TY - JOUR
T1 - High-throughput screen for poly-3-hydroxybutyrate in Escherichia coli and synechocystis sp. strain PCC6803
AU - Tyo, Keith E.
AU - Zhou, Hang
AU - Stephanopoulos, Gregory N.
PY - 2006/5
Y1 - 2006/5
N2 - 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.
AB - 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.
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U2 - 10.1128/AEM.72.5.3412-3417.2006
DO - 10.1128/AEM.72.5.3412-3417.2006
M3 - Article
C2 - 16672485
AN - SCOPUS:33646542650
SN - 0099-2240
VL - 72
SP - 3412
EP - 3417
JO - Applied and Environmental Microbiology
JF - Applied and Environmental Microbiology
IS - 5
ER -