Quantifying gene activity in individual bacterial cells

Gene expression is typically measured by averaging over millions of cells or by introducing reporters that perturb cellular function. A new method allows gene expression in individual, genetically unmodified bacterial cells to be quantified.

HFSP Young Investigator Award holder Ido Golding and colleagues
authored on Fri, 26 July 2013

Gene activity is the prime mover in the living cell, driving a cell’s function at any given time. Measuring the level of gene expression is thus the key to predicting what a given cell will do over the coming minutes and hours. Many different approaches to measuring the expression level of a given gene have been developed over the years. Unfortunately, most of these approaches are severely deficient: they either involve mixing together millions of individual cells, thus averaging over critical cell-to-cell differences; or they involve introducing “reporter genes” (such as a gene encoding for a fluorescent protein), which perturb the function of the cell.

Figure: DNA (blue) and RNA (red) labelled in individual E. coli cells.

We recently introduced a method for measuring the activity of a gene of interest in individual, genetically unmodified cells of the bacterium Escherichia coli. Building on previous work in higher organisms, the method allows the researcher to fluorescently label individual molecules of messenger RNA in the cell. The cells are then imaged under the microscope and the photos automatically processed using image analysis algorithms. The number of copies of RNA in each cell, from hundreds of cells, is obtained. These data can be used to extract the underlying kinetic parameters characterizing the activity of the gene. We have already used this method to investigate the activity of multiple genes in E. coli and to demonstrate that all genes show “bursty” (pulsatile) activity, rather than acting constantly over time. Strikingly, a very similar behavior was also observed in higher organisms, from yeast to mammals.

This work emerged from a collaboration with the groups of Ronen Segev (Israel) and Satoshi Sawai (Japan), my HFSP co-investigators. In that collaboration, we aimed to understand the way living cells process information from their environment and regulate their genes to take the right actions based on that information.  

Reference

Measuring mRNA copy number in individual Escherichia coli cells using single-molecule fluorescent in situ hybridization.  Samuel O. Skinner, Leonardo A. Sepúlveda, Heng Xu and Ido Golding. Nature Protocols 8, 1100–1113 (2013).    

Pubmed link

Nature Protocols