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Getting started

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Making PURE requires making Protein Mix, which contains the thirty-six (36) proteins in the PURE system. If you want to purify each protein individually, you can follow our 36-pot protocol (Make Protein Mix). However, if you want working protein mix with less time and effort, you can co-express and co-purify all 36 proteins at once in OnePot. This protocol shows you how. This protocol has been established to grow, purify, and characterize OnePot PURE proteins following Lavickova’s OnePot PURE protocol [Grasemann, 2021] with a few deviations.

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Materials and Equipment

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• Table 1: Reagents and Consumables
• Table 2: Equipment

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Protocol - Day 0

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• [ ] Buffer Preparation
  • [ ] Please use the Nucleus Buffer Template . Instructions on how to use template below:
  • Template Usage Note:
  • [ ] Prepare Stock Solutions:
    • [ ] Fill water to 75% of total volume.
    • [ ] Weigh and dissolve required mass of salt (check the listed molecular weight of your salts and update if necessary; hydration salts can have different molecular weights)
    • [ ] Adjust pH if specified. Use KOH (10M) or HCl (1M)
    • [ ] Adjust water to final volume.
  • [ ] Prepare Buffer Solutions:
    • [ ] Add required volume of each stock solution and adjust to final volume with water.
    • [ ] Withold TCEP until day of use.
  • Notes: Protein Storage Buffers are prepared from concentrate (4x)

<aside> <img src="/icons/iterate_gray.svg" alt="/icons/iterate_gray.svg" width="40px" /> Protocol - Day 1

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• [ ] Prepare starter cultures (night before)
  • [ ] Working under flame, Prepare the media by mixing 20 mL LB with 20 uL of Kan (50 mg/mL).
  • [ ] In a deep-well plate, add 300 uL of media into 35 wells and inoculate each well with the 35 PURE proteins (omitting EF-Tu). Seal the plate with a breathable membrane and incubate at 37C / 260 rpm / overnight.
  • [ ] For EF-Tu, inoculate 3 mL of LB + Kan media in a 14 mL cell culture tube and incubate at 37C/ 260 rpm/ (12 - 16) hrs.
• [ ] Inoculate Co-culture
  • [ ] (Optional) Confirm all strains have grown by measuring the OD600 on a 96-well plate with 10x dilutions or by visually confirming turbidity.
  • [ ] Transfer 500 mL LB + Kan into baffled 2L Erlenmeyer flask and inoculate with the starter cultures:
    • [ ] 5 mL total inoculation volume (~140x back-dilution).
      • [ ] 2.33 mL of EF-Tu culture (~300x back-dilution).
      • [ ] 75 uL of each culture from deep-well plate into reagent reservoir.
• [ ] Outgrowth & Harvest
  • [ ] Thaw IPTG at 4C.
  • [ ] Incubate culture at 37C/ 260 rpm/ ~2.5 hours or until the OD600 reaches 0.6-0.8.
    • [ ] At +2 hours, measure OD600 from 1 mL sample in a cuvette. Repeat every 20 minutes until OD600 reaches target.
    • [ ] Mark inoculation time.
  • [ ] Induce protein expression with 500 uL of 0.5 M IPTG and incubate at 37C / 260 rpm / 2.5 hours. Mark induction time.
  • [ ] Harvest the cells via centrifugation at 16,000 x g / 4C / 15 minutes.
    • [ ] Tare plastic bag on a coarse scale.
    • [ ] Decant supernatant into biohazardous waste.
    • [ ] Scoop cell pellet (”cellet”) into pre weighed ziploc bag. Weigh and record mass on bag.
    • [ ] (Optional) flash freeze plastic bag in liquid nitrogen.
• [ ] Storage
  • [ ] Store the cellet at -80C.

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Protocol - Day 2

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• [ ] Finish Buffers
  • [ ] Add TCEP (0.5M, 500x) to finish Lysis Buffer, Wash Buffer, Elution Buffer, Dialysis Buffer, and Storage Supplement Buffer. E.g., 50 mL Wash Buffer requires 100 uL TCEP.
  • [ ] Resuspend one protease inhibitor tablet per four protein preps in 4 mL Lysis Buffer in a 15 mL conical tube. Resuspend by vortexing vigorously. Add one quarter of the resuspended protease inhibitor mixture (1 mL) to your tube of Lysis Buffer and mix by inversion or vortexing.
• [ ] Cell lysis
  • [ ] Resuspend cellet in its plastic bag with 10 mL Protein Wash Buffer. Massage cellet until suspended.
  • [ ] Transfer resuspended to 15 mL conical tube and keep on ice.
  • [ ] Lyse cells by sonication using a 130-watt probe sonicator (6 mm diameter probe). Set sonicator to 25% amplitude/ 15 seconds on/30 seconds off. Sonicate samples on ice for 2 minutes on-time. It may take 2-4 cycles to sufficiently lyse cells. Do NOT let the sample heat up. If so, allow the sample to cool on ice before continuing sonication.
  • [ ] Clarify lysate by centrifugation at 16,000 x g/ 4C/ 20 min.
  • [ ] Use a 10 mL syringe with 0.22 um syringe filter to filter the supernatant into a fresh 15 mL tube.
  • [ ] Discard pellet in biohazardous waste.
    • Note: Visually inspect pellet for lysis
• [ ] Column Prep
  • [ ] Assemble columns.
    • [ ] Cut tips off of columns off using a razor blade.
    • [ ] Pack a filter into the bottom of the column (we use the back end of a cell spreader).
      • Notes: columns have ridges!
    • [ ] Assemble buffer reservoir to column.
  • [ ] Wash empty columns.
    • [ ] Put column assembly on your column holder over your flowthrough catch.
    • [ ] Wash column, buffer reservoir, and filter with ≥ 10CV (20 mL) Ultrapure water. Discard flowthrough.
      • Notes: “CV” = Column Volume
  • [ ] Load and Equilibrate Ni2+ affinity Resin into columns.
    • [ ] Resuspend Ni2+ resin (50% v/v suspension) by shaking.
    • [ ] Add 2 mL of resuspended resin by pipette to the column.
    • [ ] Wash column with ≥ 10 CV (20 mL) mL Ultrapure water. Discard flowthrough.
    • [ ] Equilibrate columns with ≥ 10 CV (20 mL) of Wash Buffer (4C). Discard flowthrough.
  • [ ] (Optional) store Columns.
    • [ ] Seal each column with a cap and bung.
    • [ ] Store columns at 4C for up to 48 hrs.
    • Notes: store columns long term in EtOH (20%)
• [ ] Gravity Column Ni-resin Purification
  • [ ] Add bung to gravity column to prevent flowthrough.
  • [ ] Add clarified lysate (supernatant) to the equilibrated column.
  • [ ] Seal top of column with cap.
  • [ ] Incubate at 4C / 1hr with rotation.
  • [ ] Mount column on a stand with a beaker below to capture flow through.
  • [ ] Remove bung and let the flow through elute off the column.
  • [ ] Wash column with 12.5 CV (25 mL) of Wash buffer and discard flow though.
  • [ ] Elute sample with 2.5 CV (5 mL) Elution Buffer and capture eluent in 15 mL tube. This is your sample.
• [ ] Buffer Exchange by diafiltration
• [ ] Concentrate buffer exchanges proteins
• [ ] Storage