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Title: Synthetic biology application and characterisation of Acinetobacter baylyi ADP1
Author: Court, Elizabeth Kathleen
ISNI:       0000 0004 7657 3198
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2018
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Acinetobacter baylyi ADP1 has great potential applications to synthetic biology in comparison to other model organisms such as E.coli due to its ease of handling, natural competency and wide range of carbon sources it can metabolise. However, the characterisation of the organism in comparison to E.coli still has far to progress and to enable successful design, construction and modelling of synthetic systems in A.baylyi ADP1, a comprehensive understanding of the bacteria is needed. This thesis is a study of Synthetic Biology applications of Acinetobacter baylyi ADP1 by characterisation as a chassis and applications to biosensors. Characterisation of A.baylyi ADP1 was carried out investigating the phosphate accumulation pathway. Polyphosphate is involved in genetic regulation and A.baylyi ADP1 has been recognised as a phosphate accumulator organism (PAO). Characterising this strain as a potential chassis for phosphate removal would be beneficial to address the phosphate problem in wastewater whereby accumulation leads to eutrophication downstream incurring high costs for treatment. Characterisation of polyphosphate accumulation in A.baylyi ADP1 was carried out by creating novel knockout mutations of the putative polyphosphate associated genes relA and ppK. These were investigated using growth, biofilm and settling assays, and confocal microscopy to characterise the phenotypes of these mutants. This work shows that both mutations have a negative effect on biofilm formation, ?relA settles from solution less and that the mutants appear to aggregate more than form a true biofilm, with reduced biofilm formation at the air-liquid interface. The application of A.baylyi ADP1 as a chassis organism for a biosensor to detect pathogens in water was investigated, specifically Vibrio cholerae. V.cholerae is a waterborne pathogen that causes the acute diarrhoeal disease cholera which is prevalent in the developing world. Current detection methods are lab based, with a timescale of hours to days, using fluorescent labelled antibodies or PCR to detect V.cholerae outer membrane proteins and DNA respectively. The principles of synthetic biology have been applied to design assembly methods for fast, simple, sensitive, mobile surveillance system that can be used in the field. The basis of the biosensor design is to detect chemicals involved in quorum sensing, the process by which bacteria communicate using secreted chemical signalling molecules to assess their population density. The biosensor consists of a reporter component, and a sensor component made up of the pathogens own intracellular quorum sensing signalling pathway genes whose proteins will detect the chemical molecule CAI-1 in a water sample and drive expression of reporter genes under control of the pathogens promoter. Here we present two designs for biosensor assembly, one a "bespoke" system where each part can be altered and another "off the shelf" method using multiple cloning sites to direct insertion of parts without needing detailed genetic knowledge to assemble. Further characterisation of A.baylyi ADP1 as a chassis was carried out by investigating intracellular networks and A.baylyi ADP1 interaction with the environment to allow the better design and development of synthetic systems in A.baylyi ADP1 in the future. The ability of A.baylyi ADP1 to interact with its environment was investigated by production of a novel knockout mutation in the putative quorum sensing transcription regulator gene from the LuxR family (YP_045866), characterisation of the transcription regulator gene using biofilm assays indicated this mutation has a negative effect on the ability to form a biofilm. Investigation into muropeptide detection in relation to antibiotic resistance gene expression control signalling networks within A.baylyi ADP1 was carried out using qPCR with additions of the muropeptides mDAP and LYS, ampicillin and lysozyme which showed that A.baylyi ADP1 can detect muropeptides in the environment and respond by change in gene expression of ampC and 1855 antibiotic resistance genes. A significant increase in expression of these genes was observed when a muropeptide not found in its own peptidoglycan structure (LYS) was applied, indicating a response to external stimuli and ability to respond to other bacteria in the environment. B.subtilis 168 was also investigated to determine if this gene regulation could be targeted for development of a beta-lactamase biosensor. This thesis successfully progressed characterisation of A.baylyi ADP1 by creating new knockout mutations for analysis, investigating A.baylyi ADP1 interaction with the environment and polyphosphate accumulation genes, and developing new constructs and plasmid designs for use in synthetic biology, specifically here for the detection of quorum sensing signalling molecules in a water environment.
Supervisor: Biggs, Catherine ; Hunt, Stuart Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available