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 built the necessary constructs. Therefore we searched for sources of the DNA parts involved and several labs kindly supported us by sending the plasmids and sequences we needed, which are listed below.  
+
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 per PCR with overhang-primers with the BioBrick prefix or suffix. If the sequence contained a EcoRI-, PstI-, XbaI-, SpeI- or NotI- restriction site, we used mutagenesis primers and fused both DNA parts by fusion PCR. Fortunately all PCRs worked 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 are of the right length. We sequenced the plasmids due to poor results from PCR products' sequencing.
+
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.
-
In parallel, we made competent cells and multiplied the ccdB (death gene)-vectors with different antibiotic resistances.  
+
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.
-
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.  
+
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 showed bands of the right size, the plasmids were extracted from overnight cultures and sequenced with forward and reverse BioBrick primers.
+
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.
-
In the end we succeeded in obtaining four sequenced BioBricks, though some BioBrick sequences turned out wrong. so that we only got 4 BioBricks although every ligation leaded to colonies after the transfection. Neither of our constructs was completed, so that we weren´t 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 a CMV-promoter we ordered from the registry was in fact a lacI gene and therefore some BioBricks just couldn´t be right.
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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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*BBa_K368019: TEV-Protease+p14*+TEVrecognition site
*BBa_K368019: TEV-Protease+p14*+TEVrecognition site
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<b>Sources, helpers and supporters:</b>
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<b>Supervisors</b>
* Prof. Dr. Angelika Böttger :  
* Prof. Dr. Angelika Böttger :  
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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
 +
* Prof. Dr. Kirsten Jung:
 +
** Helped with ideas and fundraising
 +
* Dr. Susanne Gebhard:
 +
** Helped with trouble-shooting and materials
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* Kemal Akman
 +
** Helped and supported our sub-project ProSearch
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<b> Plasmid and sequence sources </b>
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* Dr. Arnim Weber: submitted us a vector with human Bak
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* Dr. Arnim Weber: provided us with human Bak plasmid
* Dr. Philipe Soriano: <html>
* Dr. Philipe Soriano: <html>
<a href="http://www.ncbi.nlm.nih.gov/pubmed/17225864?dopt=Abstract"> (Raymond CS et al: High-Efficiency FLP and PhiC31 Site-Specific Recombination in Mammalian Cells (2007))</a>
<a href="http://www.ncbi.nlm.nih.gov/pubmed/17225864?dopt=Abstract"> (Raymond CS et al: High-Efficiency FLP and PhiC31 Site-Specific Recombination in Mammalian Cells (2007))</a>
</html>
</html>
** Sequences of attB and attP site
** Sequences of attB and attP site
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** PhiC31o was bought via addgene
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** PhiC31o was bought via Addgene
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* Knop, M (Heidelberg): <html>
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* Dr. Michael Knop:(Heidelberg): <html>
<a href ="http://www.ncbi.nlm.nih.gov/pubmed?term=Efficient%20protein%20depletion%20by%20genetically%20controlled%20deprotection%20of%20a%20dormant%20N-degron">(Knop et al.: Efficient protein depletion by genetically controlled deprotection of a dormant N-degron (2009))</a>
<a href ="http://www.ncbi.nlm.nih.gov/pubmed?term=Efficient%20protein%20depletion%20by%20genetically%20controlled%20deprotection%20of%20a%20dormant%20N-degron">(Knop et al.: Efficient protein depletion by genetically controlled deprotection of a dormant N-degron (2009))</a>
</html>
</html>
 +
** provided us with:
** 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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* Partsregistry:
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* Partsregistry provided us with:
** eGFP (BBa_I714891)
** eGFP (BBa_I714891)
** CMV-Promoter (BBa_J52034: this part was wrong: its lacI !!!)
** CMV-Promoter (BBa_J52034: this part was wrong: its lacI !!!)
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We went to the Oktoberfest, LMU Munich and the municipal secretary and asked a few questions about synthetic biology: [[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="https://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


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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

Prof. Dr. Angelika Böttger: Department Biologie II/ Cell and Developmental Biology

Prof. Dr. Kirsten Jung: Head of the Division of Microbiology

Prof. Dr. Thorsten Mascher: Department Biologie I/ Microbiology

Dr. Achim Tresch: Computational Biology and Regulatory Networks

Kemal Akman: Bioinformatics

Undergrads

Julia Bartels: expected BSc Biology, ApoControl, Pathway

Tobias Bauer: BSc Biology, Pathway

Alexander Buschle: BSc Biology, ApoControl

Benjamin Clanner: Chemistry and Biochemistry, pharmaceutical sciences, Pathway

Maria Katharina Drexler: BSc Biology, ApoControl

Erik Fumi: Chemistry and Biochemistry, Pathway

Tatyana Goldberg: BSc Bioinformatics, ApoControl

Franziska Häfele: expected BSc Biology, ApoControl

Corinna Hofer: BSc Biology, ApoControl

Nicolas Keller: Biology, Law, ApoControl, Pathway

Laura Kleinknecht: BSc Biology, ApoControl

Christina.jpg

Christina Krönauer: expected BSc Biology, Pathway

Marisa Kurz: Chemistry and Biochemistry, Pathway

Jens Popken: Biology, Pathway

Jara Radeck: Biology, ApoControl

Emanuel Stiegeler: BSc Biology, Pathway

Angelika Vetter: Biology, ApoControl

Sabine Wagner: Biology, ApoControl, Pathway

Mengzhe Wang (Grace): Biology, ApoControl

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



 

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