FACS Based High Throughput Screening Systems for Gene Libraries in Double Emulsions
Апстракт
A flow cytometry based high throughput screening system for glucose oxidase (GOx) gene libraries in double emulsions was developed. Firstly, encapsulation of yeast cells in double emulsion was optimized by changing the ABIL EM90 concentration in light mineral oil from 2.9% to 1.5%. This enabled formation of larger water droplets and more efficient yeast cell encapsulation. Several fluorescent assays for hydrogen peroxide were tested and the 3-carboxy-7-(4'-aminophenoxy)-coumarine (APCC) oxidation by horseradish peroxidase based assay best fit the requirements of the double emulsion technology. Using an optimized substrate solution consisting of 0.5 mM APCC, 40 mM glucose and 10 U/mL of horse radish peroxidase, a referent gene library containing 10(7) yeast cells was sorted in 30 min and enriched from 1% to 15% of yeast cells expressing wt GOx.
Извор:
Progress in Colloid and Polymer Science, 2012, 139, 51-Издавач:
- Springer-Verlag Berlin, Berlin
Финансирање / пројекти:
- company BRAIN AG
- Alexander von Humboldt foundation
- BMBF BiochancePlus program
DOI: 10.1007/978-3-642-28974-3_10
ISSN: 0340-255X
WoS: 000310177800010
Scopus: 2-s2.0-84870692826
Колекције
Институција/група
Hemijski fakultet / Faculty of ChemistryTY - CONF AU - Prodanović, Radivoje AU - Ostafe, Raluca AU - Blanusa, Milan AU - Schwaneberg, Ulrich PY - 2012 UR - https://cherry.chem.bg.ac.rs/handle/123456789/1542 AB - A flow cytometry based high throughput screening system for glucose oxidase (GOx) gene libraries in double emulsions was developed. Firstly, encapsulation of yeast cells in double emulsion was optimized by changing the ABIL EM90 concentration in light mineral oil from 2.9% to 1.5%. This enabled formation of larger water droplets and more efficient yeast cell encapsulation. Several fluorescent assays for hydrogen peroxide were tested and the 3-carboxy-7-(4'-aminophenoxy)-coumarine (APCC) oxidation by horseradish peroxidase based assay best fit the requirements of the double emulsion technology. Using an optimized substrate solution consisting of 0.5 mM APCC, 40 mM glucose and 10 U/mL of horse radish peroxidase, a referent gene library containing 10(7) yeast cells was sorted in 30 min and enriched from 1% to 15% of yeast cells expressing wt GOx. PB - Springer-Verlag Berlin, Berlin C3 - Progress in Colloid and Polymer Science T1 - FACS Based High Throughput Screening Systems for Gene Libraries in Double Emulsions VL - 139 SP - 51 DO - 10.1007/978-3-642-28974-3_10 ER -
@conference{ author = "Prodanović, Radivoje and Ostafe, Raluca and Blanusa, Milan and Schwaneberg, Ulrich", year = "2012", abstract = "A flow cytometry based high throughput screening system for glucose oxidase (GOx) gene libraries in double emulsions was developed. Firstly, encapsulation of yeast cells in double emulsion was optimized by changing the ABIL EM90 concentration in light mineral oil from 2.9% to 1.5%. This enabled formation of larger water droplets and more efficient yeast cell encapsulation. Several fluorescent assays for hydrogen peroxide were tested and the 3-carboxy-7-(4'-aminophenoxy)-coumarine (APCC) oxidation by horseradish peroxidase based assay best fit the requirements of the double emulsion technology. Using an optimized substrate solution consisting of 0.5 mM APCC, 40 mM glucose and 10 U/mL of horse radish peroxidase, a referent gene library containing 10(7) yeast cells was sorted in 30 min and enriched from 1% to 15% of yeast cells expressing wt GOx.", publisher = "Springer-Verlag Berlin, Berlin", journal = "Progress in Colloid and Polymer Science", title = "FACS Based High Throughput Screening Systems for Gene Libraries in Double Emulsions", volume = "139", pages = "51", doi = "10.1007/978-3-642-28974-3_10" }
Prodanović, R., Ostafe, R., Blanusa, M.,& Schwaneberg, U.. (2012). FACS Based High Throughput Screening Systems for Gene Libraries in Double Emulsions. in Progress in Colloid and Polymer Science Springer-Verlag Berlin, Berlin., 139, 51. https://doi.org/10.1007/978-3-642-28974-3_10
Prodanović R, Ostafe R, Blanusa M, Schwaneberg U. FACS Based High Throughput Screening Systems for Gene Libraries in Double Emulsions. in Progress in Colloid and Polymer Science. 2012;139:51. doi:10.1007/978-3-642-28974-3_10 .
Prodanović, Radivoje, Ostafe, Raluca, Blanusa, Milan, Schwaneberg, Ulrich, "FACS Based High Throughput Screening Systems for Gene Libraries in Double Emulsions" in Progress in Colloid and Polymer Science, 139 (2012):51, https://doi.org/10.1007/978-3-642-28974-3_10 . .