Single-cell variability in growing Saccharomyces cerevisiae cell populations measured with automated flow cytometry.
Cell cultures normally are heterogeneous due to factors such as the cell cycle, inhomogeneous cell microenvironments, and genetic differences. However, distributions of cell properties usually are not taken into account in the characterization of a culture when only population averaged values are measured. In this study, the cell size, green fluorescence protein (Gfp) content, and viability after automated staining with propidium iodide (PI) are monitored at the single-cell level in Saccharomyces cerevisiae cultures growing in a batch bioreactor using an automated flow injection flow cytometer system. To demonstrate the wealth of information that can be obtained with this system, three cultures containing three different plasmids are compared. The first plasmid is a centromeric plasmid expressing under the control of a TEF2 promoter the S65T mutant form of Gfp. The other two plasmids are 2 microm plasmids and express the FM2 mutant of Gfp under the control of either the TEF1 or the TEF2 promoter. The automated sampling, cell preparation, and analysis permitted frequent quantification of the culture characteristics. The time course of the data representing not only population average values but also their variability, provides a detailed and reproducible "fingerprint" of the culture dynamics. The data demonstrate that small changes in the genetic make up of the recombinant system can result in large changes in the culture Gfp production and viability. Thus, the developed instrumentation is valuable for rapidly testing promoter strength, plasmid stability, cell viability, and culture variability.
Kacmar
James
J
Department of Chemical Engineering and Materials Science, University of Minnesota, 151 Amundson Hall, 421 Washington Avenue S.E., Minneapolis, MN 55455-0312, USA.
Zamamiri
Abdelqader
A
Carlson
Ross
R
Abu-Absi
Nicholas R
NR
Srienc
Friedrich
F
D019149:Bioreactors / Q000382:microbiology; D002455:Cell Division; D049108:Cell Enlargement; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins; D010957:Plasmids / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000235:genetics / Q000254:growth & development*
D049452:Green Fluorescent Proteins