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.

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