Rotor robot

We recently reported a genome-wide screen in which we identified factors that exacerbate the mutator phenotype of strains expressing active-site mutant DNA polymerases (26 (link)) or that result in a mutator phenotype in strains expressing wild-type DNA polymerases. In brief, HHY5298 (MATa ura3–52 leu2Δ1 trp1Δ63 his3Δ200 lys2–10A cyh2-Q38K hom3–10.HIS3 pMFA1-klLEU2.hphNT1.lys2–10A MLH2.klURA3 POL1.natNT2) was crossed against the non-essential gene-deletion collection (MATα his3Δ1 leu2Δ0 ura3Δ lys2Δ yfg::kanMX4) using a RoToR robot (Singer Instruments). Strains containing gene deletions in the presence or absence of DNA polymerase active-site mutations were tested for mutator phenotype with two in vivo mutational reporters (lys2–10A frameshift reversion assay (37 (link)) and CAN1 inactivation assay (42 (link))). Gene deletions that have not been previously reported to cause a mutator phenotype were validated by generating these knockouts de novo in RDKY5964 and HHY6443 for further analysis.

SPI screens were performed as previously described [8 (link), 81 (link)]. Arrays of GFP strains were transformed separately with either control or experimental plasmids. For example, for the NSP1 SPI screens either pCUP1-NSP1-GBP, or as controls, pCUP1-GBP or pCUP1-NSP1 were used (pCUP1 is the promoter from the yeast CUP1 gene and GBP encodes the GFP binding protein). Selective ploidy ablation (SPA) was used to introduce plasmids into arrays of query yeast strains comprising ~4000 members of the GFP collection that represent proteins that are expressed in mitotic cells [21 (link), 77 (link)]. Briefly, the SPA method utilises a Universal Donor Strain (UDS, W8164-2B), which contains conditionally-active centromeres, transformed with each of the plasmids. These donor strains were then mated with members of the GFP collection arrayed with four replicates on 1536-colony rectangular agar plates using a pinning robot (ROTOR robot, Singer Instruments, UK). The resulting diploids were put through a series of sequential selection steps to maintain the query strain GFP genome and plasmid, while destabilising and then removing the chromosomes of the UDS by growing the cells in 5-FOA and galactose-containing media. Finally, the plates were scanned using a desktop flatbed scanner (Epson V750 Pro, Seiko Epson Corporation, Japan).

sgRNA plasmids were cloned, verified, and integrated into E. coli BW25113 as described for individual strains above. One isolate of each sgRNA recipient was stored by inoculating into 250 μl LB with chloramphenicol in deep 96-well plates, grown for 6.5 h, mixed with glycerol, and stored at −80°C. Arrayed sgRNA recipient libraries and the arrayed dcas9 donor strain were pinned from glycerol stocks to separate LB agar plates using a ROTOR robot (Singer Instruments) and grown overnight. The arrayed recipient library was then mixed with the arrayed donor strain by pinning onto a new LB agar plate and then grown for 8 h to allow conjugation. Patches were mixed and transferred to a double-selection agar plate (gentamicin and chloramphenicol) using the ROTOR robot and grown overnight. Patches were each individually struck out on double-selection plates for single-colony isolation. To store the CRISPRi library, 2 isolates of each strain were inoculated in 250 μl LB with chloramphenicol and gentamicin supplemented with 0.2% glucose in deep 96-well plates, grown for 6.5 h, mixed with glycerol, and stored at −80°C in 96-well plates.

The SPI screening process is described in detail in Berry et al. (2016) (link) and in Ólafsson and Thorpe (2018) (link). A library of GFP strains is transformed with a plasmid expressing either a fusion of a protein of interest with GBP or a control, through a mating-based method known as Selective Ploidy Ablation (SPA) (Reid et al. 2011 (link)). The plates are repeatedly copied and grown on successive rounds of selection media until a library of haploid GFP strains with the plasmid is produced. This library is assayed for colony size, giving a readout for the fitness of a given binary fusion between the GFP strain and protein of interest. Plates were scanned on a desktop flatbed scanner (Epson V750 Pro, Seiko Epson Corporation, Japan) at a resolution of 300 dpi. All plates were grown at ${30}^{\circ }$ C. All copying of yeast colonies was performed on a Rotor robot (Singer Instruments, UK).

All MA lines and ancestral isolates were streaked down to single colonies on YPD agar plates. On sporulation media (1% potassium acetate-agar), three separate colonies from each of the lines and ancestral isolates were patched (Argueso et al. 2004 (link)). To evaluate sporulation efficiency, 100 cells per sample were counted using an optical microscope at the end of 72 h. The number of dyads, triads, and tetrads divided by the total cell count was used to calculate sporulation efficiency.
Mitotic growth was determined using endpoint colony growth on solid media. MA lines were pre-cultured in YPD broth and pinned onto a solid YPD plate to a 1,536 density format using the replicating ROTOR robot (Singer Instruments). The plates were incubated for 24 h at 30°C and were scanned with a resolution of 600 dpi at 16-bit grayscale. Quantification of the colony size was performed using the R package Gitter and the growth of each of the lines was measured by estimating the growth ratio between the colony size of the MA line at bottlenecks 25, 50, 75 to the respective colony size of the MA line at bottleneck 0 (Wagih and Parts 2014 (link)).