title: Influence of Pichia pastoris cellular material on polymerase chain reaction performance as a synthetic biology standard for genome monitoring.
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 50pg (4.91×10(3) copies) of purified genomic DNA (gDNA) but the presence of cellular material reduced this sensitivity sixfold to 300pg gDNA (2.95×10(4) 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.
authors.affiliation: Department of Biochemical Engineering, University College London, Bernard Katz Building, London, WC1E 6BT, United Kingdom.
authors.forename: Alexander
authors.initials: A
authors.lastname: Templar
authors.affiliation: Department of Biochemical Engineering, University College London, Bernard Katz Building, London, WC1E 6BT, United Kingdom.
authors.forename: Stefan
authors.initials: S
authors.lastname: Woodhouse
authors.affiliation: Department of Biochemical Engineering, University College London, Bernard Katz Building, London, WC1E 6BT, United Kingdom.
authors.forename: Eli
authors.initials: E
authors.lastname: Keshavarz-Moore
authors.affiliation: Department of Biochemical Engineering, University College London, Bernard Katz Building, London, WC1E 6BT, United Kingdom. Electronic address: d.nesbeth@ucl.ac.uk.
authors.forename: Darren N
authors.initials: DN
authors.lastname: Nesbeth
mesh_terms: D017931:DNA Primers; D016681:Genome, Fungal; D023281:Genomics; D016014:Linear Models; D010843:Pichia / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D058615:Synthetic Biology / Q000379:methods*
chemical_list: D017931:DNA Primers
keywords: Absolute quantitation; Linear regression; Methylotrophic; Qpcr; Standardisation; Synthetic Biology