Lerche, Kai; Hallmann, Armin: Stable nuclear transformation of Eudorina elegans. In: BMC Biotechnology. Jg.13 H. 1. 2013
Inhalt
- Results
- Antibiotic tolerance of wild-type Eudorina algae
- Transformation experiments using an aphVIII-based selectable marker
- Antibiotic resistance of transformants
- Stable integration of plasmid DNA into the genome of transformants
- Calculation of transformation frequency
- Stable co-transformation with non-selectable reporter gene plasmids
- Transcription of co-transformed, non-selectable reporter genes
- Analysis of heterologous protein expression in co-transformants
- Inducibility of heterologous protein expression
- In-gel activity assays
- Long-term stability of DNA integration and gene expression
- Discussion
- Conclusions
- Methods
- Strains and culture conditions
- Transformation vectors
- Preparation of plasmid DNA
- Coating of microprojectiles
- Determination of cell concentration
- Stable nuclear transformation by particle bombardment
- Re-isolation of transformants
- Paromomycin-resistance assay
- Primer design
- Isolation of genomic DNA
- Genomic PCR
- Southern blotting
- Isolation of total RNA
- Reverse Transcription (RT)-PCR
- Optimum temperature for heat stress-induced expression of luciferase
- Luciferase assay
- In-gel activity assay
- Additional files
- Abbreviations
- Competing interests
- Authors’ contributions
- Acknowledgements
- References