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FACS Based High Throughput Screening Systems for Gene Libraries in Double Emulsions

  • Radivoje ProdanovicEmail author
  • Raluca Ostafe
  • Milan Blanusa
  • Ulrich Schwaneberg
Conference paper
Part of the Progress in Colloid and Polymer Science book series (PROGCOLLOID, volume 139)

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 107 yeast cells was sorted in 30 min and enriched from 1% to 15% of yeast cells expressing wt GOx.

Keywords

Yeast Cell Gene Library Horse Radish Peroxidase Double Emulsion Primary Emulsion 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Radivoje Prodanovic
    • 1
    • 2
    Email author
  • Raluca Ostafe
    • 2
    • 3
  • Milan Blanusa
    • 2
  • Ulrich Schwaneberg
    • 2
    • 3
  1. 1.University of BelgradeBelgradeSerbia
  2. 2.Jacobs University BremenBremenGermany
  3. 3.RWTH Aachen UniversityAachenGermany

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