Characterisation of anucleate cells

Abstract

PhD ThesisBacteria are beneficial in industry as they are easily genetically manipulated to express proteins, enzymes or full pathways while remaining cheap to grow in large batches with simple feed stocks. However, there are limitations associated with this as overproduction can lead to interference from complex cellular processes or metabolic burden. Metabolic burden is caused when essential cellular resources, such as energy and carbon, are diverted to the engineered pathway used for bio-production. To overcome limitations, this project aims to compartmentalise biosynthetic pathways into anucleate compartments of Bacillus subtilis, Escherichia coli and Synechococcus elongatus while regulating expression in nucleated cells. Division mutants were constructed in each chassis for the production of minicells or maxicells. Analysis of these anucleate producing strains was done to determine the properties of plasmid replicons with respect to gene expression and segregation into anucleate cells. Here the Plac/LacI regulation system was used so expression in nucleated rod cells was repressed while expression occurred within anucleate compartments. Good repression was present in the minicell and maxicell producing strains of B. subtilis, however, only 50% of anucleate cells were expressing GFP. There were efforts in B. subtilis to alter copy number to understand the pLS20 replicon and increase the amount of anucleate cells gaining a plasmid, however, there was no single mutant which worked best in minicells. There was a dramatic increase in maxicell expression with copy number mutants but there was still a cost to the regulation system in nucleated cells. In E. coli, the systems regulation does not repress as efficiently in nucleated mother cells compared to B. subtilis. Studies from this work revealed that the RSF1010 driven plasmids were better for minicell expression where ~30-35% minicells were GFP positive. Although a minicell producing mutant was constructed for S. elongatus, integration of a plasmid and repression system remained problematic. However, the minicell producing phenotype was characterised in greater detail using widefield fluorescence and transmission electron microscopy. This work contributes to the wider Portabolomics project aiming to bridge the gap between academia and industry