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We recently developed an orthogonal replication system (OrthoRep) in yeast that allows for the rapid continuous mutagenesis of a special plasmid without mutating the genome. Although OrthoRep has been successfully applied to evolve several proteins and enzymes, the generality of OrthoRep has not yet been systematically studied. Here, we show that OrthoRep is fully compatible with all Saccharomyces cerevisiae strains tested, demonstrate that the orthogonal plasmid can encode genetic material of at least 22 kb, and report a CRISPR/Cas9-based method for expedient genetic manipulations of OrthoRep. It was previously reported that the replication system upon which OrthoRep is based is only stable in respiration-deficient S. cerevisiae strains that have lost their mitochondrial genome (ρ0 strains). However, here we trace this biological incompatibility to the activity of the dispensable toxin/antitoxin system encoded on the wild-type orthogonal plasmid. Since the toxin/antitoxin system is replaced by genes of interest in any OrthoRep application, OrthoRep is a generally compatible platform for continuous in vivo evolution in S. cerevisiae.
This article was published in the following journal.
Name: ACS synthetic biology
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Macromolecular molds for the synthesis of complementary macromolecules, as in DNA REPLICATION; GENETIC TRANSCRIPTION of DNA to RNA, and GENETIC TRANSLATION of RNA into POLYPEPTIDES.
A single-stranded DNA-binding protein that is found in EUKARYOTIC CELLS. It is required for DNA REPLICATION; DNA REPAIR; and GENETIC RECOMBINATION.
Regions along polytene chromosomes that are uncondensed and active in DNA REPLICATION or RNA transcription (GENETIC TRANSCRIPTION).
Production of new arrangements of DNA by various mechanisms such as assortment and segregation, CROSSING OVER; GENE CONVERSION; GENETIC TRANSFORMATION; GENETIC CONJUGATION; GENETIC TRANSDUCTION; or mixed infection of viruses.
The discipline studying genetic composition of populations and effects of factors such as GENETIC SELECTION, population size, MUTATION, migration, and GENETIC DRIFT on the frequencies of various GENOTYPES and PHENOTYPES using a variety of GENETIC TECHNIQUES.
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