristian Müller, Dario Hermida Aponte, Normann Kilb, Robert Gawlik, Simone Weber, Veronika Götz, Kristina Brückner, Kathrin Pieper, Michael Kneib, Moritz Busacker, Sabine Jägle

How to organize the project?
The DNA origamis shall be tested on the established T-cell system. Then we will try to establish an artificial system. Extracellular fragments could be the EGFR or Fas for example. Since the Fas receptor is involved in apoptosis, signaltransduction could be detectable by cell death. Further systems for detecting signaltransduction could be NF-kappaB, the MAP-Kinase-pathway, the Jun-Fos-system or splitenzymes.
DNA-origami structure
How expensive will the synthesis of the NIP coupled Oligos be? Phillip and Michael will contact the company and ask for an calculation of costs. NIP can be ordered by the company Biosearch technology.
Controls for the DNA origamis

  • We should produce a DNA origami without NIP-coupled Oligos to check if there also a cell binding occurs (negative control)
  • To check the calculated distances between the NIP's on the origami, DNA-origami with fluorophores could be made. By using FRET the distance between the oligo's can be estimated.


May 23th 2008


Jun 2th 2008

attendees
Sabine Jägle, Normann Kilb, Robert Gawlik, Kristian Müller, Philipp Mappes

We have talked about...

  • To ensure an optimal NIP-scFV-Anti-NIP binding a longer NIP-Linker should be used
  • Calculated distances between the NIP's are 6-7 nm.
  • Integration of the TCR in the model to achieve a comparison of the sizes
  • Searching a suitable transmembraneregion for the artificial receptor
  • Calculation of costs for 10 Oligos wirh NIP coupled to the 5' or 3'-end: 3000,-US$ at uncertain coupling efficiency
  • => Order presumably from PURIMEX


Jun 13th 2008

attendees
Michael Kneib, Robert Gawlik, Philipp Mappes, Simone Weber, Kristina Brückner, Sabine Jägle, Kathrin Pieper

Ordering of NIPs
Assembly of the DNA origami with coupled NIPs was discussed and decided:

Freiburg08 NIP 13 06 08.jpg

Question: What are the exact distances between the DNA-helices/nod-points? --> Rothemund

The next steps
- searching for literature on TCR-Clustering
- searching for literature on transmembrane-helices, signal sequences
- planning of the artificial system with antibodyfragment, transmembraneregion, splitenzymes, enzyme assay
- EGFR as further test-system


Jun 16th 2008 10.00h

Preparation of intern presentation on Tuesday


Jun 17th 2008 12.00h

We have talked about...

- Until Friday we will order the NIP-coupled-Oligos. Consultation of Mahima: Philipp
- We should organize to get the vectors for cloning (Dinah/Christina: Vector with CMV-Promoter) --> Kathrin
- Reproduce Schamels experiment with the TCR --> appointment with Mahima
- Think about a cloning strategy using the BioBrick pre- and suffix
- Literature transmembranhelices WF DeGrado
- Programme for cloning: Emboss (emboss.org)


Jun 23rd 2008 12.00h

attendees

Robert Gawlik, Philipp Mappes, Michael Kneib, Kathrin Pieper, Sabine Jägle, Kristina Brückner, Simone Weber

 

We have talked about...

·    Oligos are ordered. They should be delivered until the 10th of July.

·    Transmembraneregion:

o       Literature about the transmembraneregion

o       Which transmembraneregion can be used? For example EGF-R

o       Signalpeptide?!

o       Paper: Erythropoitetin Receptor Activation by a Ligand-Induced Conformation Change

·    synthetic transmembraneregion:

o       Which parts shall build the synthetic receptor?

 

·    Vectors:

o       Genemaps for the Vectors

o       Available Vectors have to be compared to the iGEM-Systeme(Freiburg2007-WIKI)

o       possibly searching suitable vectors

 

jobs

·    Literature transmembraneregion/signalepeptide : Simone, Kristina, Sabine

·    Vectorsequences : Philipp, Kathrin, Michael

·    Registry on the official WIKI:Robert


July 3rd 12.00h

attendees
Robert Gawlik, Michael Kneib, Kathrin Pieper, Simone Weber, Daniel Hautzinger, Sabine Jägle, Philipp Mappes, Kristian Müller

1. Vectors
Philipp and Kathrin have worked with GCG. In doing so they saw that the fab fragment has iGEM restriction side. The vector from Christina, which we wanted to use, has also 7 iGEM restrictions sides.
We could mutagenesis or gensynthesis to get the iGEM restrictions sides. Genart is offering a special price for gen synthesis. If we want to use this special offer, we have to check which vector they use. It is important that their vector has not any of the iGEM restrictions sides (EcoRI, XbaI, SpeI, PstI) and non of ours restriction sides(NgoMIV, AgeI).
All of the sequences from Mahima arrived by now and they can be checked. A vector wiith CMV-promotor could be also saerched on the wiki 2007.

2. concentration of DNA structure
theoretically possible: 20 nM, maybe even 200 nM; according we need at least 2 µM to distinct between 2, 3 or 4 NIPs

3. DNA-Origami

  • binding measurement: Origami with 4 NIP molecules and 2 fluorophores
  • negative control: DNA-Origami without NIP but with fluorophores
  • Michael, Simone and Daniel: preparation of M13 phage DNA and assessment of oligo-pool

4. proof of principle: T-cell-receptor activation

  • Ca-measurement at 530 nm
  • GFP-marker: detection of product after 12 to 18 hours; maybe we can measure FACS at the universities hospital?

5. confocal microscopy

  • the MPI requires the absolvation of a training, we don´t have time for that;
  • wavelength 488nm and 568nm might be measured at another lab

6. "in general"

  • upload more data onto the intern wiki
  • meet more frequently
  • protocol/write labjournal on wiki
  • precise documentation of every generated sample


July 10th, 2008

Attendees:

Simone Weber, Sabine Jägle, Kathrin Pieper, Phillip Mappes, Norman Kilb, Robert Gawlik, Daniel Hautzinger, Michael Kneib, Kristian Müller, Kristina Brückner

 

1. Security instruction

> emergency call: 2000

2.actual state of affairs

  • M13mp18 phage DNA was preped
  • The Oligios we ordered arrived --> A poll was made, the pool contains all the Oligos except of the 10 which are to be marked with NIP or fluorophor
  • Ca2+ - Daniels experiment (making DNA-Origami 1:20 has been reproduced, but we have to check on the AFM if the Origami formed right -> AFM measurement is tomorrow.
  • We have to reorder some of the Oligo

3. We need to solve the Origami-concentration problem

  • Ca2+We could measure the calcium influx with a flourescence microscope. Therefore we have to find out which dye to use

4. Think about the synthetic receptor system

  • we could use a vector which already has the beta-chain of the TCR coupled to the NIP fab fragment and fuse half of β-Lactamase to each of it
  • if the receptors get clustered by the NIP the β-Lactamase would be functional

5. substrates for the β-Lactamase

  • have to be ordered
  • Norman will get some information about diffrent β-Lactamase and order it

6. Transfection Protocol

  • available in Kristians lab

7. Positive control for the transfections

  • Norman will read about it

8. Searching other vectores

  • Call Tollbox Freiburg

9. second system for multimerisation with different antibodieszweitesn

  • Antifluorescin-Antikalin (Arne Skerra) --> Publication in literature!
  • Andreas Blukthuhn (?) --> Dapin (> Kristian will ask him)

10. intracellular domain of EGF-R

  • http://www.ebi.ac.uk      --> SRS    --> Uniprot
  • http://www.expasy.ch     --> swissprot
  • Michael will read about it

 


July 16th 2008 12.00h

Attendees:
Robert Gawlik, Michael Kneib, Kathrin Pieper, Simone Weber, Moritz Busacker, Sabine Jägle, Philipp Mappes, Kristian Müller
1. DNA-Origami:
We reproduced the experiment of Daniel(Origami 1:20). Now we want to test, if we able to form also Origami when we use less Oligos (ratio 1:10, ratio 1:5)
We have to test, if it is possible to use Krebs-Ringer-Hepes buffer for the calcium imaging
We will also produce some Origami coupled with Alexa 488 for first binding measurements. therefore we have also to check were we could measure Alexa 488

Vectors and synthetic receptor system:
still we have not any vector which we can use for the clonings -> we have to check the vector of the Ljubljana-Projekt 2007
Those parts should be planed and ordered:
-Fab-Singlechain
-Which signal sequence coats for the transmembrane domain
-Which transmembran region will we use?
-The transmembrane region could be attached to split-fluorophors, so the receptor clustering could be detected in a fluorescence microscope
- Which linker will we use?
- We should talk to Tillmann Brunner, ZBSA about using the EGFR




July 23rd 2008 12.00h

Attendees
Robert Gawlik, Philipp Mappes, Kathrin Pieper,Sabine Jägle, Simone Weber.

EGF-Receptor (EGFR):

  • The T-cell surface has a lot of big molecules on the surface. Those molecules are much bigger than the receptor itself, therefore they could disturb the interaction between the ligand (e.g. Origami) and the receptor. -> We should think about using the EGFR as receptor for our clonings, because this one has a relatively big extra cellular domain.
  • Not only a part of the EGFR could be used but the whole receptor, which is additionally linked to the NIP fab fragment by a long linker. So the ligand binding domain should be as big as the surface molecules.
  • This 3 constructs could be cloned:
1)EGFR + Single chain + one half of beta-Lactamase
2)EGFR + Single chain + second half of beta-Lactamase
3)The whole EGFR fused to the NIP fab fragment. With this construct it might be also possible to activate the receptor by adding EGF. Hence we would have a additionally positive control. But first we would have to find a readout for the activation with EGF.

Fura 2/ Indo-1:

  • Both could be used for the Ca2+ signalling. Indo is often used for measurements on the FACS.
  • We will use Fura-2-AM for the measurements on the microscope.

    Aug. 7th 2008 Attendees:
    Robert Gawlik, Philipp Mappes, Normann Kilb, Sabine Jägle, Kristian Müller, Moritz Busacker.
    • problem: Where is the next Ca2+ measurement?
    • Take data of the Ca2+-measurement from Prof. Schamel. Titration to get the right concentration?
    • pictures from measurement should be handled with "Zeiss Software" (Sabine)
    • Carry over T-cells from Mahima (MPI) to kristians lab (Sabine)

    • ATG-Biosynthetics synthesizes all parts in a vector without unintentional iGEM restrictionsites

    • Invitrogen CCF4AM and CCF2AM for bla detection = LiveBLAzer™
      pricelist:
    • CCF2AM/CCF4AM 5mg 13.910.00 €
    • CCF2AM Loading Kit 200 μg 815.00 €
    • LiveBLAzer™-FRET B/G Loading Kit 200μg 690.80€

    brochure for further information [http://tools.invitrogen.com/content/sfs/manuals/liveblazer_FRETBGLoadingKit_man.pdf brochure_liveblazer_FRETBGLoadingKit]


    Aug. 28th 2008


    Sept. 11th 2008

    Attendees: Sabine Jägle, Philipp Mappes, Norman Kilb, Christian Müller, Wolfgang Schamel, Simone Weber

    Themes:

    1. CMV Promotor
    Sabine tried to amplificate the CMV Promotor (in the vector) -> it didn’t work!
    Improvements:     

    • Cut the CMV promoter out (of the vector) and then try again to amplificate it.
    • Prove if we used the right primer.
    • Turn the temperature down a bit.
    • Use a positive control to be sure the polymerase and buffer did work

    2. Antibodies
    Probably we could get some antibodies against NIP from Schamel and his group, so we could proof if our origamis have the NIP. Normally we could see the antibodies on the AFM.

    3. FACS
    We measured the calcium influx on the FACS.
    • Unfortunately we didn´t see a calcium influx in the cytoplasma membrane when we incubated the cells with our origami.
    • We have to try to increase the concentration of our origami
    • For the next measurement we also have to proof that TA/MgAc itself has no influence on the calcium influx.

    4. Fura-Loading
    Because the Fura stain didn´t work when we tried to measure the calcium influx at the microscope (ZBSA), we wanted to repeat the staining to see what we should change.
    o    Again the staining didn´t work -> We have to ask Nitschke about the conditions.
    o    We also have to order the Fura-2AM.

    5. NIP binding to the cells
    We also repeated the binding measurement at the microscope.
    • Like last time both the NIP origami and the control origami (without NIP) bound to our cells :-(
    • We should do the next measurement in Ringer-solution. Therefore we have to test if the origamis are stable in the Ringer-Solution with 12,5mM Mg/Ac!!!
    • We should repeat this measurement with B-Cells (with a “NIP-receptor”), to see if the origami (with and without NIP) bind to those. Maybe some surface structures of the T cells bind unspecific to our DNA.

    -> We can get the B-cells(2558Lδm/mb-1) from Schamel!


    Sept. 18th 2008

    attendees
    Philipp Mappes, Katja Arndt, Kristian Müller, Normann Kilb, Robert Gawlik, Simone Weber, Kathrin Pieper, Sabine Jägle

    organisatory

    • Bioss agrees to sponsorship
    • team picture for wiki
    • next meeting: wednesday, Sept. 24th 15.30
    • lab: new box-arrangement (Normann)
    • table for cloning strategy (Kathrin)

    Ca-measurement

    • date for measurement at the AFM (Nitschke)
    • optionally: measurement at the Henneke lab --> children's clinic (Sabine)
    • optionally: measurement at Imaging Facility in the medicin clinic --> searching for authorized contact-persons

    Wiki

    • carry over the inofficial wiki content to the official one (Robert und Philipp).
    • final report about the whole project (everyone)

    part-order

    • ask for the ATG orders (need to send the plasmid again?) (Philipp)
    • obscurities at order BCR-transmembraneregion --> control and correction(Kathrin)
    • primer-order to synthesize signalpeptide by PCR (Kathrin)

    control of DNA-Origami binding to cell-surface

    • add wash-steps after addition of DNA-origami
    • cells should keep in Mg2+ for measurement
    • possibly the measurement can be carried out by ELISA --> immobilized AK to bind origamis; fluorophors fused to the oligos can be detected or use biotin-fused Oligos to get a detection by streptavidin fused enzyme reaction.
    • control: monoclonal AK able to bind DNA-origami (Simone)



    Sept. 24th. 2008

    -Modeling
    -measurement of fura-stained T-and B-cells (ZBSA)
    -AFM-measurement at IMTEK?
    -consult MPI concerning antibody B1-8
    -cloning: CMV+YFP transfection control positive
    -order new oligos, this time modified with FluA aswell

    Sept. 29th 2008

    -forms for traveling on "company" (university) business have to be filled in
    -deadlines for short description of the project and standard variance request are close
    -can we "digest" (papain/pepsin) the B1-8 IgM pentameres to visualize the antigen pattern on the origami under the AFM?
    -received synthesis from GeneArt and ATG:biosynthetics
    -FACS still doesn´t show positive results, amount of origami seems still too low<br< -organization of hotel rooms at special rate


    Oct. 7th 2008'


    Oct. 17th 2008

    attendees:
    Philipp Mappes, Katja Arndt, Kristian Müller, Normann Kilb, Robert Gawlik, Michael Kneib

    Final report

    • structure maybe equal to scientific paper, containing Abstract, Introduction, Materials and Methods, Results
    • fusion of all sections we worked in: Origami, AFM, FACS, CA2+/Fura measuremt, cell cultures, modeling, ethics
    • quotation of literature through autor and year of publication in the text (Max Muster, 2007), full quotation in references in the end
    • deadline Friday Oct. 24th 2008

    official wikipediasite

    • continue the labjournal on the official page only
    • not too much linking: maintenance of the flow of information
    • all embedded files should start with "Freiburg2008"

    iGEM parts

    • uploading the parts starting on Monday Oct. 20th 2008



    Oct. 21th 2008

    attendees:
    Moritz Busacker, Philipp Mappes, Kristian Müller, Normann Kilb, Robert Gawlik, Michael Kneib, Kathrin Pieper, Simone Weber


    Cloning:
    At least - All the constructes are cloned!!!

    construcs:

    pMA - signal peptide – scFv-anti-NIP – gggs-Linker – transmembrane region(BCR/EGFR) - X – pMA

    X :

    • bla1(1/2 beta-Lactamase)-
    • bla2 (1/2 beta-Lactamase) –
    • split-linker-C-CFP(Cerutan) –
    • N-CFP –
    • split-linker-C-GFP (split venus)–
    • N-GFP –
    • luciferase 1 (58) –
    • luciferase 2 (57) –



    Next steps:
    1) We want to test wheather the transmembrane region really is located in the membrane
    Therefore we want to clone a construct expressing YFP on the cytoplasmic side of the receptor.
    Construct:
    - singal peptide – scFv-anti-NIP – gggs-Linker – transmembrane region - split-linker - bla1 - YFP -

    2) We also want to test if we able to bring the receptors together using a NIP coupled peptide.
    Therefore we need the peptides from Schamels group -> Norman will ask them.

    3) Origami-We want to try if the Origami are able to bring two or more receptors together.
    Have to produce new Origami -> Michael and Norman


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