Knocking out multigene redundancies via cycles of sexual assortment and fluorescence selection

Abstract

Phenotypes that might otherwise reveal a gene’s function can be obscured by genes with overlapping function. This phenomenon is best-known within gene families, where an important shared function may only be revealed by mutating all family members. Here we describe the ‘Green Monster’ technology enabling the precise deletion of many genes. In this method, a population of deletion strains with each deletion marked by an inducible green fluorescent protein (GFP) reporter gene, is subjected to repeated rounds of mating, meiosis, and flow-cytometric enrichment. This results in the aggregation of multiple deletion loci within single cells. The Green Monster strategy is potentially applicable to assembling other engineered alterations in any species with sex or alternative means of allelic assortment. To demonstrate the technology, we generated a single broadly drug-sensitive strain of Saccharomyces cerevisiae bearing precise deletions of all 16 adenosine triphosphate-binding cassette transporters within clades associated with multi-drug resistance.

Multiplex assay for condition-dependent changes in protein–protein interactions

“…Poor cell permeability can be an obstacle of cellbased assays in yeast, whose cell wall and elaborate chemical defense mechanisms represent a formidable barrier to many chemical compounds. Although it is unclear how many compounds were actually able to penetrate into yeast, prescreening compounds for growth inhibition of a wild-type strain, applying computational models to prioritize cell-permeable bioactive compounds (34), or genetically disabling yeast's chemical defense mechanisms (35,36) could each increase the number of compounds effectively entering the cell in future studies. Indeed, in the current study our "hit rate" was much higher for growth inhibitory compounds, as 13 of the 14 growth inhibitors identified at least one change in our multiplex assay.…”

“…The utility of yeast as a model for studying multidrug resistance is supported by its vast repertoire of drug transporters (29,35) and by the homology of both Pdr5 and Snq2 to human Mdr1, a major contributor to tumor resistance (43,44). Of the 80 chemicals screened in this study, 21 (26.25%) yielded an interaction score >10 in at least one of three drug-pump strains (Pdr5:Pdr5, Snq2:Snq2, or Tpo1: Pdr5).…”

Reiterative Recombination for the in vivo assembly of libraries of multigene pathways

“…To meet the first of these needs, altering the genetic background, classic mutagenesis techniques such as mutator strains can be useful for phenotypic optimization, but they do not provide control over the extent and location of mutations (49), unlike several notable, recently reported techniques. Exploiting sexual reproduction in S. cerevisiae, Suzuki et al have used iterative cycles of mating and sporulation along with a quantitative GFP marker to gather up to 16 deletion mutations in an individual strain (50). Using an E. coli strain engineered with the λ Red recombination system (36), multiplex automated genome engineering automates the transformation of mutagenic singlestranded oligonucleotides ∼90 bases in length to create a powerful method for introducing specified deletions, point mutations, and very short insertions of ≤30 base pairs anywhere in the chromosome, but it has not been shown to be capable of efficient whole-gene insertion in its current form (see Note.)…”

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

“…Because the number of markers is limited, methods have been developed for removing and reusing the same marker 5,6,7,8,9,10 . However, sequentially engineering multiple mutations using these methods is time-consuming because the time required scales linearly with the number of deletions to be generated.Here we describe the Green Monster method for routinely engineering multiple deletions in yeast 11 . In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1).…”

“…Here we describe the Green Monster method for routinely engineering multiple deletions in yeast 11 . In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1).…”