Transcript JEFFS - Northeastern University College of Engineering

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
– 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
Start with bacteria grown in
96-well plates
Mini-Prep:
DNA Yield (30 PSIG)Lysis
Complete Filtration
Alkaline
Start with bacteria grown in
96-well plates
Centrifuge
16
min
(First separation step)
Transfer to custom plate
14000
Yield (ng)
6
min12000
• Previous Capstone Goals
Add & mix
Solution 1
10000
6 8000
min 6000
Add & mix
Solution 2
4000
2 2000
min
0
Add & mix
Solution 3
0
16
min
2
4
– Replace centrifugation with
positive pressure filtration
that is easier to automate
– Maintain purity and yield
– Decrease time per sample
Total 64
Minutes
8
6
Centrifuge
10
12
14
Filter
(First separation step)
1.5
min
Add & mix
Solution 1
4
min
Add & mix
Solution 2
6
min
Add & mix
Solution 3
2
min
Filter
2.5
min
Test #
(Second separation step)
Filtration
18
min
1.5
min
Average Centrifugation
Transfer to lysateclearing plate and
centrifuge
(Second separation step)
Capture plasmid DNA
Capture plasmid DNA
Total 20
Minutes
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
Filtration
Assembly
Clamping and Pressurization
Dry Dispensing
Liquid Dispensing
Mixing/Resuspension
Plasmid
DNA
System: Purifies Plasmid DNA
Filtration Assembly Requirements
• Prevent cross contamination
(purity)
Through
Plate
• Light weight (mixing)
Gasket
• Occupy a minimum footprint
(OEM compatibility)
Filter
• Minimize complexity
(simplify automation)
• Reduce consumables (goal)
Support
Transfer
Plate
Well
Plate

2
WWm1m1 GG2 PP22bbmmPP
44
Forcerequired
requiredtotoprevent
preventleakage
leakage
WWm1m1Force
WW
RR m1m1
AA
Required
quiredpressure
pressure
RRRe
42
psi
RR42
.3.3psi
Design
Tolerance Range
•
Simple assembly motion
– 1 planar motion
•
•
Weighs 2-3 lbs
Smallest footprint
– 4.5” x 5.25”
Interlocking
Bolt Design
•
Rail Design
Standard well plate
– 3.25’’ x 4.9’’
We ight
Foot
P rint
Ease o f
A ut o m a t io n
N um be r
o f P a rt s
T o tal (30)
Int e rlo c k ing B o lt D e s ign
5
5
5
5
20
R a il D e s ign
5
4
5
4
18
N ut a nd B o lt D e s ign
3
3
1
1
8
La t c h D e s ign
4
4
2
1
11
C le v is P in D e s ign
4
4
2
1
11
0 = Poor
5=Excellent
Material Study: Assembly
Flexural
Strength
Flexural
Modulus
Density
Cost
Chemical
Resistance
to 1%
NaOH
Chemical
Resistance
to 70%
Ethyl
Alcohol
Total
Material
Autoclave
Tensile
Yield
Strength
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
F


48 EI
L3
Displacement
Calculation
ymax  Max Displacem ent
5wl 4
ymax 
384EI
bh3
I
12
lbs
w  Distributed Load  267
in
l  3"
E  Elastic Mod.  1.3e6 psi
b  base  .052*15  .78"
h  height  2.0"
ymax  .004"
Numerical Design Analysis
(CosmosWorks)
Max Stress: 2050 psi
Total Displacement: .0024”
Design Verification – Leak Test
Bromophenol Blue Dye
Test Setup
Interlocking Bolt Design
Leak = more than 1% well
to well fluid transfer
Rail Design
Leak Testing:
Bromophenol Blue Dye
98.75%
100.00%
92.08%
90.00%
80.00%
Percentage of Wells
70.00%
60.00%
50.00%
No Leak
Leak
40.00%
30.00%
20.00%
7.92%
10.00%
1.25%
0.00%
2 Rail
Interlocking Pin
Design
Design Trade Study
Mixing
Robot
Liftable
Resistant
to
Failure
Minimal
Tolerance
Stack-up
Ease of
Machining
Cost
Leaking
Total
5
5
5
4
3
5
5
3
68
5
5
4
5
5
4
3
5
72
Weight
Foot
Print
Number
of Parts
C Channel
Design
5
5
Interlocking
Bolt Design
5
4
MOLECULAR WEIGHTS (particle size)
Bromophenol Blue: 670 Daltons
FITC DEXTRAN: 150,000 Daltons
Plasmid DNA: 55,000,000 Daltons
Leak Testing Results
120.00%
Percent Occurance
100.00%
99.58%
98.75%
80.00%
No Leak
60.00%
Leak
40.00%
20.00%
1.25%
0.42%
0.00%
Bromophenol Blue
FITC Dextran
Test Media
Clamping and Pressurization
Requirements
Clamping
Pressurization
• 800lbs force capacity
• Compact and economical
• Rapid actuation time
•
•
– Less than 10 seconds
0 = Poor
5=Excellent
800lbs
Economical
of
Force
Constant pressure
application of 30psi
Must be safe
Automatable Compact
Actuation Contamination
Total (30)
Time
Possibility
Piston Design
Yamaha TM Linear Actuator
5
5
5
5
5
5
30
5
0
5
5
5
Promess Ball Screw
Ball Screw Custom
Hydraulic Jack
Automotive Jack Adaption
0
0
0
5
5
0
5
0
5
5
0
0
5
5
0
0
5
5
5
0
5
5
5
0
5
25
25
20
10
10
Gear Box Design
0
5
5
0
0
0
10
Bimba Piston
®
• 1130lbs of applied force at 90 psi supply
• Easy to implement into the lab
Piston Assembly Concept
Pneumatic
Pneumatic Piston
Piston
Ball
JointHolding Plate
Holding
Plate
Pressure Head
Pressure
Head
96 Well
Plate
Assembly
96 Well
Plate
Assembly
Structural Stress Analysis
Applied
Force
Zero
Displacement
Supports
Max Displacement: 4.2e-5 in
Max Stress: 430 psi
Pressurization Method
Gasket
R  APH P  2 AG P
R  Re quired forceto preventleaking
APH  Surfacearea of pressurehead
P  InternalP r essure
AG  Surfacearea of gasket
R  500lbs
Dry Dispensing
Lid
Screen
Container
Slide Plate
• Proprietary compound
• 4g ± 25% over the 96 wells
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
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
testing
Dispense Volume = 250 µL
Fill time = 27.6 seconds
Station Setup
Solution 0
378 mL/min
Peristaltic
Pump
Solution 1
Solution 2
Solution 3
Solution 4
Solution 5
Solution 6
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
Maximum Exit Velocity
= 0.6526 ft/s
378 mL/min
Peristaltic
Pump
BASIN
WellMate
Electrical Schematic
Common relay circuit between the clamping and pressurization and liquid
distribution stations
User Control Interface
Liquid
Distribution
Clamping and
pressurization
Mixing
Requirements
• Programmable
• Range of high speeds
• Accept the assembly
• Ability to mix
– Resuspend bacteria pellet
– High viscosity fluid
Type
Speed
Range
(RPM)
Timer
Number 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 Mixer
Pellet Resuspension
• Resuspension Speed
Hand Pipetting
– 2000 RPM for 5
minutes in pulse mode
• Bacteria Pellet
RPM
– Angle Test
Hand Mixed
Flat
10 Degree
40 Degree
Full Alkaline Lysis with Assembly
DNA Yield
DNA Type
Solution 2-3 Mixing Times
1500 rpm
Qaigen Maxiprep
5 sec x 2
DNA Yield (ng)
25000
20000
20 Sec
15000
10 sec
10000
5 Sec
5000
No Mix
0
0
By Hand
5
10
20
5 sec
2 times
Size Markers
Mix Time (sec)
•Average yield of 21,000 ng
Desired Type
•Obtained desired Plasmid
DNA
Mixer Adapter Plate
• Holds assembly on Talboy
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
Optimization:
Using ONE of All Stations
Plate Throughput vs Stagger Time into the System
55 Assemblies!
60
Throughput (#plates)
50
22 Assemblies
40
30
Series1
20
10
0
20
19
18
17
16
15
14
13
12
11
10
9
8
7
Stagger Time into the System (min)
Goal: 20 Assemblies
6
5
4
3
2
1
Optimum Configuration
Ratio of Throughput to Capital Cost
Ratio of Throughput to Capital Cost for Various Station Configurations
165 Plates per Day
Configuration:
2 Clamping and
Pressurization Stations
1 Liquid Distribution Station
3 Mixing Stations
0.008
0.007
0.006
0.005
0.004
0.003
0.002
0.001
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Configuration Number
(Varying Number of Liquid Distributing, Mixing, and Filtration Stations)
23
24
25
26
27
• 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