Improved DNA/protein delivery in microalgae – A simple and reliable method for the prediction of optimal electroporation settings
| Autor: | Lenny de Jaeger, Gerrit Eggink, Camilo F. Muñoz, Ka Y.F. Lip, Emil J.H. Wolbert, René H. Wijffels, Dirk E. Martens, Justus W.J. Olijslager, Ruud A. Weusthuis, Mark H. J. Sturme, Jan Springer |
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| Rok vydání: | 2018 |
| Předmět: |
0106 biological sciences
0301 basic medicine Bio Process Engineering Propidium iodide Chlorella vulgaris Chlamydomonas reinhardtii Labelled DNA 01 natural sciences Cell membrane 03 medical and health sciences chemistry.chemical_compound medicine VLAG Labelled protein Green microalgae biology Chemistry Electroporation biology.organism_classification Neochloris oleoabundans Sytox Green Transformation (genetics) BBP Bioconversion 030104 developmental biology medicine.anatomical_structure Nucleic acid Biophysics Agronomy and Crop Science 010606 plant biology & botany |
| Zdroj: | Algal Research, 33, 448-455 Algal Research 33 (2018) |
| ISSN: | 2211-9264 |
| Popis: | Genetic transformation of microalgae remains a challenge due to poor intracellular delivery of exogenous molecules. This limitation is caused by the structure and composition of the cell wall and cell membrane of each species. Moreover, successful delivery of proteins or nucleic acids cannot be assessed by determining transformability since their functionality is not always known in the studied microorganism. We propose a quick and effective screening tool for the prediction and optimization of electroporation settings by monitoring cell permeability and viability using Sytox Green and propidium iodide respectively. We determined voltage settings for the microalgae Chlamydomonas reinhardtii, Chlorella vulgaris, Neochloris oleoabundans and Acutodesmus obliquus. To evaluate the predicted settings, we delivered labelled DNA and proteins into the cells. We demonstrated that high transformation efficiencies can be accomplished when predicted values were applied with functional plasmids. Additionally, we increased transformation efficiencies by testing cell concentrations, light intensities and fragment sizes. This method can be used to determine suitable transformation conditions for non-transformed microalgae species and to increase the insight on established transformation protocols. |
| Databáze: | OpenAIRE |
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