Mapping of Chromosome Dynamics over the Bacterial Cell Cycle

Abstract: The replication of DNA, its compaction and segregation in dividingcells are challenges which all organisms are faced with. Ensuringthat these processes occur without error is essential for thesurvival of the organism. While the major mechanisms governingchromosome replication and segregation have been elucidated ineukaryotic organisms, analogous processes and their details inprokaryotic organisms have been more challenging to analyse. Inorder to understand the processes behind the localisation of thechromosome during cell division, this project has aimed atanalysing the dynamics of 13 fluorescently labelled loci over thecell cycle of Escherichia coli. The results of this project can beused for further analysis of the chromosome in a large-scale studywhere more loci are analysed to map the dynamics of the wholechromosome.The fluorescent labelling was achieved by introducing the parSsequence at the chosen sites with lambda-Red recombination andexpression of ParB fused to the fluorescent protein mCherry. Thesequence was successfully introduced at eight different positionsin eight separate strains. The introduced parS/ParB system wasconfirmed to result in fluorescent foci by fluorescence microscopyimaging of the strains on agarose pads. Three of these strainswere analysed in a microfluidic PDMS chip platform withfluorescence microscopy. Microfluidic systems provide an advantageof capturing large amounts of cells and making it possible toanalyse them continuously in the same conditions. Combining thesesystems with bright-field, phase contrast and fluorescenceimaging, the growth rates of the cells and dynamics of thefluorescent foci were successfully analysed over several hours.