Cargando…

Superloser: A Plasmid Shuffling Vector for Saccharomyces cerevisiae with Exceedingly Low Background

Here we report a new plasmid shuffle vector for forcing budding yeast (Saccharomyces cerevisiae) to incorporate a new genetic pathway in place of a native pathway – even an essential one – while maintaining low false positive rates (less than 1 in 10(8) per cell). This plasmid, dubbed “Superloser,”...

Descripción completa

Detalles Bibliográficos
Autores principales: Haase, Max A. B., Truong, David M., Boeke, Jef D.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Genetics Society of America 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6686923/
https://www.ncbi.nlm.nih.gov/pubmed/31213518
http://dx.doi.org/10.1534/g3.119.400325
_version_ 1783442641755570176
author Haase, Max A. B.
Truong, David M.
Boeke, Jef D.
author_facet Haase, Max A. B.
Truong, David M.
Boeke, Jef D.
author_sort Haase, Max A. B.
collection PubMed
description Here we report a new plasmid shuffle vector for forcing budding yeast (Saccharomyces cerevisiae) to incorporate a new genetic pathway in place of a native pathway – even an essential one – while maintaining low false positive rates (less than 1 in 10(8) per cell). This plasmid, dubbed “Superloser,” was designed with reduced sequence similarity to commonly used yeast plasmids (i.e., pRS400 series) to limit recombination, a process that in our experience leads to retention of the yeast gene(s) instead of the desired gene(s). In addition, Superloser utilizes two orthogonal copies of the counter-selectable marker URA3 to reduce spontaneous 5-fluoroorotic acid resistance. Finally, the CEN/ARS sequence is fused to the GAL1-10 promoter, which disrupts plasmid segregation in the presence of the sugar galactose, causing Superloser to rapidly be removed from a population of cells. We show one proof-of-concept shuffling experiment: swapping yeast’s core histones out for their human counterparts. Superloser is especially useful for forcing yeast to use highly unfavorable genes, such as human histones, as it enables plating a large number of cells (1.4x10(9)) on a single 10 cm petri dish while maintaining a very low background. Therefore, Superloser is a useful tool for yeast geneticists to effectively shuffle low viability genes and/or pathways in yeast that may arise in as few as 1 in 10(8) cells.
format Online
Article
Text
id pubmed-6686923
institution National Center for Biotechnology Information
language English
publishDate 2019
publisher Genetics Society of America
record_format MEDLINE/PubMed
spelling pubmed-66869232019-08-11 Superloser: A Plasmid Shuffling Vector for Saccharomyces cerevisiae with Exceedingly Low Background Haase, Max A. B. Truong, David M. Boeke, Jef D. G3 (Bethesda) Investigations Here we report a new plasmid shuffle vector for forcing budding yeast (Saccharomyces cerevisiae) to incorporate a new genetic pathway in place of a native pathway – even an essential one – while maintaining low false positive rates (less than 1 in 10(8) per cell). This plasmid, dubbed “Superloser,” was designed with reduced sequence similarity to commonly used yeast plasmids (i.e., pRS400 series) to limit recombination, a process that in our experience leads to retention of the yeast gene(s) instead of the desired gene(s). In addition, Superloser utilizes two orthogonal copies of the counter-selectable marker URA3 to reduce spontaneous 5-fluoroorotic acid resistance. Finally, the CEN/ARS sequence is fused to the GAL1-10 promoter, which disrupts plasmid segregation in the presence of the sugar galactose, causing Superloser to rapidly be removed from a population of cells. We show one proof-of-concept shuffling experiment: swapping yeast’s core histones out for their human counterparts. Superloser is especially useful for forcing yeast to use highly unfavorable genes, such as human histones, as it enables plating a large number of cells (1.4x10(9)) on a single 10 cm petri dish while maintaining a very low background. Therefore, Superloser is a useful tool for yeast geneticists to effectively shuffle low viability genes and/or pathways in yeast that may arise in as few as 1 in 10(8) cells. Genetics Society of America 2019-06-18 /pmc/articles/PMC6686923/ /pubmed/31213518 http://dx.doi.org/10.1534/g3.119.400325 Text en Copyright © 2019 Haase et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Investigations
Haase, Max A. B.
Truong, David M.
Boeke, Jef D.
Superloser: A Plasmid Shuffling Vector for Saccharomyces cerevisiae with Exceedingly Low Background
title Superloser: A Plasmid Shuffling Vector for Saccharomyces cerevisiae with Exceedingly Low Background
title_full Superloser: A Plasmid Shuffling Vector for Saccharomyces cerevisiae with Exceedingly Low Background
title_fullStr Superloser: A Plasmid Shuffling Vector for Saccharomyces cerevisiae with Exceedingly Low Background
title_full_unstemmed Superloser: A Plasmid Shuffling Vector for Saccharomyces cerevisiae with Exceedingly Low Background
title_short Superloser: A Plasmid Shuffling Vector for Saccharomyces cerevisiae with Exceedingly Low Background
title_sort superloser: a plasmid shuffling vector for saccharomyces cerevisiae with exceedingly low background
topic Investigations
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6686923/
https://www.ncbi.nlm.nih.gov/pubmed/31213518
http://dx.doi.org/10.1534/g3.119.400325
work_keys_str_mv AT haasemaxab superloseraplasmidshufflingvectorforsaccharomycescerevisiaewithexceedinglylowbackground
AT truongdavidm superloseraplasmidshufflingvectorforsaccharomycescerevisiaewithexceedinglylowbackground
AT boekejefd superloseraplasmidshufflingvectorforsaccharomycescerevisiaewithexceedinglylowbackground