QSB: Variability in Cell Populations

本项目的总体目标是确定大肠杆菌和沙门氏菌群体中表型变异与相关的全基因组转录模式之间的关系。酿酒酵母细胞在明确定义的、可再现的条件下培养。 这将在根据培养模式区分的三种类型的实验中进行。 第一种类型的实验将涉及细胞的简单分批培养。 将从培养物的不同生长阶段采集样品。 第二类实验将涉及在不同稳态下操作的恒化器中连续培养细胞。 第三类实验将涉及将细胞暴露于环境挑战。 此类挑战将包括将稳态恒化器培养转变为新的稀释率。 将分析对这种营养上调或下调的反应。 在所有类型的实验中，将通过流式细胞术测定培养物的表型变异性，并通过细胞分选分离细胞亚群。 分选的细胞将进行微阵列分析方案，以确定全基因组转录谱。 为了使这成为可能，将开发扩增方案以仅从少数细胞可重复地产生足够量的RNA以允许定量微阵列分析。

The overall objective of this project is to determine the relationship between phenotypic variations and the associated whole genome transcriptional pattern in populations of E.coli and S. cerevisiae cells cultivated under well defined, reproducible conditions. This will be done in three types of experiments distinguished according to the cultivation mode. The first type of experiments will involve simple batch cultivation of cells. Samples will be taken from different growth stages of the culture. The second type of experiments will involve continuous cultivation of cells in a chemostat operated at different steady states. The third type of experiments will involve exposure of the cells to an environmental challenge. Such challenges will include the shifting of a steady state chemostat culture to a new dilution rate. The response to such a nutritional upshift or downshift will be analyzed. In all types of experiments the phenotypic variability of the culture will be determined by flow cytometry and subpopulations of cells will be isolated via cell sorting. Sorted cells will be subjected to protocols for microarray analysis to determine the whole genome transcriptional profile. To make this possible an amplification protocol will be developed to reproducibly generate a sufficient amount of RNA from only a few cells to permit quantitative microarray analysis.