Meetings

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

Aug. 28th 2008

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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
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Sept. 29th 2008
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Oct. 7th 2008'
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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

@@ Line 20: / Line 20: @@
 <ul> <li> CCF2AM Loading Kit 200 μg  '''815.00 €''' </ul>
 <ul> <li> LiveBLAzer™-FRET B/G Loading Kit 200μg '''690.80€''' </ul>
-brochure for further information [http://tools.invitrogen.com/content/sfs/manuals/liveblazer_FRETBGLoadingKit_man.pdf Broschüre]<br>
+brochure for further information [http://tools.invitrogen.com/content/sfs/manuals/liveblazer_FRETBGLoadingKit_man.pdf brochure_liveblazer_FRETBGLoadingKit]<br>