The overall organization of our team has as in previous years been in the responsibility of Prof. Dr. Roland Eils. The team is in this year further assisted by Dr. Dirk Grimm, who is a fellow at the Bioquant institute with a strong expertise on adeno-associated viruses and RNA interference. We consider iGEM as a sophisticated research project with the additional ambition to involve students into such work, which is usually hard to access at this age. We believe this will allow them to learn at an early stage the difficulties of such research and thus enable them to plan their future curriculum with regard to engineering aspects. To achieve this high ambition, it requires sometimes a close supervision and an intensive support, but our utmost clause is that all work is done by students themselves, in any case physically and in the ideal case including the experimental design. We think that we very closely achieved this high aim and declare in the following for the critical achievements of our project individually the attributions with particular regard to sources and conductance.
1) Main Project Idea: The initiative for the project idea (i.e. viral therapy with adeno-associated viruses, which is special for its safety level 1) has come in a very early stage from students of our team. Encouraged by Dr. Grimm, we further decided to focus on RNA interference as instrument for gene regulation.
In the following we distinguish between students, advisors/instructors, external advisors, and service providers.
2) miMeasure (siRNA Measurement Standard Construct):
The design of the measurement standard construct for shRNA or miRNA silencing strengths has been supported by advisors of the team. This has been accompanied by the documentation on the measurement standard for synthetic promoters (Team Heidelberg 2009, RFC 41) and the prokaryotic promoter measurement kit (Jason Kelly, RFC 19). The design in annotated sequence format of the final construct has been conducted by students. The basic construct has been synthesized by Geneart, and the following replacements of parts (in particular binding sites) within the construct have been cloned by students. This includes the design of cloning strategies.
4) miTuner (Tuning Construct for regulation of target genes via siRNA):
The design of the tuning construct to silence target genes via shRNA has been supported by advisors. The final design in annotated sequence format has been conducted by students. Source DNA (e.g. luciferase gene, promoter sequences, terminators) has been provided by advisors. The DNA has been modified/reassembled via PCR/cloning by students.
4) Design of shRNA sequences:
The design of imperfect shRNA sequences to achieve various silencing strengths has been supported by advisors. The design in annotated sequence format and cloning of these sequences into the miMeasure construct has been done by students.
5) Measurement of shRNA silencing strengths:
Measurements have been closely supervised by advisors of the team to avoid the risk of damages to the expensive devices such as a wide-field and a confocal microscope, a flow cytometer, and a microplate reader. The measurements themselves have been taken by students (i.e. they explained the instructor their needs and could in this way be guided to use the measurement device). Measurement settings have also been designed in agreement with instructors/advisors.
6) miBEAT:
The design of the model to predict silencing strengths of imperfect shRNAs has been given by advisors, the implementation itself has been done by students. The design of the miBEAT-GUI to access the gene silencing model has been supported by alumni of the Heidelberg 2009 team, the implementation has been done by students from our team.
7) Hepatocyte Identification via miR-122:
The strategy to design a miRNA binding site pattern within the miMeasure construct using random assembly PCR (Heidelberg 2009 team, RFC 42) has been developed by students. Theoretical background on miRNA expression profiles has been given by advisors.
8) Virus capsid shuffling:
We have followed two approaches for the capsid shuffling: (1) it has been conducted using the Alberta approach from 2009 (RFC 47) in all respects by the students; (2) it has been conducted using a protocol published previously by Grimm et al. in close supervision by instructors/advisors. The digestion, ligation, and cloning of the capsid gene have been conducted by students, but partially also instructors/advisors in the presence of students.
9) Cell Culture:
Taught by instructors/advisors and conducted completely and independently by students themselves.
10) Virus selection:
Virus clones which are selective for hepatocytes have been selected under close supervision of instructors in a safety level 2 laboratory. The picking of the evolved clone itself has been done by a student.
11) Mouse infection:
The infection of a mouse to test a potential virus clone on its selectivity for hepatocytes has been done by an instructor in the presence of a student who had had already experience with mice and therefore could have done it herself.
12) Human Practice:
The philosophical reflection, the psychological survey, and the dance performance have been done solely by students.
13) Documentation:
The complete documentation has been done by students except the introduction, conclusion and attribution, which have been written by an advisor. The complete documentation has been proofread and criticized by advisors.
14) Logo, wiki, poster, and presentation design:
The team logo, the poster and the presentation have been done by students. Advice has been given by Lange+Pflanz, a marketing agency and sponsor of our team. The layout of the wiki has been done solely by students.
15) Funding:
A research proposal to apply for funding has been written by students after discussions involving the complete team.
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