(571bb) Construction and Analysis of Cell Population Heterogeneity of Negative Feedback Gene Auto-Regulatory Systems In Escherichia Coli

The accumulation of genetic information and the development in molecular biologic techniques enable us to design and construct the artificial gene regulatory networks. Since there is a tight relationship between the regulation of gene expression and cell population heterogeneity, analysis of cell population heterogeneity of well-designed artificial gene regulatory networks can provide insights into the underlying gene regulation mechanisms. In the current work we are interested in examining the role of negative feedback gene regulatory systems on gene expression patterns, particularly the degree of heterogeneity within a population as indicated by a reporter protein. We created a low copy number cloning vector pMHLZ1 with two lac operators using GeneTailor Site-Directed Mutagenesis system method. We designed and constructed negative feedback gene regulatory systems by placing the gene encoding the green fluorescent protein (gfp), which serves as a reporter, and the gene encoding the LacI repressor protein under the control of lac promoter systems with one or two lac operators in Escherichia coli. Strains carrying various genetic constructions were cultivated aerobically in shake flasks at 37°C in LB medium. We investigated the variation of cell population heterogeneity of these systems under the induction of IPTG. We also studied the temperature effect of cell population heterogeneity at 32, 37 and 40 °C. GFP expressions at the single cell level were monitored by a flow cytometer. Time profiles of both mean value and coefficient of variation (CV) were analyzed by sampling every 20 min. In this study, we mainly studied the mean value and CV at the reference state. Both cultures and sample measurements were performed in triplicate.