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