Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.779097
Title: Industrially robust synthetic biology standards for the polymerase chain reaction
Author: Templar, A.
ISNI:       0000 0004 7964 7973
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2017
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Abstract:
Synthetic Biology is ushering in a new era where reengineered genomes can enhance the capacity of host cells to produce biologic and chemical products. Standardisation is a key component of synthetic biology as it enables effective implementation (Müller and Arndt, 2012). This project has successfully generated synthetic biology standards for the quantitative polymerase chain reaction (qPCR), a highly specific and sensitive analytical platform, in order to increase its robustness for monitoring of host cell processes in an industrial setting. This project has also increased the assay throughput to allow for at-line analysis, in accordance with initiatives such as Process Analytical Technology (PAT) (Gnoth et al., 2007). Analysis was conducted on three commonly used host cell chassis and industrial contamination was also simulated by the addition of plasmid proxies. All assays were optimised by primer design and screening to ensure accuracy. End point PCR (e -pPCR) and quantitative PCR (qPCR) was conducted in the presence and absence of cellular material disrupted by a mild sonication procedure. We found that, whilst cellular material reduces assay sensitivity for a genomic locus, the presence of contaminating species can be accurately quantified. We also employed LRE-qPCR, which uses the CAL1 standard for quantification. LRE-qPCR matched the accuracy of a conventional standard curve qPCR method and we propose it as a Synthetic Biology standard. We next developed a modified standard curve method that streamlined methodology and bypassed errors inherent to the gold standard methodology to, for the first time, enable quantification of multiple targets from a single standard curve. The CyCal curve is a standard curve constructed from the CAL1 standard combined with the Cy0 data analysis. The approach was validated against 6 bioprocess targets and it was found that CyCal was able to replicate the accuracy of the gold standard approach. We then used CyCal to accurately determine how host cell plasmid copy number (PCN) evolves during fermentation. The combination of rapid sample preparation and a universal standard means that CyCal is capable of becoming the basis of an at-line qPCR assay when conducted on modern ultra-rapid qPCR thermocycler technology.
Supervisor: Nesbeth, D. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.779097  DOI: Not available
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