Growth and Protein Expression of Saccharomyces Cerevisiae

流式细胞术和狭缝扫描测量技术将在单细胞水平上用于分析酿酒酵母的生长群体。传统的流式细胞术可以快速提供特定单细胞特性的联合分布数据，而狭缝扫描测量技术可以提供某些特性（如细胞大小、DNA或蛋白质含量）在单个细胞内的低分辨率空间分布信息。利用该技术，可以在细胞周期和不对称细胞分裂期间定量测定母细胞和生长中的子细胞的性质。特别是，通过这种新颖的实验方法，获得了总细胞群体内、分裂细胞亚群体内和由细胞分裂形成的细胞群体内属性的频率分布函数的信息。这些实验数据与群体平衡方程相结合，可以快速确定流式细胞术检测到的任何成分的单细胞积累率。这项工作包括改进测量技术、改进细胞制备和免疫荧光染色程序，以检测酿酒酵母细胞中特定的细胞内成分。特别地，利用一种适用于细菌>-半乳糖苷酶流式细胞定量的免疫荧光技术作为模型系统，研究了酿酒酵母在单细胞水平和不同环境条件下单个蛋白的表达动态。此外，正在开发单细胞生长模型来支持实验工作。对于利用微生物的生产工艺的设计，了解微生物的组成以及这种组成如何随时间变化是很重要的。流式细胞术是一种复杂的、基于激光的测量技术，可以以每秒5000个细胞的速度测定单个细胞的组成。在很短的时间内，人们就可以获得单细胞组成在细胞群中如何分布的信息。这种分布的知识提供了对整个细胞群体属性的详细了解，这些属性是由该群体中每个细胞的贡献决定的。例如，由于遗传差异和单个细胞的不同发育阶段，在不稳定的重组细胞群中，细胞间的异质性尤为明显。

Flow cytometry and slit scanning measuring technology will be developed and applied to analyze growing populations of Saccharomyces cerevisiae cells at the single cell level. While conventional flow cytometry provides data on the joint distribution of specific single- cell properties at rapid rate, the slit scanning measuring technique can provide information on low resolution spatial distribution within individual cells of certain properties such as cell size, DNA, or protein content. With this technique, properties of mother cell and growing daughter cell can be quantitated during the cell cycle and during asymmetric cell division. In particular, with this novel experimental approach, information is obtained on frequency distribution functions of properties within the total cell population, within the subpopulation of dividing cells, and within the group of cells that has been formed by cell division. These experimental data, in combination with population balance equations, allow rapid determination of single-cell rates of accumulation of any component detectable by flow cytometry. The work includes refinement of the measuring technique, refinement of cell preparation, and immunofluorescence staining procedures to detect specific intracellular components in Saccharomyces cerevisiae cells. In particular, an immunofluorescence technique suitable for flow cytometric quantification of bacterial >- galactosidase is used as a model system to study the dynamics of expression of a single protein in S. cerevisiae at the single cell level and under varying environmental conditions. In addition, single cell growth models are being developed to support the experimental work. For the design of a production process that utilizes microorganisms, it is important to know the composition of the microorganism and how this composition changes over time. Flow cytometry represents a sophisticated, laser based measuring technology that permits determination of the composition of single cells at rates up to 5000 cells per second. Within a fraction of time one obtains the information on how the single cell composition is distributed within a cell population. Knowledge of such distributions provides a detailed insight into the properties of entire cell populations that are determined by the contribution from each individual cell present in that population. For instance, cell to cell heterogeneities are particularly pronounced in the case of unstable recombinant cell populations because of genetic differences and because of different developmental stages of individual cells.