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In this thesis, I present three microscopy studies of surface growing Escherichia coli (E. coli ) microcolonies. All experiments were carried out by growing microcolonies on agarose pads, and imaging their growth using phase contrast, fluorescence and confocal microscopy. In the first project, the importance of spatial structure and growth strategies between competing populations of E. coli was studied. An agarose pad was seeded with bacterial cells and their colonisation success tracked. Cell lag-times and local cell density were found to play important roles in determining the eventual success of a colony. Arrangements of neighbouring cells were found to be partially responsible at high cell densities. These results were reproduced using a simple simulation, which also highlighted the importance of exponential expansion in determining colonisation success. The second project investigates the effect of confinement on growing microcolonies restricted to one plane (2d growth). Colonies were grown in agarose microchannels with different aspect ratios, and in unconfined environments. In particular internal physical colony structure and genealogical structure was studied by using single-cell tracking. Results showed that relatedness between cells was directionally biased (cells tended to be more closely related to cells at their poles, than to their side) regardless of the amount of spatial restriction. Furthermore, confinement caused cells to align with each other more, and induced high cell velocities at the colony edges driven by cell expansion. In the final project, growth of secondary layers in growing colonies of E. coli was studied. Cells initially grew as a monolayer, before invading the agarose bulk, producing a secondary layer. By analysing time-lapse movies, this layer was found to initially expand rapidly well in excess of cell growth rates and initial colony expansion rates, before slowing down. The initial secondary growth rate likely depends on the colony area at agarose invasion. Furthermore, the colony area when colonies invaded the agarose depended on their rate of growth, suggesting a complex interplay between forces exerted by the agarose, and by the colony.

In this study a library of EcoRI fragments encompassing the entire TOL region of the Pseudomonas putida TOL plasmid pDK1 was constructed in the Escheria coli cloning vector pBR325.