Ozdemir, Tanel; (2018) Design and construction of therapeutic bacterial sensors in Escherichia coli Nissle 1917. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The human microbiota refers to the ecosystem of microorganisms living on or within the human body, and is increasingly being implicated as a regulator of health and disease. Abnormal alterations in the development or composition of the intestinal microbiota are referred to as dysbiosis. However, the reciprocal interactions between the microbiota and the host’s diet, immunology and genetics can in turn make it extremely difficult to distinguish the cause and effect of dysbiosis in pathologies. Synthetic biology has facilitated the design and creation of more complex and clinically relevant genetic circuits. The commensal nature of the intestinal microbiota and its constituents provide a number of well tolerated microorganisms such as Escherichia coli NISSLE 1917 (EcN) that could be used as powerful investigative tools. The use of antibiotic selection within synthetic circuits would eventually hinder their investigative power during microbiota experiments. Toxin-Antitoxin (TA) post segregational killing systems are a naturally occurring bacterial mechanism to maintain plasmid stability. Here we show that the Axe/Txe TA system from Enterococcus faecium was able to significantly outperform the more widely used Hok/Sok system to maintain the stability of fluorescent and luminescent reporter plasmids in EcN without antibiotic selection during both liquid culture and in vivo animal experiments. In addition, we created a sensor circuit that in EcN could detect both exogenous and bacterial-derived reactive nitrogen species, which are thought to play a crucial role in the human host inflammatory response. We also developed biosensors for pH and the short-chain fatty acid propionate. Finally, we demonstrated that an EcN sensor could detect and report on environmental signals in vivo from the intestines of the Caenorhabditis elegans worm. Synthetic biological tools such as these could help further elucidate the underlying role of the microbiota in conditions such as inflammatory bowel disease and cancers of the gasterointestinal tract.

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