Use this URL to cite or link to this record in EThOS:
Title: Novel in vivo biosensors for monitoring of mammalian cell cultures
Author: Goers, Lisa
ISNI:       0000 0004 6061 467X
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2015
Availability of Full Text:
Access from EThOS:
Access from Institution:
Mammalian cell cultures are used for production of biopharmaceuticals, e.g. monoclonal antibodies. Only mammalian hybridoma cells contain the pathways for antibody production, but due to their multicellular origin the cells have complex nutrient requirements. Cell growth and antibody production are limited by supply of essential nutrients such as glutamine and accumulation of toxic waste products such as lactate. Many attempts have been made at tackling these challenges, e.g. by optimising growth media to keep metabolite concentrations at optimal levels. These approaches have been hampered by our ability to monitor relevant cell culture parameters such as metabolite concentration dynamics in real time. The aim of this study is to develop a solution to this problem using a synthetic biology approach. Whole-cell bacterial biosensors for important culture parameters, glutamine, leucine, alanine and lactate, were designed, built and characterised. The biosensors were designed from natural metabolite-sensing systems, specifically the Escherichia coli Ntr regulon, Lrp regulon and lldPRD operon and the Bacillus subtilis GlnK-GlnL system. Characterisation of the biosensors in defined medium using known lactate concentrations was followed by validation in mammalian cell culture media and using cell culture samples. A lactate sensor based on the lldPRD operon showed a reliable lactate-response during initial characterisation and was chosen to determine lactate concentrations in cell culture samples in parallel with lactate analysis using a bioprofiler. Generally, the lactate concentrations from the two methods showed a good match. Data points where the results differed showed that there are some sources of error in the usage of the biosensor that could be addressed in future. The results of this study also highlight the many challenges of applying synthetic biology constructs to complex industrial contexts. The biosensors presented in this study are more generally applicable in any experimental context that requires sensing of metabolites.
Supervisor: Polizzi, Karen Marie ; Freemont, Paul Sponsor: Biotechnology and Biological Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral