Team:Freiburg Bioware/NoteBook/Labjournal

Get to know our virus

Our project starts with first sequence analysis.

Checking iGEM compatibility

Scanning for iGEM restriction sites:
• Vector plasmid
• Thymidine kinase
• Cytosindeaminase

First practical steps

We took a closer look to the theoretical DNA- and amino acid sequence of the ITRs, questioned the role of the β-globin intron, had a closer look to the CMV promoter and performed our first cloning attempt.

First fluorescing HT1080 cells

We conducted our first site-directed mutagenesis.

First cell culture steps:
• Splitting and seeding of AAV 293 and HT1080 cells
• Calcium phosphate transfection
• Transduction with virus particles containing YFP encoding sequence
• Microscopy: Fluorescent transduced HT1080 cells

We planned to determine infectious virus titer via quantitative real-time PCR. For this purpose primers were designed, transduced HT1080 cells were harvested and prepared.

Theoretical studies of the AAV structure: Impressions.

Modifying Rep/Cap & Modularizing Vector plasmid

We developed a plan for modifying the surface exposed loops of the virus capsid. For this purpose not only 5 iGEM restriction sites, but also the so called ViralBrick restriction sites located in the Rep/Cap sequence needed to be deleted: BamHI, SalI

BioBrick production of the inverted terminal repeats (ITRs) turned out to be difficult. Therefore the alternative “ITR fancy method” was developed. We successfully produced AAV2 left and right ITR fused to the RFC10 prefix.

Further on we received first quantitative transduction results via flow cytometry.

mGMK_TK30 was successfully cloned into the vector plasmid.

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