The initiative for the project idea (i.e. viral therapy with adeno-associated viruses, which is special for its low safety level 1) has been developed by our students. Encouraged by the work of Dr. Grimm and others, we further decided to focus on RNA interference as potent instrument for gene regulation.
In the following we distinguish between students, advisors/instructors, external advisors, and service providers.
The design of the measurement standard construct for shRNA or miRNA silencing strengths has been done by the students with support by advisors. This standard has been inspired by 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.
The design of the tuning construct to silence target genes via shRNA has been made by the students together with instructors and 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/instructors. The DNA has been modified/reassembled via PCR/cloning by students.
The design of imperfect shRNA sequences to achieve various silencing strengths has been designed by students with support by advisors/instructors. The design in annotated sequence format and cloning of these sequences into the miMeasure construct has been done by students.
Measurements were done by the students under close supervision 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. Measurement settings have also been designed in agreement with instructors/advisors.
The design of the model to predict silencing strengths of imperfect shRNAs has been done by the students with support by advisors. The implementation itself has been done by students. The design of the miBEAT-GUI to access the gene silencing model has been inspired by alumni of the iGEM Heidelberg 2009 team, the implementation has been done by students from our team.
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.
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, partially also supported by instructors/advisors.
Taught by instructors/advisors and conducted completely and independently by students themselves.
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.
The infection of a mouse to test a potential virus clone on its selectivity for hepatocytes has been done by an instructor for legal reasons in the presence of a student who had had already experience with mice and therefore could have done it herself.
The philosophical reflection, the psychological survey, and the dance performance have been done solely by students.
The complete documentation has been done by students except the introduction, conclusion and attribution, which have been written together with an advisor. The complete documentation has been proofread and criticized by advisors.
The team logo, the poster and the presentation have been designed 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.
A research proposal to apply for funding has been written by students after discussions involving the complete team.
The iGEM Team Heidelberg.