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Automation of Plasmid DNA Purification
Faculty AdvisorProf. Ruberti
Natalie BloomhardtJeffrey PatenaudeZachary Withrow
David Schiavoni-ExmanStefanie McGuckian
SponsorHarlow Laboratory
Harvard Medical School
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
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
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
Mini-Prep:Alkaline Lysis
• Previous Capstone Goals– Replace centrifugation with
positive pressure filtration that is easier to automate
– Maintain purity and yield– Decrease time per sample
16 min
6 min
6 min
2 min
16 min
Centrifuge(Second
separation step)
Centrifuge(Second
separation step)
Capture plasmid DNA
Capture plasmid DNA
Start with bacteria grown in 96-well
plates
Start with bacteria grown in 96-well
plates
Centrifuge(First separation
step)
Centrifuge(First separation
step)
Add & mix
Solution 1
Add & mix
Solution 1
Add & mix
Solution 2
Add & mix
Solution 2
Add & mix
Solution 3
Add & mix
Solution 3
Transfer to lysate- clearing
plate and centrifuge
Transfer to lysate- clearing
plate and centrifuge
18 min
Total 64 Minutes
1.5 min
4 min
6 min
2 min
2.5 min
Filter(Second
separation step)
Filter(Second
separation step)
Capture plasmid DNA
Capture plasmid DNA
Start with bacteria grown in 96-well
plates
Start with bacteria grown in 96-well
plates
Filter(First separation
step)
Filter(First separation
step)
Add & mix
Solution 1
Add & mix
Solution 1
Add & mix
Solution 2
Add & mix
Solution 2
Add & mix
Solution 3
Add & mix
Solution 3
Total 20 Minutes
Transfer to custom plate
Transfer to custom plate
1.5 min
Complete Filtration DNA Yield (30 PSIG)
0
2000
4000
6000
8000
10000
12000
14000
0 2 4 6 8 10 12 14
Test #
Yie
ld (n
g)
Filtration Average Centrifugation
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
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
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
System Overview
Clamping and Pressurization
Dry Dispensing
Liquid Dispensing
Mixing/Resuspension
Filtration Assembly
System: Purifies Plasmid DNA
Plasmid DNA
Filtration Assembly Requirements• Prevent cross contamination
(purity)
• Light weight (mixing)
• Occupy a minimum footprint (OEM compatibility)
• Minimize complexity
(simplify automation)
• Reduce consumables (goal)
Gasket
Support
Transfer Plate
Well Plate
Through Plate
Filter
psiR
pressurequiredRA
WR
leakagepreventtorequiredForceW
mPbPGW
m
m
m
3.42
Re
24
1
1
21
psiR
pressurequiredRA
WR
leakagepreventtorequiredForceW
mPbPGW
m
m
m
3.42
Re
24
1
1
21
DesignTolerance Range
Interlocking Bolt Design 5 5 5 5 20
Rail Design 5 4 5 4 18
Nut and Bolt Design 3 3 1 1 8
Latch Design 4 4 2 1 11
Clevis P in Design 4 4 2 1 11
Total (30)0 = P oor 5=Excellent WeightFoot Print
Ease of Automation
Number of Parts
• 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
Material Study: Assembly
Material Autoclave
Tensile Yield
StrengthFlexural Strength
Flexural Modulus Density Cost
Chemical Resistance
to 1% NaOH
Chemical Resistance
to 70% Ethyl
Alcohol Total
Ultem 2300 (PEI) 5 5 5 5 3 1 3 3 47
Polysulfone 30% Glass Filled (PSU) 5 2 2 2 4 2 2 2 31
Nylon 30% Glass Filled (PA6/12) 5 5 5 4 5 3 5 5 62
Nylon 33% Glass Filled (PA6/6) 1 4 3 3 5 4 2 2 35
Polystyrene 36% Glass Filled (PS) 1 1 1 3 5 4 2 2 28
High Impact Polystyrene 20% Glass
Filled 1 1 1 1 5 5 2 2 27
Solvay AvaSpire Polyethertherketone
(PEEK) 5 4 5 5 3 1 5 5 58
Solvay Torlon (Polyamide-imide) 5 5 5 5 3 1 3 3 47
Material Autoclave
Tensile Yield
StrengthFlexural Strength
Flexural Modulus Density Cost
Chemical Resistance
to 1% NaOH
Chemical Resistance
to 70% Ethyl
Alcohol Total
Ultem 2300 (PEI) 5 5 5 5 3 1 3 3 47
Polysulfone 30% Glass Filled (PSU) 5 2 2 2 4 2 2 2 31
Nylon 30% Glass Filled (PA6/12) 5 5 5 4 5 3 5 5 62
Nylon 33% Glass Filled (PA6/6) 1 4 3 3 5 4 2 2 35
Polystyrene 36% Glass Filled (PS) 1 1 1 3 5 4 2 2 28
High Impact Polystyrene 20% Glass
Filled 1 1 1 1 5 5 2 2 27
Solvay AvaSpire Polyethertherketone
(PEEK) 5 4 5 5 3 1 5 5 58
Solvay Torlon (Polyamide-imide) 5 5 5 5 3 1 3 3 47
Displacement Calculation
3
48
L
EIF
3
48
L
EIF
"0.2
"78.15*052.
63.1.
"3
267
max
heighth
baseb
psieModElasticE
lin
lbsLoaddDistributew
ntDisplacemeMaxy
"004.max y12
384
5
3
4
max
bhI
EI
wly
Numerical Design Analysis(CosmosWorks)
Max Stress: 2050 psiTotal Displacement: .0024”
Design Verification – Leak Test
Bromophenol Blue Dye
Test Setup
Interlocking Bolt Design
Rail Design
Leak = more than 1% well to well fluid transfer
92.08%
98.75%
7.92%
1.25%
0.00%
10.00%
20.00%
30.00%
40.00%
50.00%
60.00%
70.00%
80.00%
90.00%
100.00%
2 Rail Interlocking Pin
Pe
rce
nta
ge
of
We
lls
Design
Leak Testing:Bromophenol Blue Dye
No Leak
Leak
Design Trade Study
WeightFootPrint
Number of Parts Mixing
Robot Lift-able
Resistant to
Failure
Minimal Tolerance
Stack-upEase of
MachiningCost
Leaking Total
C ChannelDesign 5 5 5 5 5 4 3 5 5 3 68
Interlocking Bolt Design 5 4 5 5 4 5 5 4 3 5 72
WeightFootPrint
Number of Parts Mixing
Robot Lift-able
Resistant to
Failure
Minimal Tolerance
Stack-upEase of
MachiningCost
Leaking Total
C ChannelDesign 5 5 5 5 5 4 3 5 5 3 68
Interlocking Bolt Design 5 4 5 5 4 5 5 4 3 5 72
MOLECULAR WEIGHTS (particle size)
Bromophenol Blue: 670 Daltons
FITC DEXTRAN: 150,000 Daltons
Plasmid DNA: 55,000,000 Daltons
Leak Testing Results
98.75% 99.58%
1.25% 0.42%0.00%
20.00%
40.00%
60.00%
80.00%
100.00%
120.00%
Bromophenol Blue FITC Dextran
Test Media
Pe
rce
nt
Oc
cu
ran
ce
No Leak
Leak
Clamping and Pressurization
Clamping• 800lbs force capacity• Compact and economical• Rapid actuation time
– Less than 10 seconds
Piston Design 5 5 5 5 5 5 30
YamahaTM Linear Actuator 5 0 5 5 5 5 25
Promess Ball Screw 0 5 5 5 5 5 25
Ball Screw Custom 0 0 5 5 5 5 20
Hydraulic Jack 0 5 0 0 5 0 10
Automotive Jack Adaption 5 0 0 0 0 5 10
Gear Box Design 0 5 5 0 0 0 10
Total (30)0 = Poor
5=ExcellentEconomical
800lbs of
ForceAutomatable Compact
Actuation Time
Contamination Possibility
Pressurization• Constant pressure
application of 30psi• Must be safe
Requirements
Bimba® Piston
• 1130lbs of applied force at 90 psi supply
• Easy to implement into the lab
Pneumatic Piston
Holding Plate
Pressure Head
96 Well Plate Assembly
Piston Assembly Concept
Pneumatic Piston
Holding Plate
Pressure Head
96 Well Plate
Assembly
Ball Joint
Structural Stress Analysis
Zero Displacement Supports
Applied Force
Max Displacement: 4.2e-5 inMax Stress: 430 psi
Pressurization Method
lbsR
gasketofareaSurfaceA
essureInternalP
headpressureofareaSurfaceA
leakingpreventtoforcequiredR
PAPAR
G
PH
GPH
500
Pr
Re
2
Gasket
Dry Dispensing
• Proprietary compound• 4g ± 25% over the 96 wells
Lid
Screen
Container
Slide Plate
Liquid Dispensing
• 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
Requirements
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
Thermo Scientific - WellMate
• Movable dispenser up to 3.5 in.
• 2 ml dispensing capability
• Autoclavable cartridges
• Small and lightweight• Already one in lab for
testingDispense Volume = 250 µLFill time = 27.6 seconds
Station SetupSolution 0 Solution 6Solution 5Solution 4Solution 3Solution 2Solution 1
378 mL/min Peristaltic
Pump
116 mL/min Peristaltic
Pump
116 mL/min Peristaltic
Pump
116 mL/min Peristaltic
Pump
116 mL/min Peristaltic
Pump
378 mL/min Peristaltic
Pump
378 mL/min Peristaltic
Pump
378 mL/min Peristaltic
Pump
BASIN WellMate
Maximum Exit Velocity = 0.6526 ft/s
Electrical Schematic
Common relay circuit between the clamping and pressurization and liquid distribution stations
User Control Interface
Clamping and pressurization
Liquid Distribution
Mixing
Requirements• Programmable• Range of high speeds• Accept the assembly• Ability to mix
– Resuspend bacteria pellet– High viscosity fluid
TypeSpeed Range (RPM)
TimerNumber of Well Plates
Max Load
Automatable Total
Orbital Shaker Tables 2 5 5 2 5 19
Magnetic Mixers 5 5 2 3 1 11
Vortex 4 5 5 4 5 23
Talboy MixerPellet Resuspension
• Resuspension Speed– 2000 RPM for 5
minutes in pulse mode
RPM
Hand Pipetting
Hand Mixed Flat 10 Degree 40 Degree
• Bacteria Pellet– Angle Test
Full Alkaline Lysis with Assembly
•Average yield of 21,000 ng
Solution 2-3 Mixing Times1500 rpm
0
5000
10000
15000
20000
25000
0 By Hand 5 10 20 5 sec 2 times
Mix Time (sec)
DN
A Y
ield
(ng)
DNA Type
Size Markers
No Mix
5 Sec
10 sec
20 Sec
5 sec x 2
Qaigen Maxiprep
Desired Type
DNA Yield
•Obtained desired Plasmid DNA
Mixer Adapter Plate
• Holds assembly on Talboy
• Creates a 40° incline
• Holds assembly on Talboy
• Creates a 40° incline
System Optimization
• Goal: Maximize Plasmid DNA Throughput
• Process Includes:– 2 Clamping and Pressurization Steps– 3 Liquid Dispensing Steps– 4 Mixing Steps
Optimization Analysis
• Simulated process in Arena
• Variation– Rate that assemblies entered the system– Number of each station
Plate Throughput vs Stagger Time into the System
0
10
20
30
40
50
60
20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1
Stagger Time into the System (min)
Th
rou
gh
pu
t (#
pla
tes)
Series1
Optimization: Using ONE of All Stations
55 Assemblies!
22 Assemblies
Goal: 20 Assemblies
Optimum ConfigurationRatio of Throughput to Capital Cost for Various Station Configurations
0
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0.008
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
Configuration Number(Varying Number of Liquid Distributing, Mixing, and Filtration Stations)
Rat
io o
f Thr
ough
put t
o C
apita
l Cos
t
165 Plates per DayConfiguration:2 Clamping and Pressurization Stations 1 Liquid Distribution Station3 Mixing Stations
• 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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