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Automation of Plasmid DNA Purification

Automation of Plasmid DNA Purification. Natalie Bloomhardt Jeffrey Patenaude Zachary Withrow David Schiavoni-Exman Stefanie McGuckian. Faculty Advisor Prof. Ruberti. Sponsor Harlow Laboratory Harvard Medical School. Background. Dr. Harlow’s Lab at Harvard Medical School

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Automation of Plasmid DNA Purification

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  1. Automation of Plasmid DNA Purification Natalie Bloomhardt Jeffrey Patenaude Zachary Withrow David Schiavoni-Exman Stefanie McGuckian Faculty Advisor Prof. Ruberti Sponsor Harlow Laboratory Harvard Medical School

  2. Background • Dr. Harlow’s Lab at Harvard Medical School • Determine individual gene function of genes mapped by the Human Genome Project • Identify genetic influence on cancer expression • Achieve through gene suppression technology

  3. Gene Suppression 1. Using the gene library, different types of Plasmid DNA are replicated in E.coli 2. Plasmid DNA is separated from bacteria through Alkaline Lysis and collected 3. Plasmid DNA is then transfected into mammalian cells via a virus 4. Interference is run in the RNA transcription process, effectively silencing a targeted gene

  4. Market Demand for Plasmid DNA • Academia • Thousands of labs across the country are performing mini-preps • Compile genetic library • Pharmaceutical companies • Profit driven • Develop new treatments and cures for diseases • Need 100,000 samples per genomic screen • Qiagen has sold over 1 Billion Mini-Prep kits

  5. Start with bacteria grown in 96-well plates Centrifuge (First separation step) Add & mix Solution 1 Add & mix Solution 2 Add & mix Solution 3 Centrifuge (Second separation step) Transfer to lysate- clearing plate and centrifuge Capture plasmid DNA Mini-Prep:Alkaline Lysis Start with bacteria grown in 96-well plates 16 min 1.5 min Transfer to custom plate • Previous Capstone Goals • Replace centrifugation with positive pressure filtration that is easier to automate • Maintain purity and yield • Decrease time per sample 6 min Filter (First separation step) 1.5 min 6 min Add & mix Solution 1 4 min 2 min Add & mix Solution 2 6 min 16 min Add & mix Solution 3 2 min 18 min Filter (Second separation step) 2.5 min Total 20 Minutes Total 64 Minutes Capture plasmid DNA

  6. Project Objective Design a fully automated system that can achieve a throughput of at least 2000 (20 assemblies) highly-purified Plasmid DNA samples per day

  7. Market Competitors and Patent Search Vacuum Beckman Coulter 80 minutes per plate DNA yield 8000-10000 ng Centrifugation Tecan 30 minutes per plate DNA yield 2500-3000 ng

  8. Design Challenges • Scale-up to run 96 samples in parallel • Prevention of cross contamination • Providing uniform filtration pressure • Delivering fluids and filtration aid • Mixing Single Well Design

  9. System Overview Filtration Assembly Clamping and Pressurization Dry Dispensing Liquid Dispensing Mixing/Resuspension Plasmid DNA System: Purifies Plasmid DNA

  10. Filtration Assembly Requirements • Prevent cross contamination (purity) • Light weight (mixing) • Occupy a minimum footprint (OEM compatibility) • Minimize complexity • (simplify automation) • Reduce consumables (goal) Through Plate Gasket Filter Support Transfer Plate Well Plate

  11. Design Tolerance Range

  12. Simple assembly motion • 1 planar motion • Weighs 2-3 lbs • Smallest footprint • 4.5” x 5.25” • Standard well plate • 3.25’’ x 4.9’’ Interlocking Bolt Design Rail Design

  13. Material Study: Assembly

  14. Displacement Calculation

  15. Numerical Design Analysis(CosmosWorks) Max Stress: 2050 psi Total Displacement: .0024”

  16. Design Verification – Leak Test Bromophenol Blue Dye Test Setup Interlocking Bolt Design Leak = more than 1% well to well fluid transfer Rail Design

  17. Design Trade Study

  18. MOLECULAR WEIGHTS (particle size) Bromophenol Blue: 670 Daltons FITC DEXTRAN: 150,000 Daltons Plasmid DNA: 55,000,000 Daltons

  19. Clamping and Pressurization Requirements Clamping • 800lbs force capacity • Compact and economical • Rapid actuation time • Less than 10 seconds • Pressurization • Constant pressure application of 30psi • Must be safe

  20. Bimba® Piston • 1130lbs of applied force at 90 psi supply • Easy to implement into the lab

  21. Pneumatic Piston Pneumatic Piston Pressure Head Pressure Head Ball Joint 96 Well Plate Assembly Holding Plate 96 Well Plate Assembly Holding Plate Piston Assembly Concept

  22. Structural Stress Analysis Applied Force Zero Displacement Supports Max Displacement: 4.2e-5 in Max Stress: 430 psi

  23. Pressurization Method Gasket

  24. Dry Dispensing Proprietary compound 4g ± 25% over the 96 wells Lid Screen Container Slide Plate

  25. Liquid Dispensing Requirements • Fit the filtration assembly • Volume accuracy of ±5% • Total dispense time: ≤ 1 minute • Liquid dispenser: ≤ 35 seconds • Pumps: ≤ 20 seconds • Computer controlled • Prevent cross contamination • Reduce consumables Peristaltic Pump

  26. Liquid Handlers vs Distributors • Liquid Handler • Current pipette tips = $73,000 per year • Liquid Distributor • Replaceable cartridge tubing at: • $0.16 per plate • $1,200 per year • Savings = $71,800

  27. Movable dispenser up to 3.5 in. 2 ml dispensing capability Autoclavable cartridges Small and lightweight Already one in lab for testing Thermo Scientific - WellMate Dispense Volume = 250 µL Fill time = 27.6 seconds

  28. Station Setup Maximum Exit Velocity = 0.6526 ft/s

  29. Electrical Schematic Common relay circuit between the clamping and pressurization and liquid distribution stations

  30. User Control Interface Liquid Distribution Clamping and pressurization

  31. Mixing Requirements • Programmable • Range of high speeds • Accept the assembly • Ability to mix • Resuspend bacteria pellet • High viscosity fluid

  32. Talboy MixerPellet Resuspension • Resuspension Speed • 2000 RPM for 5 minutes in pulse mode Hand Pipetting • Bacteria Pellet • Angle Test RPM Hand Mixed 40 Degree Flat 10 Degree

  33. Full Alkaline Lysis with Assembly DNA Yield DNA Type Qaigen Maxiprep 5 sec x 2 20 Sec 10 sec 5 Sec No Mix Size Markers • Average yield of 21,000 ng Desired Type • Obtained desired Plasmid DNA

  34. Mixer Adapter Plate • Holds assembly on Talboy • Creates a 40° incline • Holds assembly on Talboy • Creates a 40° incline

  35. System Optimization • Goal: Maximize Plasmid DNA Throughput • Process Includes: • 2 Clamping and Pressurization Steps • 3 Liquid Dispensing Steps • 4 Mixing Steps

  36. Optimization Analysis • Simulated process in Arena • Variation • Rate that assemblies entered the system • Number of each station

  37. Optimization: Using ONE of All Stations 55 Assemblies! 22 Assemblies Goal: 20 Assemblies

  38. Optimum Configuration 165 Plates per Day Configuration: 2 Clamping and Pressurization Stations 1 Liquid Distribution Station 3 Mixing Stations

  39. FINAL ACHIEVEMENTS • Proven New Filtration Assembly • Materials study, leak testing, gasket analysis, stress analysis • Pressurization and Filtration Station • Frame, piston, alignment • Liquid Dispensing Station • Pumps, enclosure, reservoir • Mixing Station • Optimized mixing times/configurations • User Control • LabVIEW Virtual Interface • Process Optimization

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