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Sample GSM8244563

Query DataSets for GSM8244563

Status

Public on May 13, 2024

Title

fyn_permissivity_input1

Sample type

SRA

Source name

yeast cells

Organism

Saccharomyces cerevisiae

Characteristics

lab strain: BY4741
cell type: yeast cells

Treatment protocol

abundancePCA and bindingPCA (High-resolution mapping of protein concentration reveals principles of proteome architecture and adaptation, Levy and Michnick, 2014; Mapping the energetic and allosteric landscapes of protein binding domains, Faure et. Al, Nature 2022)

Growth protocol

The bottlenecked library pool containing randomly subsampled fractions of the FYN SH3, CI-2A and CspA DTS core libraries in pGJJ162, the full complexity FYN SH3 DTS core library in pGJJ159-PRD1super and the FYN SH3 permissivity library in pGJJ162 were transformed in BY4741 yeast cells by heat shock. The number of transformation reactions was scaled depending on the final selection volume, which was in turn scaled to achieve a 100X coverage at the most stringent selection step in terms of cell count bottlenecking (inoculation in selection medium, see below). An individual transformation was performed per 200 mL of selection culture. For each individual transformation, 25 mL YPDA were inoculated with BY4741 colonies from a freshly streaked YPDA plate and grown overnight at 30ºC. The process was performed in parallel for each independent biological replicate, starting from a different BY4741 colony (2 replicates for the subsample DTS core library pool, 3 replicates for the other experiments). The precultures were used to inoculate 180 mL of YPDA to an OD600 of 0.3, which was grown for 4 h at 30ºC. Cells were harvested by 5 min centrifugation at 3,000 g, washed in water and treated with 8.6 mL SORB for 30 min. 3.5 μg of plasmid DNA were then added along with 175 μL of 10 mg/mL sonicated salmon sperm DNA (Santa Clara, CA, US), and the mixture was agitated at room temperature for 10 minutes followed by addition of 35 mL of plate mixture plus another 30 min incubation. 3.5 mL of DMSO were then added and the mixture was heat-shocked at 42ºC for 20 min. After the heat-shock, cells were harvested by 5 min centrifugation at 3,000 g, resuspended in recovery media and incubated at 30ºC for 1 h. Cells were then harvested again and resuspended in 200 mL of SC -URA selective medium. A 10 μL aliquot was serially diluted and plated in SC -URA to assess transformation efficiency. All biological replicate transformation cultures were grown in parallel at 30ºC for ~24 h. Then, the selection input cultures were inoculated by resuspending water washed harvested cell pellets in SC -URA -ADE to an OD600 of 0.1 (800 mL in 5 L flasks in the cases of the subsampled DTS core library pool and FYN SH3 binding library, 400 mL in 2L flasks in the case of the FYN SH3 permissivity library). The input cultures were grown at 30ºC and harvested upon reaching an OD600 of 1.6 (5 generations), which typically occurred from 12 to 15 h after inoculation. Cell pellets were stored at -20ºC. Aliquots of the input culture were used to inoculate the selection (output) cultures (SC -URA -ADE, 200 μg/mL methotrexate) at an OD600 of 0.05. The output cultures were grown at 30ºC for ~30 h (subsampled DTS core library pool and FYN SH3 binding library) or until they reached an OD600 of 1.6. At that time, cells were harvested by 5 min centrifugation at 3,000 g, thoroughly washed in water and stored at -20ºC.

Extracted molecule

genomic DNA

Extraction protocol

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 62ºC 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, US) 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 -80ºC. The mixture was kept at -20 ºC for 30 min and spun down at 3,300 g for 30 min at 4ºC. 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 (Waltham, MA, US). 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 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 37ºC, then for 20 min at 80ºC. Samples were cleaned-up using MinElute columns, eluted in 30 μL of pre-warmed water (55ºC) and 1.5 μL were used as template for each PCR2 reaction.16x50 μL 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. Samples were quality controlled by the CRG Genomics core facility using a TapeStation instrument (Agilent, Santa Clara, CA, US), quantified by qPCR and sequenced in either a NextSeq500 (FYN SH3 permissivity library) or NextSeq2000 (subsampled core DTS library pool and FYN SH3 binding library) instrument (Illumina, San Diego, CA, US).
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 were all obtained from IDT, Coralville, IA, US). Each of the genes encoding the FYN SH3 domain, CI-2A and CspA were codon optimized for expression in Saccharomyces cerevisiae and divided in two segments smaller than 200 bp each, which was the size limit for commercial synthetic degenerate oligo production at the time of performing this study. Both the 5’ and the 3’ gene segments contained a homology region of at least 25 bp to the linearized pGJJ191 mutagenesis vector, plus an internal homology region of at least 25 bp. 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 ). The pGJJ191 vector was linearized via a 25-cycle PCR reaction using primers oGJJ311 and oGJJ406, followed by 1h DpnI (NEB) treatment at 37ºC plus inactivation at 80ºC for 20 minutes (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 permissivity library was purchased as an oligo pool from Twist Bioscience (San Francisco, CA, US). 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 50ºC for 8h, dialyzed against water using 0.025 μm filters (Millipore, Burlington, MA, US) for 1.5h, concentrated using a SpeedVac and transformed into E. coli C3020 cells (NEB, Ipswich, MA, US). 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. For sparse sampling of combinatorial core libraries (FYN SH3, CI-2A and CspA in pGJJ162), the full-complexity libraries were transformed into E. coli C3020 electrocompetent cells and bottlenecked by outgrowth medium serial dilution into overnight selective medium (LB supplemented with 50 μg/mL ampicillin) assessed in parallel through colony count of plated aliquots, aiming at ~10,000 transformants per library. Bottlenecked libraries were extracted by mini-prep, qPCR quantified and mixed to equivalent molar ratios.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina NextSeq 500

Description

FYN-SH3_core_permissivity_scores.csv

Data processing

DiMSum (Demultiplex with Cutadapt)
DiMSum (QC raw reads with FastQC)
DiMSum (Trim reads with Cutadapt)
DiMSum (Process and analyse variants with DiMSum)
Assembly: NA
Supplementary files format and content: All in tabular format. sparse_DTS_core_mutagenesis_scores.csv contains a list of variants, each linked to the protein they are related to (Protein), its Uniprot identifier (UniprotCode), their complete amino acid sequence (aa_seq), the sequence in core positions (core), the hamming distance to the WT protein they are related to (Nham_aa), whether they were used as the reference in DiMSUM processing (WT), and their fitness and error values as obtained directly from DiMSum (fitness_raw and sigma_raw) and after scaling in the 0-1 range (fitness_scaled and sigma_scaled). The FYN-SH3_core_DTS_binding_scores_PDR1super.csv file contains equivalent aa_seq, core, Nham_aa, WT, fitness_scaled, sigma_scaled, fitness_raw and sigma_raw fields for variants all related to FYN SH3 binding to PDR1super. The FYN-SH3_core_permissivity_scores contains equivalent aa_seq, WT, fitness_scaled, sigma_scaled, fitness_raw and sigma_raw fields for variants in the permissivity library.
Library strategy: PCR amplicon

Submission date

Apr 30, 2024

Last update date

May 13, 2024

Contact name

Albert Escobedo

Phone

+34933160209

Organization name

CRG

Department

Systems and synthetic biology