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Applied and Environmental Microbiology, May 2006, p. 3412-3417, Vol. 72, No. 5
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.5.3412-3417.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

High-Throughput Screen for Poly-3-Hydroxybutyrate in Escherichia coli and Synechocystis sp. Strain PCC6803

Keith E. Tyo, Hang Zhou, and Gregory N. Stephanopoulos*

Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, Massachusetts 02139

Received 16 November 2005/ Accepted 8 March 2006

A novel, quantitative method for detecting poly-3-hydroxybutyrate (PHB) amounts in viable cells was developed to allow for high-throughput screening of mutant libraries. The staining technique was demonstrated and optimized for the cyanobacterium Synechocystis sp. strain PCC6803 and the eubacterium Escherichia coli to maximize the fluorescence difference between PHB-accumulating and control cells by flow cytometry. In Synechocystis, the level of nonspecific dye binding was reduced by using nonionic stain buffer that allowed quantitation of fluorescence levels. In E. coli, the use of a mild sucrose shock facilitated uptake of Nile red without significant loss of viability. The optimized staining protocols yielded a linear response for the mean fluorescence against (chemically measured) PHB. The staining protocols are novel methods useful in the high-throughput evaluation of combinatorial libraries of Synechocystis and E. coli using fluorescence-activated cell sorting to identify mutants with increased PHB-accumulating properties.

* Corresponding author. Mailing address: Department of Chemical Engineering, MIT 56-469, 77 Massachusetts Ave., Cambridge, MA 02139. Phone: (617) 253-4583. Fax: (617) 253-3122. E-mail: gregstep{at}mit.edu.

Applied and Environmental Microbiology, May 2006, p. 3412-3417, Vol. 72, No. 5
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.5.3412-3417.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.





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