Team:LMU-Munich/Team

From 2010.igem.org

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'''Team LMU iGEM 2010'''
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==<font color="#9933CC">'''Team LMU iGEM 2010'''</font>==
[[Image:LMU-Munich_team.png|center|600px|Our team picture]]
[[Image:LMU-Munich_team.png|center|600px|Our team picture]]
[[Image:TeamLMU.JPG|center|600px|Also our team]]
[[Image:TeamLMU.JPG|center|600px|Also our team]]
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<!-- |align="center"|[[Team:LMU-Munich | Team Example]] -->
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<!--- The Mission, Experiments --->
<!--- The Mission, Experiments --->
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== '''Who is in charge of what?''' ==
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== '''Who is in charge of what''' ==
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| Coordination/Organisation
| Coordination/Organisation
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|  Jara Radeck
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|  Jara Radeck, Angelika Vetter
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| Contact
| Contact
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| igemlmu@yahoo.de
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| igemlmu(at)yahoo(dot)de
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To establish our two systems “Cut’N’Survive” and “Jump-Or-Die” for selecting cells by apoptosis, we first set out to build the necessary BioBricks. We searched for sources of the DNA parts involved and several labs kindly provided us with the plasmids and sequences in need, as listed at the end.  
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The creation of certain constructs was necessary for our two systems for cell selection by means of apoptosis: “Cut’N’Survive” and “Jump-Or-Die”. We searched for sources of the DNA sequences we needed and found several supporters which are listed below.
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Most genes and promoters were amplificated per PCR with overhang-primers with the BioBrick prefix or suffix. If the sequence contained a EcoRI-, PstI-, XbaI-, SpeI- or NotI- restriction site, mutagenesis primers were used and the amplified DNA parts were then fused by fusion PCR. All PCRs worked in the end in spite of several problems with the touch down PCR and the fusion PCR.The PCR products were then tested by agarose gel electrophoresis if they were of the right lengths.
+
Most genes and promoters were amplificated via PCR with overhang-primers with the BioBrick prefix or suffix. If the sequence contained a EcoR1-, Pst1-, Xba1-, Spe1- or Not1- restriction site, we used mutagenesis primers and fusioned both DNA parts by fusion PCR. All PCRs worked out, even the fusion PCRs.
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In parallel, we made competent cells and multiplied the ccdB (death gene)-vectors with different antibiotic resistances.  
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The length of the PCR products were tested by agarose gel electrophoresis. We tried to sequence our PCR products, but obtained poor results and resorted to sequencing the plasmids.
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All components were then digested with the appropriate restriction enzymes. The PCR products were cleaned with a PCR purification kit and the ccdB-vectors were dephosphorylated to reduce false ligations.
+
In parallel, we made competent cells and multiplied ccdB (death gene)-vectors with different antibiotic resistances. All components were digested with the appropriate restriction enzymes. The samples were cleaned with a PCR clean up kit or dephosphorylated to reduce false ligations.
-
We ligated our constructs and several intermediates with the 3A-assembly according to the schedule. The ligations were transformed to E.coli DH5α strains and selected by antibiotics. The colonies were later picked and the insertion of the construct was confirmed by colony PCR.  
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We ligated our constructs and several interim stages with the 3A-assembly according to our schedule. The ligations were transformed to E.coli DH5α strains and selected by antibiotics. Afterwards, some colonies were picked and we tested the insertion of the construct by colony PCR.  
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If the colony PCR showed bands of the right size, the plasmids were extracted from overnight cultures and sequenced with forward and reverse BioBrick primers.
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If the colony PCR resulted in bands of the right size, we extracted the plasmids from overnight cultures and sequenced the samples with forward and reverse BioBrick primers.
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In the end we succeeded in obtaining four correctly sequenced BioBricks. But since the sequences of other necessary parts turned out wrong, neither of our systems could be completed and we were not able to test them in eukarytic cell lines.  
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Unfortunately, not all BioBricks were cloned succesfully. However, we were able to produce 4 BioBricks, one of which represents a full construct while the other three are intermediates. The system wasn't completed on time, so we weren´t able to test them in eukarytic cell lines.  
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In addition, we found out that the CMV-promoter we ordered from the BioBrick registry was in fact a lacI gene, which resulted in the procrastination of our plan. We have sent the correction to the registry to facilitate future teams.
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Besides, we found out that the CMV-promoter we ordered from the BioBrick registry (<html><a href="http://partsregistry.org/Part:BBa_J52034">BBa_J52034</a href></html>) was in fact a lacI gene, which resulted in the procrastination of our plan. We have sent the correction to the registry to facilitate future teams.
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<b>The protocols we used are listed here: </b> [[Team:LMU-Munich/Notebook#Protocols|Protocols]]  
<b>The protocols we used are listed here: </b> [[Team:LMU-Munich/Notebook#Protocols|Protocols]]  
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<b>The Biobricks we could submit: </b>
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<b>The Biobricks we submitted: </b>
*BBa_K368004: attP+eGFP+SV40PA
*BBa_K368004: attP+eGFP+SV40PA
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** SV40PA (Polyadenylation site): gave us a vector containing it
** SV40PA (Polyadenylation site): gave us a vector containing it
** Human Bak:  her assistant Erika Clement gave us appropriate cDNA
** Human Bak:  her assistant Erika Clement gave us appropriate cDNA
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* Prof. Dr. Thorsten Mascher:
 +
** Helped with primer design, agarose gel electrophoresis apparatuses and trouble shooting
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* Prof. Dr. Kirsten Jung:
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** Helped with ideas and fundraising
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* Dr. Susanne Gebhard:
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** Helped with trouble-shooting and materials
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* Kemal Akman
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** Helped and supported our sub-project ProSearch
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<b> Plasmid and sequence sources <b>
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<b> Plasmid and sequence sources </b>
* Dr. Arnim Weber: provided us with human Bak plasmid
* Dr. Arnim Weber: provided us with human Bak plasmid
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** TEVrecognition site+N-degron+SF3b155
** TEVrecognition site+N-degron+SF3b155
** TEV-Protease+p14*+TEVrecognition site
** TEV-Protease+p14*+TEVrecognition site
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* Prof. Dr. Thorsten Mascher:
 
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** Helped with primer design, agarose gel electrophoresis apparatuses and trouble shooting
 
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* Prof. Dr. Kirsten Jung:
 
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** Helped with ideas and fundraising
 
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* Dr. Susanne Gebhard:
 
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** Helped with trouble-shooting and materials
 
* Prof. Dr. Andreas Brachmann:  
* Prof. Dr. Andreas Brachmann:  
** Sequenced our samples
** Sequenced our samples
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As our project for human practice, we carried out a survey about synthetic biology on the general public at the Oktoberfest, LMU Munich and the Municipal Secretary: [[Team:LMU-Munich/Human practice|Human practice]]
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As our project for human practice, we carried out a survey about synthetic biology on the general public at the Oktoberfest, LMU Munich and the Municipal Secretary: [[Team:LMU-Munich/HumanPractice|Human practice]]
== '''Where we are from''' ==
== '''Where we are from''' ==
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[[Image:lmu2.jpg|center|760px|Biozentrum]]our bio-center (photos courtesy of www.stbam2.bayern.de)
[[Image:lmu2.jpg|center|760px|Biozentrum]]our bio-center (photos courtesy of www.stbam2.bayern.de)
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There's a second University in our beautiful Munich, even though it is "only" a technical University. Also check out their
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<html><a href="http://2010.igem.org/Team:TU_Munich">wiki</a href></html>.
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[[Image:Tu.jpg]]
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Latest revision as of 21:10, 10 January 2011


Team LMU iGEM 2010

Our team picture
Also our team


-->

Who we are

We are a team constisting of biologists, biochemists, chemists and bioinformatics and four faculty advisors... The LMU team is competing for the first time.

Advisors

Undergrads

Who is in charge of what

Press Nicolas Keller, Marisa Kurz
Sponsoring Sabine Wagner, Franziska Haefele
Pathway Tobias Bauer, Emanuel Stiegeler, Benny Clanner
ApoControl Julia Bartels, Jara Radeck, Mengzhe Grace Wang
Homepage Tatyana Goldberg, Jens Popken
Coordination/Organisation Jara Radeck, Angelika Vetter
Contact igemlmu(at)yahoo(dot)de

What we did

Short description of our work, our results and our supporters


The creation of certain constructs was necessary for our two systems for cell selection by means of apoptosis: “Cut’N’Survive” and “Jump-Or-Die”. We searched for sources of the DNA sequences we needed and found several supporters which are listed below.

Most genes and promoters were amplificated via PCR with overhang-primers with the BioBrick prefix or suffix. If the sequence contained a EcoR1-, Pst1-, Xba1-, Spe1- or Not1- restriction site, we used mutagenesis primers and fusioned both DNA parts by fusion PCR. All PCRs worked out, even the fusion PCRs.

The length of the PCR products were tested by agarose gel electrophoresis. We tried to sequence our PCR products, but obtained poor results and resorted to sequencing the plasmids.

In parallel, we made competent cells and multiplied ccdB (death gene)-vectors with different antibiotic resistances. All components were digested with the appropriate restriction enzymes. The samples were cleaned with a PCR clean up kit or dephosphorylated to reduce false ligations.

We ligated our constructs and several interim stages with the 3A-assembly according to our schedule. The ligations were transformed to E.coli DH5α strains and selected by antibiotics. Afterwards, some colonies were picked and we tested the insertion of the construct by colony PCR.

If the colony PCR resulted in bands of the right size, we extracted the plasmids from overnight cultures and sequenced the samples with forward and reverse BioBrick primers.

Unfortunately, not all BioBricks were cloned succesfully. However, we were able to produce 4 BioBricks, one of which represents a full construct while the other three are intermediates. The system wasn't completed on time, so we weren´t able to test them in eukarytic cell lines.

Besides, we found out that the CMV-promoter we ordered from the BioBrick registry (BBa_J52034) was in fact a lacI gene, which resulted in the procrastination of our plan. We have sent the correction to the registry to facilitate future teams.


Our notebook with detailed descriptions can be found here: ApoControl notebook

The protocols we used are listed here: Protocols

The Biobricks we submitted:

  • BBa_K368004: attP+eGFP+SV40PA
  • BBa_K368011: eGFP+SV40PA
  • BBa_K368016: TEVrecognition site+N-degron+SF3b155
  • BBa_K368019: TEV-Protease+p14*+TEVrecognition site

Supervisors

  • Prof. Dr. Angelika Böttger :
    • prevTRE (tet-on CMV promoter; inducible by doxycycline in special cell lines)
    • supported the construction ideas and would have given us the cells and mediums we would have needed
    • SV40PA (Polyadenylation site): gave us a vector containing it
    • Human Bak: her assistant Erika Clement gave us appropriate cDNA
  • Prof. Dr. Thorsten Mascher:
    • Helped with primer design, agarose gel electrophoresis apparatuses and trouble shooting
  • Prof. Dr. Kirsten Jung:
    • Helped with ideas and fundraising
  • Dr. Susanne Gebhard:
    • Helped with trouble-shooting and materials
  • Kemal Akman
    • Helped and supported our sub-project ProSearch

Plasmid and sequence sources


As our project for human practice, we carried out a survey about synthetic biology on the general public at the Oktoberfest, LMU Munich and the Municipal Secretary: Human practice

Where we are from

We are students at Ludwigs-Maximilians-University in Munich, Germany.


Biozentrum
our bio-center (photos courtesy of www.stbam2.bayern.de)

There's a second University in our beautiful Munich, even though it is "only" a technical University. Also check out their wiki.

Tu.jpg