Team:Oxford/Experiments
Experiments
Introduction
Our enzymatic approach to the treatment of urinary tract infections (UTIs) is centred on the design of a "pathogen killing" engineered microbial host containing three key features:
- Constant secretion of biofilm-degrading enzymes - degrading the biofilms of the pathogenic bacteria reduces their resistance towards antibiotics
- Production and intracellular accumulation of enzymes that can kill both the pathogenic bacteria and our engineered microbial host upon release into the extracellular medium
- A quorum sensing mechanism that triggers the release of the antibacterial enzymes in the presence of pathogenic bacteria
Due to constraints in time and resources, we focused our experimental efforts towards the development of proof-of-concepts for only the first two features.
Through our experimental work with secretion assays, biofilm assays, and cell-killing assays we were able to obtain preliminary data suggesting that the BioBrick parts which we designed to allow our microbial host to produce the relevant biofilm-degrading enzymes and bacteria-killing enzymes are indeed able to function as expected individually.
Bacterial Strains and Growth Cultures
E. coli DH5α was used for all cloning purposes. The E. coli strains MG1655 and RP437 ∆FliC, as well as the multi-effector knockout BSL-1 strain of Y. enterocolitica, IML421asd, were used as expression hosts. Cultures for cloning were grown in Lysogeny Broth (LB) at 37°C. The E. coli expression host cultures were grown in Lysogeny Broth (LB) at 37°C, while cultures of Y. enterocolitica IML421asd was grown in Brain Heart Infusion (BHI) media supplemented with diaminopimelic acid (DAP) at 30°C.