Quantitative detection of low abundance gene expression products in individual E. coli cells
Taylor, Hannah Louise
Stochastic fluctuations in mRNA and protein copy number between cells are inevitable during the process gene expression, even when cells carry identical chromosomes. Such fluctuations are able to impact the phenotypic fate of the cell, and are known to have greater impact when the copy number of the molecule involved is low. Additionally, up to 50% of proteins in Escherichia coli are present in the cell at a level of 10 molecules per cell or fewer (Taniguchi et al. 2010). As such, quantification of low copy number gene expression products and their distribution in cellular populations is key in understanding the process of gene expression. Currently, there are few techniques that allow investigation with the single cell and single molecule resolution required to study low copy number gene expression products. This work presents a novel method for protein quantification at the single molecule level, Quantitative HaloTag-TMR labelling, and uses the technique to quantify the absolute numbers of the low copy number RecB, RecC and RecD subunits of the bacterial DNA repair enzyme RecBCD, finding each subunit is present at between two and eight molecules per cell with mean numbers per cell of 4.9, 4.7 and 4.5 respectively. Additionally single molecule mRNA FISH was used to quantify the mRNA levels of recB and recD within cells, with means of 0.21 and 0.31 mRNA per cell being observed respectively. Finally this work presents a new method for use detecting both mRNA and protein simultaneously in individual cells by combining the HaloTag and FISH protocols to give HaloFISH. This work introduces two novel techniques that allow for single cell examination of gene expression, and investigates RecBCD expression at the single molecule level.