GSM8267373: TFO Grown library plasmid, BioReplicate 1; Escherichia coli; OTHER
1.2mL of oil phase was prepared fresh for each condition: 54µL Span 80, 4.8µL Tween 80, 0.6µL Triton X-100, and 1140.6µL light mineral oil (all from Sigma Aldrich). Oil phase was aliquoted into 2mL tubes, to each of which a rubber stopper from a 1mL syringe was added, vortexed, and chilled on ice.0.3mL of aqueous phase was prepared fresh for each condition and chilled on ice: 30µL 10x ThermoPol buffer, 7.8µL of (10mM each) dNTPs, 15µL 10µM CSR-DMS forward primer, 15µL 10µM CSR-DMS reverse primer, 50µL 6x GuSCN (water for conditions with no GuSCN), and 182.2µL water.The previously aliquoted cells were pelleted at 1500g for 15 min. Supernatant was removed and cells were resuspended in 1mL tris-buffered saline (TBS). Cells were then pelleted and resuspended in 0.3mL of ice-cold aqueous phase. The cell-aqueous phase mixtures were then added to the 1.2mL oil phase aliquots and emulsified on a Qiagen TissueLyser LT at 42 Hz for 4 min at 4°C. We ordered a library saturated with all possible single-residue mutants of Thin Fingers polymerase from Twist Bioscience. To synthesize this library, the vendor needed to codon-optimize the wild-type sequence, and provided us with the optimized sequence (which we are calling “TFO”, for “Thin Fingers Optimized”). We validated that TFO-WT can be expressed and purified at yields comparable to TF-WT. The TFO library was delivered dry in 96-well plates, with each well containing 50ng of DNA saturated at a single codon, for total of 540 wells. We resuspended each well in 20uL of IDTE buffer (10mM Tris, 0.1mM EDTA, pH 8.0) (Integrated DNA Technologies) for a final concentration of 2.5ng/µL. 10µL of each well was sampled and pooled to mix all variants. The pooled library was cloned into 2B-T plasmid (Addgene plasmid #29666) using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs E2621S) following the manufacturer-recommended protocol for assembling two dsDNA fragments (2:1 molar ratio of TFO library insert to 2B-T vector, 0.03pmol total fragments, 1x NEBuilder master mix in 20µL reactions incubated at 50°C for 60 minutes followed by a 4°C hold). 2µL of the chilled NEBuilder products were then transformed into NEB Stable cells following the manufacturer-recommended high efficiency transformation protocol. Following outgrowth, varying dilutions of cells in NEB Stable outgrowth medium (1µL cells plus 99µL outgrowth medium, 1µL previous dilution plus 99µL outgrowth medium) were plated on LB+carb agar plates and incubated at 30C overnight while the remaining 999µL of cells were added to 9mL LB+carb medium and incubated at 30C, 250rpm overnight. The following day, plasmid was purified from the liquid culture using a Monarch Plasmid Miniprep Kit (New England BioLabs T1010) (culture was split into eight replicate plasmid preps). Colony counts from plates were estimated using OpenCFU45 and the results were extrapolated to estimate a total transformation yield of roughly 7e5 colony-forming units in the liquid culture (well in excess of the total number of synthesized variants and therefore expected to maintain coverage for all but the least abundant variants). The TFO saturated variant library was barcoded in a similar manner to the mock selection TFO-WT and TFO-XX constructs, but with additional care taken to minimize the number of PCR cycles and the fraction of unbarcoded variants. The library was linearized by PCR at the desired barcode insertion site using barcoding primers, 10ng TFO library in 2B-T plasmid, and Q5 HotStart High-Fidelity DNA Polymerase (New England Biolabs M0493S), following the manufacturer-recommended recipe for 50µL reactions with 1x Q5 High GC Enhancer. The reaction was supplemented with 1x EvaGreen intercalating dye (Biotium), monitored during amplification on a Bio-Rad CFX96 real-time PCR thermal cycler, and stopped before the PCR plateau phase. Thermal cycling parameters were 98°C for 30 seconds, 18 cycles of (98°C for 10 seconds, 72°C for 5 minutes). PCR products were purified using an Invitrogen PureLink PCR Cleanup Kit (using B3 high-molecular-weight-cutoff binding buffer), digested with DpnI (New England Biolabs R0176S) in 1x rCutSmart buffer for 15 minutes at 37°C to remove carryover circular plasmid template, and purified once more with the PureLink PCR Cleanup Kit (B3 buffer). Barcode oligos (ordered as Ultramer ssDNA oligos from Integrated DNA Technologies) were then cloned into the linearized plasmid PCR products using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs E2621S) following the manufacturer-recommended protocol for bridging double-stranded DNA with a single-stranded DNA oligo (1pmol oligo, 0.005 pmol vector, 1x NEBuilder master mix in 20µL reactions incubated at 50°C for 60 minutes followed by a 4°C hold). 2µL of the chilled NEBuilder products were then transformed into NEB Stable cells following the manufacturer-recommended high efficiency transformation protocol. Following 1 hour of outgrowth, varying dilutions of cells in NEB Stable outgrowth medium (1µL cells plus 99µL outgrowth medium, or 100µL of (1µL cells plus 999µL outgrowth medium)) were plated on LB+carb agar plates and incubated at 30C overnight while the remaining 998µL of cells were added to 9mL LB+carb medium and incubated at 30C, 250rpm overnight. The following day, plasmid was purified from the liquid culture using a Monarch Plasmid Miniprep Kit (New England BioLabs T1010) (culture was split into eight replicate plasmid preps). Colony counts from plates were estimated using OpenCFU and the results were extrapolated to estimate a total transformation yield of roughly 1.1e5 barcoded variants in the liquid culture.