Meetings

From 2008.igem.org

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'''28. August 2008 14:00 Uhr'''<br>
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<br>
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......<br>
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'''Sept. 11th 2008'''<br>
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<span style="font-weight: bold;">Attendees:</span>
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Sabine J&auml;gle, Phillip, Norman Kilb, Christian M&uuml;ller,
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Wolfgang Schamel, Simone Weber<br>
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<br>
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<span style="font-weight: bold;">Themes:<br>
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<br>
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</span><span style="font-weight: bold;">1. CMV
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Promotor</span><br>
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Sabine tried to amplificate the CMV Promotor (in the vector) -&gt;
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it didn&rsquo;t work!<br>
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Improvements: &nbsp;&nbsp; &nbsp;<br>
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<ul>
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  <li>Cut the CMV promoter out (of the vector) and then try again
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to amplificate it.<br>
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  </li>
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  <li>Pove, if we used the right primer.</li>
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  <li>Turn the temperature down a bit.</li>
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  <li>Use a positive control to be sure the polymerase and buffer
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did work<span style="font-weight: bold;"></span></li>
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</ul><br>
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<span style="font-weight: bold;">2. Antibodies</span><br>
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Probably we could get some antibodies against NIP from Schamel and his
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group, so we could proof if our origamis have the NIP. Normally we
 +
could see the antibodies on the AFM.<br>
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<br>
 +
<span style="font-weight: bold;">3. FACS</span><br>
 +
We measured the calcium influx on the FACS. <br>
 +
<ul>
 +
  <li>Unfortunately we didn&acute;t see a calcium influx in
 +
the cytoplasma membrane when we incubated the cells with our origami.</li>
 +
  <li>We have to try to increase the concentration of our origami</li>
 +
  <li>For the next measurement we also have to proof that TA/MgAc
 +
itself has no influence on the calcium influx. <br>
 +
  </li>
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</ul><br>
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<span style="font-weight: bold;">4. Fura-Loading</span><br>
 +
Because the Fura stain didn&acute;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.<br>
 +
o&nbsp;&nbsp; &nbsp;Again the staining didn&acute;t work<small>&nbsp;</small>-&gt;<small>
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</small>We have to ask Nitschke about the conditions.<br>
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o&nbsp;&nbsp; &nbsp;We also have to order the Fura-2AM.<br>
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<br>
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<span style="font-weight: bold;">5. NIP binding to the
 +
cells</span><br>
 +
We also repeated the binding measurement at the microscope.<br>
 +
<ul>
 +
  <li>Like last time both the NIP origami and the control origami
 +
(without NIP) bound to our cells :-(</li>
 +
  <li>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!!!</li>
 +
  <li>We should repeat this measurement with B-Cells (with a
 +
&ldquo;NIP-receptor&rdquo;), 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.<br>
 +
  </li>
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</ul><br>
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-> We can get the B-cells(2558L&delta;m/mb-1) from Schamel!<br>
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</body>
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</html>

Revision as of 15:53, 29 October 2008


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_meetings





28. August 2008 14:00 Uhr

......

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.
  • Pove, 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

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