Propagation of an amplifiable recombinant plasmid in Saccharomyces cerevisiae: flow cytometry studies and segregated modeling

Abstract

Efficient expression of a foreign protein product by the yeast Saccharomyces cerevisiae requires a stable recombinant vector present at a high number of copies per cell. A conditional centromere yeast plasmid was constructed which can be amplified to high copy number by a process of unequal partitioning at cell division, followed by selection for increased copy number. However, in the absence of selection pressure for plasmid amplification, copy number rapidly drops from 25 plasmids/cell to 6 plasmids/cell in less than 10 generations of growth. Copy number subsequently decreases from 6 plasmids/cell to 2 plasmids/cell over a span of 50 generations. A combination of flow cytometric measurement of copy number distributions and segregated mathematical modeling were applied to test the predictions of a conceptual model of conditional centromere plasmid propagation. Measured distributions of plasmid content displayed a significant subpopulation of cells with a copy number of 4-6, even in a population whose mean copy number was 13.5. This type of copy number distribution was reproduced by a mathematical model which assumes that a maximum of 4-6 centromere plasmids per cell can be stably partitioned at cell division. The model also reproduces the observed biphasic kinetics of plasmid number instability. The agreement between simulation and experimental results provides support for the proposed model and demonstrates the utility of the flow cytometry/segregated modeling approach for the study of multicopy recombinant vector propagation.

Programmable bacteria detect and record an environmental signal in the mammalian gut

“…One class of artificial gene-based memory systems uses bistable transcriptional switches to record transient environmental signals transmitted either directly through one of the transcription factors in the switch or indirectly through a distinct trigger element (12)(13)(14)(15)(16). Such memory devices may be used as sophisticated reporters to record a biological event in an organism and interrogate the system at a later time.…”

Mathematical model for aerobic culture of a recombinant yeast

“…The yeast, Saccharomyces cerevisiae, has been an important workhorse in industrial biotechnology and many valuable pharmaceutical proteins have been expressed in this host. Since the early 1980's, several mathematical models have been proposed to describe plasmid instability kinetics of recombinant S. cerevisiae [1,2,3,4]. These models often described plasmid loss without considering the metabolic pathways.…”

Recent advances of flow cytometry in fundamental and applied microbiology

“…FCM has been used to analyse the behaviour of cell populations in fermenters or bioreactors in various fermentation systems such as batch, fed-batch or continuous cultivation under different growth conditions. The physiological behaviour of several industrial microorganisms, with or without recombinant plasmids, has been analysed: the bacteria Escherichia coli [36, 71,170,172], Klebsiella pneumoniae [36], and Micrococcus luteus [99], the yeasts Saccharomyces cerevisiae [4, 118,129,171,193,208], Kluyveromyces lactbs [ 157], and Hansenula polymorpha [37], and the green algae Chlorella vulgaris [97,98]. Some unrecognized aspects of the physiology of microorganisms, such as cellular heterogeneity, have come to light.…”