User:Catherine Backus/sandbox

SAT1 Flipping refers to the excision of a target gene and integration of a new different gene to create a mutant genome different than the wild type genome. This method uses the effects of a cassette. In genetics, a cassette is a movable piece of genetic material that includes a recombination site and a specific gene about 500 - 1000 base pairs long. In the SAT1 flipping method, the specific gene within the cassette is a dominant nourseothricin resistance marker, also known as a caSAT1 gene, which is used to select and excise the target gene within the string of DNA being examined. Cassettes are the optimal choice of methodology for SAT1 gene flipping because they are known to carry antibiotic resistant genes within their genetic material. The SAT1 flipping method is preferred over other knock-out methods for gene excision when other said methods provide less than optimal results. One example of a successful use of the SAT1 flipping method came from a group of scientists within the Elsevier company in the Netherlands. The method was used in Candida albicans, a species of yeast that is very commonly used to examine fungal virulence. The choice to attempt SAT1 flipping to create new mutant phenotypes of C. albicans was made when the regular knock-out method of using a repeated URA3 marker was discovered to potentially cause issues in reading the new mutant phenotype. In the sample of C. albicans used, transformants from a selective medium and integrated into the genome at the locus of the target gene. The integrated transformant, along with a C. albicans-adapted FLP gene allowed for the successful excision of the OPT1 gene. The deletion of the gene within the yeast’s genome allowed for the resistance of a toxic peptide KLLth. Proof that the use of SAT1 flipping of the OPT1 gene was confirmed by the reintroduction of the OPT1 gene back into the new strain of mutant C. albicans, which in turn restored susceptibility to the toxic peptide.