Зеленый монстр процесса генерации штаммов дрожжей проведение нескольких удалений Гена
Summary
Зеленый монстр метод позволяет быструю сборку нескольких удалений отмечены кодирования гена-репортера зеленого флуоресцентного белка. Этот метод основан на вождение штаммов дрожжей через повторяющиеся циклы сексуальных ассортимент удалений и флуоресценции на основе обогащения клеток, несущих более удалений.
Abstract
Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition1,2. There are examples where many genes need to be deleted to unmask hidden gene functions3,4. Despite the potential for important discoveries, genetic interactions involving three or more genes are largely unexplored. Exhaustive searches of multi-mutant interactions would be impractical due to the sheer number of possible combinations of deletions. However, studies of selected sets of genes, such as sets of paralogs with a greater a priori chance of sharing a common function, would be informative.
In the yeast Saccharomyces cerevisiae, gene knockout is accomplished by replacing a gene with a selectable marker via homologous recombination. Because the number of markers is limited, methods have been developed for removing and reusing the same marker5,6,7,8,9,10. 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 yeast11. 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). Repeated rounds of assortment of GFP-marked deletions via yeast mating and meiosis coupled with flow-cytometric enrichment of strains carrying more of these deletions lead to the accumulation of deletions in strains (Figure 2). Performing multiple processes in parallel, with each process incorporating one or more deletions per round, reduces the time required for strain construction.
The first step is to prepare haploid single-mutants termed ‘ProMonsters,’ each of which carries a GFP reporter in a deleted locus and one of the ‘toolkit’ loci—either Green Monster GMToolkit-a or GMToolkit-α at the can1Δ locus (Figure 3). Using strains from the yeast deletion collection12, GFP-marked deletions can be conveniently generated by replacing the common KanMX4 cassette existing in these strains with a universal GFP–URA3 fragment. Each GMToolkit contains: either the a– or α-mating-type-specific haploid selection marker1 and exactly one of the two markers that, when both GMToolkits are present, collectively allow for selection of diploids.
The second step is to carry out the sexual cycling through which deletion loci can be combined within a single cell by the random assortment and/or meiotic recombination that accompanies each cycle of mating and sporulation.
Protocol
Representative Results
Discussion
Как мы разработали подход Зеленый Монстр, мы были обеспокоены возможностью рекомбинации между различными кассетами замены GFP, что приводит к перестройке генома. Смягчение против этой возможности нашего выбора для клеток, которые успешно прошли несколько раундов спаривания и мейоза. К…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Эта работа была поддержана США Defense Advanced Research Projects Agency контракту N66001-12-C-4039, чтобы Ю.С., грант от Alfred P. Sloan Foundation в РГБ и американского Национального института здравоохранения грантов R01 и R21 HG003224 CA130266 в FPRFPR было Также поддерживается общение от Канадского института передовых исследований и исследований Канады программы мастерства председателей.
Materials
| Name of the reagent or instrument | Company | Catalogue number | Comments (optional) |
| G418 | Sigma-Aldrich | A1720 | Dissolve in water and filter-sterilize (0.2-μm filter). Stock concentration: 200 mg/ml. Store at 4 °C. |
| ClonNAT (nourseothricin) | WERNER BioAgents | 5001000 | Dissolve in water and filter-sterilize (0.2-μm filter). Stock concentration: 100 mg/ml. Store at 4 °C. |
| Doxycycline | Sigma-Aldrich | D9891 | Dissolve in 50% ethanol and filter-sterilize (0.2-μm filter). Stock concentration: 10 mg/ml. Make fresh every four weeks. Shield from light using aluminum foil and store at 4 °C. |
| Zymolyase | ZymoResearch | E1005 | |
| Difco yeast nitrogen base w/o amino acids | BD | 291940 | |
| Revolver (rotator for tubes) | Labnet | H5600 | |
| Enduro Gel XL electrophoresis unit | Labnet | E0160 | |
| Sonifier 450 | Branson | 101-063-198 | |
| Microtip for Sonifier 450 | Branson | 101-148-062 | |
| FACSAria cell sorter | BD | ||
| MoFlo cell sorter | Beckman-Coulter | ||
| Biomek FX or equivalent robot | Beckman Coulter | Optional. For setting up genotyping PCRs. |
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