Genetics Problem Spaces

Introduction Problem Spaces Arabinose Operon Overview Intro Exercise Research Questions Literature Resources Assignments Student Projects

Arabinose Operon Research Questions Question I Siegele and Hu (1997) analyzed expression from a GFP reporter gene fused to the araBAD promoter much like that in pGLO. They found that low concentrations of arabinose induced low levels of GFP expression in a population of cells. However, when they analyzed individual cells within this population, they found an interesting pattern of expression. Instead of all the cells expressing low levels of GFP, they found a few cells expressing high levels of GFP, while other cells expressed no GFP. This is termed “all or nothing expression” and had been seen before with the lac operon. Their paper discusses what might be causing all or nothing expression. Siegele and Hu varied the arabinose concentration to alter araBAD driven GFP expression. Because of catabolite repression, glucose can also alter expression from this promoter by a different mechanisms than arabinose. Will low levels of glucose lead to low levels of GFP expression? Does glucose repression result in an all or nothing pattern of expression. Can low levels of GFP expression result from low levels of catabolite repression? Question II Siegele and Hu (1997) speculate in their methods that expression of GFP in E. coli may be toxic to the cells. Jiu et al have reported similar toxicity for eukaryotic cells. How can GFP toxicity be measured? Can you design an experiment to measure the toxic effects of GFP in E. coli? Can your experiment differentiate whether the toxicity acts by killing the cells or slowing their growth? Can you design an experiment to differentiate whether the toxicity is due simply to the presence of GFP in cells or is its toxicity dependent on active synthesis of GFP in cells? Question III Siegel and Hu (1997) measure the kinetics of induction of GFP from an araBAD-GFP fusion in a population of cells. Given their observation concerning mixed expression in individual cells, can you compare induction kinetics of a population of cells to induction kinetics of individual cells? Question IV The introductory exercise investigated whether non-PTS sugars can repress expression of the ara operon. Students in previous semesters have often observed that while glycerol doesn’t repress the operon to the degree that glucose does, there does seem to be some reduction in gfp expression. This seems to be consistent with the observations of Eppler and Boos (1999) where glycerol caused a 2-5 fold reduction in the expression of the maltose operon. Can you design an experiment to detect whether glycerol has the weak repressive effect? Do other non-PTS sugars have a weak catabolite repression effect?