ABSTRACT
We present a teaching protocol suitable for demonstrating the use of EasyClone and CRISPR/Cas9 for metabolic engineering of industrially relevant yeasts Saccharomyces cerevisiae and Yarrowia lipolytica, using ß-carotene production as a case study. The protocol details all steps required to generate DNA parts, transform and genotype yeast, and perform a phenotypic screen to determine ß-carotene production. The protocol is intended to be used as an instruction manual for a two-week practical course aimed at M.Sc. and Ph.D. students. The protocol details all necessary steps for students to engineer yeast to produce ß-carotene and serves as a practical introduction to the principles of metabolic engineering including the concepts of boosting native precursor supply and alleviating rate-limiting steps. It also highlights key differences in the metabolism and heterologous production capacity of two industrially relevant yeast species. The protocol is divided into daily experiments covering a two-week period and provides detailed instructions for every step meaning this protocol can be used 'as is' for a teaching course or as a case study for how yeast can be engineered to produce value-added molecules.
Subject(s)
CRISPR-Cas Systems , Gene Editing/methods , Metabolic Engineering/methods , Saccharomyces cerevisiae/genetics , Yarrowia/genetics , Education, Graduate/methods , Humans , Industrial Microbiology , Saccharomyces cerevisiae/metabolism , Teaching , Yarrowia/metabolismABSTRACT
A statistical analysis of the anomalous particle flux in the edge region of the RFX experiment has revealed that laminar times between bursts, which account for more than 50% of the losses, have a power law distribution and that flux fluctuations are not self-similar. These properties are found in contrast with a wide class of self-organized-criticality models so that it is concluded that there is no experimental evidence of avalanchelike process occurrence in the plasma of RFX.
ABSTRACT
In order to test the self-organized criticality (SOC) paradigm in transport processes, a novel technique has been applied for the first time to plasmas confined in reversed field pinch configuration. This technique consists of an analysis of the probability distribution function of the times between bursts in density fluctuations measured by microwave reflectometry and electrostatic probes. The same analysis has also been applied to intermittent events sorted out from the Gaussian background. In both cases, the experimental results disagree with the predictions for a SOC system.
