PCR on yeast colonies: an improved method for glyco-engineered Saccharomyces cerevisiae

Abstract

BackgroundSaccharomyces cerevisiae is extensively used in bio-industries. However, its genetic engineering to introduce new metabolism pathways can cause unexpected phenotypic alterations. For example, humanisation of the glycosylation pathways is a high priority pharmaceutical industry goal for production of therapeutic glycoproteins in yeast. Genomic modifications can lead to several described physiological changes: biomass yields decrease, temperature sensitivity or cell wall structure modifications. We have observed that deletion of several N-mannosyltransferases in Saccharomyces cerevisiae, results in strains that can no longer be analyzed by classical PCR on yeast colonies.FindingsIn order to validate our glyco-engineered Saccharomyces cerevisiae strains, we developed a new protocol to carry out PCR directly on genetically modified yeast colonies. A liquid culture phase, combined with the use of a Hot Start DNA polymerase, allows a 3-fold improvement of PCR efficiency. The results obtained are repeatable and independent of the targeted sequence; as such the protocol is well adapted for intensive screening applications.ConclusionsThe developed protocol enables by-passing of many of the difficulties associated with PCR caused by phenotypic modifications brought about by humanisation of the glycosylation in yeast and allows rapid validation of glyco-engineered Saccharomyces cerevisiae cells. It has the potential to be extended to other yeast strains presenting cell wall structure modifications.

Bootleg Biology: a Semester-Long CURE Using Wild Yeast to Brew Beer

“…Equipment for subsequent experiments includes the following: methylene blue and/or Gram stain reagent packs (1 bottle of each for every 2 to 3 groups), 1 500-mL bottle of bacteriological-grade agar and sterile petri dishes (3 to 4 plates per group for the semester), one bottle of yeast nutrient, 500 mL of 33% sucrose, mason jars (1 to 2 jars per group), cheesecloth, a yeast genomic DNA prep kit (1 per class), primers (1 set per class), master mix (500 μL), and an account for Sanger sequencing. Yeast genomic DNA was purified using a yeast colony PCR protocol ( 10 ) modified for this lab, and internal transcribed spacer (ITS) DNA was amplified using the SC1 and SC2 primers ( 11 ). Samples were purified with the GeneJet PCR purification kit according to the manufacturer’s instructions and sequenced at Eurofins Genomics.…”

“…Once their organisms had been identified as strains of brewer’s yeast, we moved to a standard BSL-1 room; eye protection, lab coats, and gloves were worn at all times, and the bench space was disinfected before and after each session. In order to minimize any issues, the syllabus indicated that wild yeast should be isolated first and colony PCR modified using methods outlined elsewhere ( 10 ) to make sure it is a brewer’s yeast isolate before any beer is tasted.…”

Design and Construction of a Designed Ankyrin Repeat Protein (DARPin) Display Library

“…15. For analysis of enriched libraries after multiple selection rounds, use the same PCR reaction parameters as for the naïve library but PCR-amplify the DARPinencoding region from individual colonies or a cell suspension by colony PCR (Bonnet et al, 2013; see Current Protocols article: Woodman et al, 2016) using the DARP.Amplicon.F/DARP.Amplicon.R primer pair (Table 2) to generate a ∼365-bp PCR product.…”

Rapid and robust squashed spore/colony PCR of industrially important fungi

“…However, this technique is sometimes hampered by insufficient genomic DNA released into the PCR reaction mix and the presence of PCR inhibitors [ 17 ]. As a consequence, contradictory results have been observed and colony PCR efficiency in different yeast species and strains can vary due to different cell wall thickness and cell components [ 16 , 18 ].…”