Templar, A;
Woodhouse, S;
Keshavarz-Moore, E;
Nesbeth, DN;
(2016)
Influence of Pichia pastoris cellular material on polymerase chain reaction performance as a synthetic biology standard for genome monitoring.
Journal of Microbiological Methods
, 127
pp. 111-122.
10.1016/j.mimet.2016.05.013.
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Abstract
Advances in synthetic genomics are now well underway in yeasts due to the low cost of synthetic DNA. These new capabilities also bring greater need for quantitating the presence, loss and rearrangement of loci within synthetic yeast genomes. Methods for achieving this will ideally; i) be robust to industrial settings, ii) adhere to a global standard and iii) be sufficiently rapid to enable at-line monitoring during cell growth. The methylotrophic yeast Pichia pastoris (P. pastoris) is increasingly used for industrial production of biotherapeutic proteins so we sought to answer the following questions for this particular yeast species. Is time-consuming DNA purification necessary to obtain accurate end-point polymerase chain reaction (e-pPCR) and quantitative PCR (qPCR) data? Can the novel linear regression of efficiency qPCR method (LRE qPCR), which has properties desirable in a synthetic biology standard, match the accuracy of conventional qPCR? Does cell cultivation scale influence PCR performance? To answer these questions we performed e-pPCR and qPCR in the presence and absence of cellular material disrupted by a mild 30s sonication procedure. The e-pPCR limit of detection (LOD) for a genomic target locus was 50 pg (4.91 × 103 copies) of purified genomic DNA (gDNA) but the presence of cellular material reduced this sensitivity sixfold to 300 pg gDNA (2.95 × 104 copies). LRE qPCR matched the accuracy of a conventional standard curve qPCR method. The presence of material from bioreactor cultivation of up to OD600 = 80 did not significantly compromise the accuracy of LRE qPCR. We conclude that a simple and rapid cell disruption step is sufficient to render P. pastoris samples of up to OD600 = 80 amenable to analysis using LRE qPCR which we propose as a synthetic biology standard.
| Type: | Article |
|---|---|
| Title: | Influence of Pichia pastoris cellular material on polymerase chain reaction performance as a synthetic biology standard for genome monitoring |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1016/j.mimet.2016.05.013 |
| Publisher version: | http://dx.doi.org/10.1016/j.mimet.2016.05.013 |
| Language: | English |
| Additional information: | Copyright © 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) (CC BY 4.0). You may copy and distribute the article, create extracts, abstracts and new works from the article, alter and revise the article, text or data mine the article and otherwise reuse the article commercially (including reuse and/or resale of the article) without permission from Elsevier. You must give appropriate credit to the original work, together with a link to the formal publication through the relevant DOI and a link to the Creative Commons user license above. You must indicate if any changes are made but not in any way that suggests the licensor endorses you or your use of the work. |
| Keywords: | Absolute quantitation; Synthetic Biology; Qpcr; Standardisation; Methylotrophic; Linear regression |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > UCL BEAMS UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering |
| URI: | https://discovery.ucl.ac.uk/id/eprint/1493674 |
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