GSM9107679: fyn_randomization_output2; Saccharomyces cerevisiae; OTHER
Input and output culture cell pellets were resuspended in DNA extraction buffer (10 mM Tris-HCl, 100 mM NaCl, 2% Triton-X, 1% SDS, pH 8). Volumes were scaled depending on the volume of cell pellets to process. For pellets from 100- ml cultures harvested at OD600 1.6, 1 ml of DNA extraction buffer was used. Cells were lysed by cyclically freezing in liquid nitrogen followed by incubation in a 62°C water bath twice. Total DNA was extracted by adding 1 ml of a 25:24:1 phenol:chloroform:isoamyl alcohol mixture (Merck, Darmstadt, Germany) along with 1 g of acid washed glass beads (Sigma-Aldrich, Saint Louis, MO, USA) and vortexing for 10 min. The mixture was centrifuged at room temperature for 30 min at 3,300 g and the aqueous phase (upper layer) was recovered in a separate tube. 1 ml of the phenol:chloroform:isoamyl alcohol was added to it and the mixture was vortexed for 2 min followed by 45 min centrifugation at 3,300 g. The aqueous phase was again transferred to a new tube and mixed with 0.1 volumes of 3 M sodium acetate and 2.2 volumes of pure ethanol previously cooled down at −-80°C. The mixture was kept at −-20°C for 30 min and spun down at 3,300 g for 30 min at 4°C. The DNA pellets were dried overnight in a fume extraction hood. Plasmid DNA from total DNA extracts was quantified by qPCR using the oGJJ152- oGJJ153 oligo pair that anneals at the origin of replication of all pGJJ vector series. The qPCR reaction was performed in a LightCycler 400 instrument (Roche, Basel, Switzerland) using the SYBR green qPCR 2X master mix from Thermo Fisher Scientific (Waltham, MA, USA). Quantification was achieved by measuring a standard curve on a serial dilution of a control sample of known concentration in the same qPCR run as the query samples, and subtracting blank measurements. Library preparation for sequencing in Illumina instruments followed a two-step PCR protocol. Frameshifts and a segment of the Illumina adapter overhangs were introduced in PCR1. For each sample, 8x50 μl PCR1 reactions were performed using 31.25 million plasmid molecules as a template as quantified by qPCR. PCR1 used the oGJJ595 and the oGJJ748 frameshifting oligo pools (table S1) and consisted of 11 cycles. Next, all reactions corresponding to the same sample were pooled and treated with 16 μl ExoSAP for 30 min at 37°C, then for 20 min at 80°C. Samples were cleaned-up using MinElute columns, eluted in 30 μl of pre-warmed water (55°C) and 1.5 μl were used as template for each PCR2 reaction.16x50 μl 8-cycle PCR2 reactions were performed per sample to introduce the rest of the Illumina adapters and the dual index barcodes. PCR2 reactions corresponding to the same sample were pooled, concentrated using a MinElute column, and run in a 2% agarose gel. Bands of the correct sizes were excised from the gel and sequencing-ready DNA was recovered by using a centrifugal filter unit followed by MinElute clean-up. Oligonucleotides, plasmids and media used are listed in tables S1, S2 and S3, respectively. Combinatorial core libraries using a reduced alphabet of hydrophobic amino acids (F, L, I, M, V) were built by multiple segment Gibson assembly of oligonucleotides bearing the DTS degenerate codon at core residue-encoding positions (oligos used in this study are detailed in table S1 and were all obtained from IDT, Coralville, IA, USA). Each of the genes encoding the FYN SH3 domain, CI-2A and CspA were codon optimized for expression in S cerevisiae and divided in two segments smaller than 200 bp each. These two segments had internal homology regions of at least 25 nt. FYN-SH3 buried and exposed residue randomization libraries were instead purchased as 233- nt single-stranded DNA oligonucleotides, with degenerate codons optimized ad hoc for each randomized position. For all libraries, both the 5′ and the 3′ gene segments contained a homology region of at least 25 nt to the linearized pGJJ191 mutagenesis vector. Double stranded Gibson assembly inserts were produced from single stranded commercial oligonucleotides via a single cycle PCR reaction using a 3′ annealing reverse primer specific to each segment (see table S1). The pGJJ191 vector was linearized via a 25-cycle PCR reaction using primers oGJJ311 and oGJJ406, followed by 1 hour DpnI (NEB) treatment at 37°C plus inactivation at 80°C for 20 min (1 μl enzyme per 50 μl PCR reaction). Both inserts and vector PCR reactions were cleaned-up in MinElute columns (Qiagen, Hilden, Germany). Gibson assembly mixtures contained 300 ng of linearized pGJJ191 plus the two insert segments at a 5:1 molar ratio each, along with a Gibson enzyme mixture produced in house by the Protein Technologies core facility at the CRG. Conversely, the FYN SH3 suppression library was purchased as an oligo pool from Twist Bioscience (San Francisco, CA, USA). The oligo pool was amplified in a 13- cycle PCR reaction with primers oAE018 and oAE227, which was cleaned up using a MinElute column. The insert amplified oligo pool contained 25-bp homology regions at both ends and was mixed at a 5 to 1 molar ratio with 300 ng of linearized pGJJ191 and subsequently with the Gibson enzyme mixture. All Gibson assembly reactions were incubated at 50°C for 8 hours, dialyzed against water using 0.025- μm filters (Millipore, Burlington, MA, USA) for 1.5 hours, concentrated using a SpeedVac and transformed into E. coli C3020 cells (NEB, Ipswich, MA, USA). The number of transformants was assessed by colony count in serial dilutions of transformation outgrowth aliquots in LB plates supplemented with 50 μg/ml spectinomycin, and 1 to 5 equivalent transformation reactions were pooled to reach at least 20X variant coverage per library as required. Plasmid DNA was extracted using the Qiagen Plasmid Plus Midi kit and the library inserts were obtained through double endonuclease digestion (HindIII-HF and NheI-HF, NEB) followed by agarose gel purification achieved by a combined use of centrifugal filter units (Millipore) and MinElute clean-up. Inserts were introduced in double-restricted, Quick CIP- trated (NEB) pGJJ162 (AbundancePCA) or pGJJ159-PRD1super (BindingPCA) vectors by overnight thermal-cyclic ligation using T4 DNA ligase. The pGJJ159-PRD1super vector was previously obtained by introducing the S. cerevisiae codon-optimized PRD1super encoding gene from commercial oligo synthesis (IDT) into BamHI-SpeI double restriction-linearized pGJJ159 via Gibson assembly. Ligation mixtures were dialyzed against water, SpeedVac-concentrated and transformed into E. coli C3020 electrocompetent cells. Equivalent transformations were combined to reach at least 20X variant coverage, and selection-ready libraries in PCA vectors were Midi-prep extracted.