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Apr. 24th 2008

Apr. 30th 2008

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 a 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$ by unknown coupling efficiency
• => Ordering presumably by 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 wiht the coupled NIPs was discussed and decided:

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

The next steps
- searching for literature of TCR-Clustering
- searching for literature transmembranhelices, signalesequence
- planning the artificial system with antibodyfragment, transmembraneregion, splitenzymes, enzyme assay
- EGFR as further testsystem

Jun 16th 2008 10.00h

Preparation for the presentation on Tuesday 12.00h

Jun 17th 2008 12.00h

We have talked about...

- Until Friday we will order the NIP-coupled-Oligos. Consultation with Mahima --> Phillip
- We should organize to get the vectors for cloning (Dina/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

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.Dabei hat sich gezeigt, dass in der Fab Fragment Sequenz eine IGEM Standard Schnittstelle befindet. In dem Vektor von Christina befinden sich 7 Schnittstellen.
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.
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?).
Man könnte auch noch mal auf der bei den IGEM parts nach Vektoren mit CMV Promotor schauen.

2. Konzentration der DNA Struktur
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.

3. DNA Strukturen

• 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.
• Negativkontrolle. DNA Struktur ohne NIP's, mit 2 Fluorophoren. Nachweis dass diese Struktur alleine nicht zu einer Stimulation des T-Zellrezeptors führt.
• Michael, Simone und Daniel machen M13 Phagen Prep und den Oligopool.
  

4. Nachweis der T-Zellrezeptor-Stimulation

• Ca-Messung: Bei 530 nm. Gerät im MPI ist allerdings kaputt?
• 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.
  

5. Konfokale Mikroskopie

• Im MPI muss man zuerst ein Trainingsprogramm absolvieren, worauf man allerdings auch 3 Wochen warten muss
• Wir könnten das konfokale Mikroskop im Fischbach Labor nutzen. Da stehen die Wellenlängen 488nm und 568nm zur Verfügung.Ansprechpartner ist Gerit.
  

6. Allgemeines

• Rezepte von Puffern usw auf's Wiki schreiben
• Wenn man etwas Neues in Erfahrung gebracht hat Rundmail schreiben, später dann aber auch ins Wiki
• Laborjournal wird chronologisch geführt
• 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
  

10. Juli 2008 12:00 Uhr 10. Juli 2008 12:00 Uhr

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 the 10 which could 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 sovle the Origami concentration problem

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

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

16 Juli 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




23 Juli 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:

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 Teilnehmer:
Robert Gawlik, Philipp Mappes, Normann Kilb, Sabine Jägle, Kristian Müller, Moritz Busaker.

• 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, Phillip, 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




Sept. 29th 2008




Oct. 7th 2008'




Oct. 17th 2008

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

Final report

• structure equal to a scientific paper containing the parts 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 Busaker, Philipp Mappes, Kristian Müller, Normann Kilb, Robert Gawlik, Michael Kneib, Kathrin Pieper, Simone Weber



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

construcs:

pMA - singalpeptide – scFv-anti-NIP – gggs-Linker – transmembran region - 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, if the transmembran region really is located in the membrane
Therefore we want to clone a constructe which expresses a YFP on the cytoplasmic side of the receptor.
Construct:
- singalpeptide – scFv-anti-NIP – gggs-Linker – transmembran region - split-linker - bla1 - YFP -

2) We also want to test, if we able to bringe the receptors together by 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

Next meeting: Oct. 23th 2008