Team:Freiburg/Project/attributions

Attributions

Broad Institute of MIT and Harvard

Zhang lab

We used the pX334a plasmid (Addgene plasmid 42333) provided by the Zhang lab (Cong et al., Science 2013). This plasmid contains a human codon-optimized Cas9 with a nickase function (humanized S. pyogenes Cas9 (D10A) nickase). This hSpCas9 is flanked by two NLS and fused N-terminally to an HA-tag. Because of the D10A mutation, hSpCas9 is only able to introduce single strand breaks. We used this hSpCas9 as a template to introduce a second mutation that removed the nickase function (H840A, Jinek et al., Science 2012) and for our standardization mutagenesis. Our registry dCas9 device contains a different promoter, HA-tag, NLS and backbone. The pX334a plasmid also includes tracrRNA and crRNA loci which we amplified for further cloning of the RNAimer plasmid.

University of Freiburg

AG Weber

The lab of AG Weber is an extremely experienced lab in the field of synthetic biology. Prof. Wilfried Weber holds the first professorship in synthetic biology in Germany. Not only was he supervising us in our project but also providing us with a lot of information and materials and we received great support from the whole group. Additionally we want to point out the support of our PI, Prof. Gerald Radziwil as well as many members of the lab:

• Dr. Matias Zurbriggen for providing us with knowledge, plasmids and support.
• Konrad Müller and Hannes Beyer for many plasmids, introduction to light systems and a lot of know-how.
• Maximilian Hörner for providing us plasmids and discussing our project with us.
• Desiree Hövermann for all the help concerning the uniBAss (binding assay).
• Balder Rebmann for great input concerning the VEGF experiments.
• Sabrina Wend for general advices and plasmids.
• Frauke Bartels-Burgahn, Dr. Natasha Sprossmann, Susanne Knall for all the technical support.

BIOSS administration and Toolbox

BIOSS is an excellence cluster of the University of Freiburg. This institute was founded in 2008 and provides an interdisciplinary environment for as well fundamental and synthetic signaling research.

One division of this institute is the Toolbox, that provides plasmids, cell lines and antibodies for all groups associated with BIOSS. The facility provided us with lab room, a lot of equipment, support and funding of our project. We want to thank the whole BIOSS administration and especially we would like to thank Dr. Johannes Kaiser and Kari Reulecke who provided us with a lot of support concerning legal and financial questions.

The group of Prof. Dr. Jens Timmer is working on quantitative and model approaches in life sciences.

Dipl. Phys. Raphael Engesser helped us choosing the components for our system. Especially he helped us setting up and programming our model.

AG Driever

We want to thank Prof. Dr. Wolfgang Driever for discussing our project with us and Theresa Schredelseker for supporting us in questions of testing the system in vivo.

AG Ulbrich

We thank AG Ulbrich for kindly providing us cells when we were in need.

AG Eimer

We thank AG Eimer for the opportunity to use their fluorescence microscope.

Great thanks we owe to our advisors Bea Kaufmann, Hanna Wagner and Adrian Fischer. Besides working on their diploma or PhD thesis they always took time to help us solving problems or celebrating achievements. They joined our meetings giving wise advises and knocked us out of the skies when we had ideas that were not realistically practical. They paid attention not to influence us in the process of deciding what project to choose for iGEM.

When we had questions or problems concerning cloning strategies Bea and Hanna always suggested solutions or good tips. Adrian helped us to establish our mammalian cell culture by teaching us how to work sterile and how to handle the cells. He supported us with his protein-biochemical knowledge. Without Bea, Hanna and Adrian we would have been totally lost.

University of Stuttgart

AG Jeltsch

Prof. Albert Jeltsch from the University of Stuttgart is an expert on epigenetic modifications and supported us with know-how and plasmids concerning our epigenetic modification project. His group focusses on enzymes catalyzing epigenetic modifications. Especially we want to thank Dr. Srikanth Kuditipudhi who helped us troubleshooting our cloning strategy.