iGEM 2010 — Team Freiburg_Bioware

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# Team:Freiburg Bioware/Notebook

### From 2010.igem.org

* [Home](https://2010.igem.org/Team%3AFreiburg_Bioware)
* [Team](https://2010.igem.org/Team%3AFreiburg_Bioware/Team/Team_2010)
  :   [Team 2010](https://2010.igem.org/Team%3AFreiburg_Bioware/Team/Team_2010)
  :   [Collaboration](https://2010.igem.org/Team%3AFreiburg_Bioware/Team/Collaboration)
  :   [Cuckoo Clock Competition](https://2010.igem.org/Team%3AFreiburg_Bioware/Team/Cuckoo_Clock)
  :   [Photo Gallery](https://2010.igem.org/Team%3AFreiburg_Bioware/Team/Photo_Gallery)
  :   [Statistics](https://2010.igem.org/Team%3AFreiburg_Bioware/NoteBook/Statistics)
  :   [Contribution](https://2010.igem.org/Team%3AFreiburg_Bioware/Team/Contribution)
* [Project](https://2010.igem.org/Team%3AFreiburg_Bioware/Project/Results#highlights)
  :   [Summary](https://2010.igem.org/Team%3AFreiburg_Bioware/Project/Results)
  :   [Highlights](https://2010.igem.org/Team%3AFreiburg_Bioware/Project/Results#highlights)
  :   [Introduction](https://2010.igem.org/Team%3AFreiburg_Bioware/Project/Project_Description)
  :   [Results](https://2010.igem.org/Team%3AFreiburg_Bioware/Project/Results)
  :   [Modularization](https://2010.igem.org/Team%3AFreiburg_Bioware/Project/Results#modularization)
  :   [Targeting](https://2010.igem.org/Team%3AFreiburg_Bioware/Project/Results#targeting)
  :   [Arming](https://2010.igem.org/Team%3AFreiburg_Bioware/Project/Results#arming)
  :   [Tumor Killing](https://2010.igem.org/Team%3AFreiburg_Bioware/Project/Results#superconstruct)
  :   [Modeling](https://2010.igem.org/Team%3AFreiburg_Bioware/Modeling)
  :   [Methods](https://2010.igem.org/Team%3AFreiburg_Bioware/Project/Methods)
  :   [Virus Construction Kit Manual](https://2010.igem.org/Team%3AFreiburg_Bioware/Project/Virus_Construction_Kit)
* [BioBricks](https://2010.igem.org/Team%3AFreiburg_Bioware/BioBricks)
  :   [Overview](https://2010.igem.org/Team%3AFreiburg_Bioware/BioBricks)
  :   [Capsid Coding Parts](https://2010.igem.org/Team%3AFreiburg_Bioware/BioBricks/capsid_coding)
  :   [Vector Plasmid Parts](https://2010.igem.org/Team%3AFreiburg_Bioware/BioBricks/vector_plasmid)
* [Notebook](https://2010.igem.org/Team%3AFreiburg_Bioware/Notebook)
  :   [Labjournal](https://2010.igem.org/Team%3AFreiburg_Bioware/NoteBook/Labjournal)
  :   [Cloning Material](https://2010.igem.org/Team%3AFreiburg_Bioware/NoteBook/Materials)
  :   [Methods](https://2010.igem.org/Team%3AFreiburg_Bioware/Project/Methods)
  :   [Meetings](https://2010.igem.org/Team%3AFreiburg_Bioware/NoteBook/Meetings)
  :   [Statistics](https://2010.igem.org/Team%3AFreiburg_Bioware/NoteBook/Statistics)
* [Sponsors](https://2010.igem.org/Team%3AFreiburg_Bioware/Sponsors)
* [Safety](https://2010.igem.org/Team%3AFreiburg_Bioware/Safety)
* [Modeling](https://2010.igem.org/Team%3AFreiburg_Bioware/Modeling)
  :   [Modeling Overview](https://2010.igem.org/Team%3AFreiburg_Bioware/Modeling)
  :   [Virus Production](https://2010.igem.org/Team%3AFreiburg_Bioware/Modeling/Virus_Production)
  :   [Virus Infection](https://2010.igem.org/Team%3AFreiburg_Bioware/Modeling/Virus_Infection)
  :   [Structure Modeling](https://2010.igem.org/Team%3AFreiburg_Bioware/Modeling/Structure_Modeling)

# Notebook

[## Labjournal](https://2010.igem.org/Team%3AFreiburg_Bioware/NoteBook/Labjournal)

Slide through all our Labwork from April till Oktober.

[## Materials](https://2010.igem.org/Team%3AFreiburg_Bioware/NoteBook/Materials)

Materials we used for all our labwork.

[## Methods](https://2010.igem.org/Team%3AFreiburg_Bioware/NoteBook/Methods)

Have a look on the protocols and work methods.

[## Experiments](https://2010.igem.org/Team%3AFreiburg_Bioware/NoteBook/Experiments)

See all the Experiments we made.

[## Meetings](https://2010.igem.org/Team%3AFreiburg_Bioware/NoteBook/Meetings)

Lots of meetings we had.

[## Statistics](https://2010.igem.org/Team%3AFreiburg_Bioware/NoteBook/Statistics)

Our lab and project statistics, just for fun.

Retrieved from "[http://2010.igem.org/Team:Freiburg\_Bioware/Notebook](http://2010.igem.org/Team%3AFreiburg_Bioware/Notebook)"

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[![](https://2010.igem.org/images/wiki.png)"](/Main_Page "Main Page")

* [Page](/Team%3AFreiburg_Bioware/Project/Results)
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* [View source](/wiki/index.php?title=Team:Freiburg_Bioware/Project/Results&action=edit)
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# Team:Freiburg Bioware/Project/Results

### From 2010.igem.org

* [Home](https://2010.igem.org/Team%3AFreiburg_Bioware)
* [Team](https://2010.igem.org/Team%3AFreiburg_Bioware/Team/Team_2010)
  :   [Team 2010](https://2010.igem.org/Team%3AFreiburg_Bioware/Team/Team_2010)
  :   [Collaboration](https://2010.igem.org/Team%3AFreiburg_Bioware/Team/Collaboration)
  :   [Cuckoo Clock Competition](https://2010.igem.org/Team%3AFreiburg_Bioware/Team/Cuckoo_Clock)
  :   [Photo Gallery](https://2010.igem.org/Team%3AFreiburg_Bioware/Team/Photo_Gallery)
  :   [Statistics](https://2010.igem.org/Team%3AFreiburg_Bioware/NoteBook/Statistics)
  :   [Contribution](https://2010.igem.org/Team%3AFreiburg_Bioware/Team/Contribution)
* [Project](https://2010.igem.org/Team%3AFreiburg_Bioware/Project/Results#highlights)
  :   [Summary](https://2010.igem.org/Team%3AFreiburg_Bioware/Project/Results)
  :   [Highlights](https://2010.igem.org/Team%3AFreiburg_Bioware/Project/Results#highlights)
  :   [Introduction](https://2010.igem.org/Team%3AFreiburg_Bioware/Project/Project_Description)
  :   [Results](https://2010.igem.org/Team%3AFreiburg_Bioware/Project/Results)
  :   [Modularization](https://2010.igem.org/Team%3AFreiburg_Bioware/Project/Results#modularization)
  :   [Targeting](https://2010.igem.org/Team%3AFreiburg_Bioware/Project/Results#targeting)
  :   [Arming](https://2010.igem.org/Team%3AFreiburg_Bioware/Project/Results#arming)
  :   [Tumor Killing](https://2010.igem.org/Team%3AFreiburg_Bioware/Project/Results#superconstruct)
  :   [Modeling](https://2010.igem.org/Team%3AFreiburg_Bioware/Modeling)
  :   [Methods](https://2010.igem.org/Team%3AFreiburg_Bioware/Project/Methods)
  :   [Virus Construction Kit Manual](https://2010.igem.org/Team%3AFreiburg_Bioware/Project/Virus_Construction_Kit)
* [BioBricks](https://2010.igem.org/Team%3AFreiburg_Bioware/BioBricks)
  :   [Overview](https://2010.igem.org/Team%3AFreiburg_Bioware/BioBricks)
  :   [Capsid Coding Parts](https://2010.igem.org/Team%3AFreiburg_Bioware/BioBricks/capsid_coding)
  :   [Vector Plasmid Parts](https://2010.igem.org/Team%3AFreiburg_Bioware/BioBricks/vector_plasmid)
* [Notebook](https://2010.igem.org/Team%3AFreiburg_Bioware/Notebook)
  :   [Labjournal](https://2010.igem.org/Team%3AFreiburg_Bioware/NoteBook/Labjournal)
  :   [Cloning Material](https://2010.igem.org/Team%3AFreiburg_Bioware/NoteBook/Materials)
  :   [Methods](https://2010.igem.org/Team%3AFreiburg_Bioware/Project/Methods)
  :   [Meetings](https://2010.igem.org/Team%3AFreiburg_Bioware/NoteBook/Meetings)
  :   [Statistics](https://2010.igem.org/Team%3AFreiburg_Bioware/NoteBook/Statistics)
* [Sponsors](https://2010.igem.org/Team%3AFreiburg_Bioware/Sponsors)
* [Safety](https://2010.igem.org/Team%3AFreiburg_Bioware/Safety)
* [Modeling](https://2010.igem.org/Team%3AFreiburg_Bioware/Modeling)
  :   [Modeling Overview](https://2010.igem.org/Team%3AFreiburg_Bioware/Modeling)
  :   [Virus Production](https://2010.igem.org/Team%3AFreiburg_Bioware/Modeling/Virus_Production)
  :   [Virus Infection](https://2010.igem.org/Team%3AFreiburg_Bioware/Modeling/Virus_Infection)
  :   [Structure Modeling](https://2010.igem.org/Team%3AFreiburg_Bioware/Modeling/Structure_Modeling)

# Summary

Gene delivery is a very promising approach, which yet has to live up to its potentials. Despite several clinical trials, no modular and at the same time exhaustive line of attack has been published. We took advantage of current knowledge to generate a fully modular recombinant Adeno-associated virus (rAAV) based system, which incorporates an extensive set of known surface modifications. In addition, we utilize recently developed binding molecules to generate and demonstrate a novel tumor-targeting approach for rAAV. Our modules have been first extensively tested individually and then in combination for viral production and infectivity. They compared well or exceeded performance of existing systems. The BioBrick-compatible viral vectors demonstrated their ability of tissue-specific delivery of genes coding for fluorescent protein reporters as well as prodrug-activating enzymes for tumor therapy. Using these viruses, we demonstrate specific prodrug-mediated killing of human tumor cells overexpressing a tumor-specific receptor while a reference cell line was unaffected.

During the development of our system, we mastered several hurdles and developed the know-how to manipulate the viral genome. We had to generate a new version of the standard iGEM backbone, since we need additional singular restriction sites to enable swapping of sequences coding for the viral loop structures, which can be used to determine the tropism. Our systems allows for the exchange of two loops of the virus for either a His-tag, which enables purification, or a biotinylation-tag, or an antibody binding module. We demonstrated the functional assembly of viruses with all three modifications.

In addition, we established two N-terminal fusions to the capsid proteins using the binding scaffolds of DARPins and Affibodies. To our knowledge, neither the use of these motifs in a viral setting nor the tumor targeting of rAAV by N-terminal fusion have been demonstrated so far.

We applied a wide range of techniques to achieve our goals. Cloning was performed in E. coli, viruses were produced in a human kidney cell line, and viral infections were tested in further human cell lines. Viruses were purified by liquid chromatography and virus samples were analyzed by quantitative real-time PCR and ELISA. Expression of virally delivered reporter genes was analyzed by fluorescence microscopy including time-lapse imaging. Expression and cell viability upon infection was also assessed by flow cytometry and MTT-assays. Viral samples were additionally analyzed by atomic force microscopy and electron microscopy.

Last but not least, we provide an extensive lab journal, a well documented set of over 100 BioBricks and a detailed manual for the Virus Construction Kit.

Our key achievements are:

* A BioBrick set and assembled plasmids for gene delivery.
  This set comprises the AAV ITRs, a CMV and a hTERT promoter, as well as the genes of interest mVenus, mCherry as reporters and thymidine kinase and cytosin deaminase constructs for prodrug activation, as well as a beta-globin intron and a hGH polyadenylation tag to control gene expression.
* A Biobrick set and plasmids for viral capsid production and modification.
  This set comprises an Affibody and a DARPin targeting the EGF-receptor and a set of linkers which can be utilized for N-terminal fusions. A modified gene coding for viral capsid proteins, which is modified for the fusion. In addition we provide a viral capsid gene for loop modification and the fitting loop modifications coding for a His-tag, biotinylation sequence, and antibody binding site.
* An extensive set of experiments demonstrating the functionality of our constructs.

Retrieved from "[http://2010.igem.org/Team:Freiburg\_Bioware/Project/Results](http://2010.igem.org/Team%3AFreiburg_Bioware/Project/Results)"

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* [Recent changes](/Special%3ARecentChanges "Recent changes")
* [What links here](/Special%3AWhatLinksHere/Team%3AFreiburg_Bioware/Project/Results "List of all wiki pages that link here [j]")
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* [Special pages](/Special%3ASpecialPages "List of all special pages [q]")
* [My preferences](/Special%3APreferences)

* [Printable version](/wiki/index.php?title=Team:Freiburg_Bioware/Project/Results&printable=yes "Printable version of this page [p]")
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