DESIGN TEAM:Forrest HarringtonAlexandre LessisAshley MattisonJason McDermottMichael Zabbo
Sponsored by:Ed Harlow
Harvard Medical School
Advised by:Professor Jeff Ruberti
Northeastern University
Plasmid DNA PurificationCapstone Design Project
The Harlow laboratory at Harvard Medical School would like to automate their plasmid DNA purification process to increase throughput, improve purity, and reduce cost.
Our focus was to design and demonstrate the feasibility of an improved single unit operation which meets the purity and yield requirements, setting the stage for scale-up to meet the throughput and cost requirements.
Problem Statement
The goal of the Harlow lab is to understand gene function by using shRNA to see the phenotype associated with the loss of gene function. - This is expected to lead to an improved understanding of
biochemical processes and ultimately the development of new drugs.
Genes are “transcribed” to create mRNA, which produce proteins. The Harlow lab uses shRNA, a form of RNAi, to prevent the production of the protein (“protein expression”).
Scientists analyze the impact of the loss of the protein.
Up to 100,000 genes are needed to complete one full genome screen(20,000 genes in human genome) x (5 tests each)
Harlow Lab and DNA Research
In order to have large quantities of each gene to be researched, a gene is inserted into a plasmid DNA, and the plasmid is inserted into an E. coli bacterium, which can be grown easily and quickly
In order to analyze the plasmid, it must be extracted from the bacteria, or “purified”- Meaning 100,000 purifications required per
genome screen
Why Plasmid DNA?
Plasmid DNA
Gene to be studied
E. coli bacterium
Chromosomal DNA
Current Purification Process -
A “Mini-prep”Separate out bacteria in
centrifuge- Draw off supernatant (throw away 96 pipette tips)
3 Step alkaline lysis- Add Solution #1 (throw away 96 pipette tips)
Resuspends bacteria Prevent degradation
- Add Solution #2 (throw away 96 pipette tips) Breaks open cell wall to release plasmid Very alkaline
- Add Solution #3 (throw away 96 pipette tips) Neutralizes mixture Precipitates out everything but plasmid Very acidic
Separate out plasmid in centrifuge- Draw off plasmid in solution (throw away 96 pipette
tips)- Transfer to lysate-clearing plate (throw away first
plate)Centrifuge to clean plasmid
- Collect plasmid in standard well plate (throw away lysate-clearing plate)
16 min
6 min
6 min
2 min
16 min
TOTAL over
1 hour
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
Current ProblemsTime Consuming
- 8 hours to do 800 purifications by mini-prep
- Cuts into research timeHeavily dependent on
human interaction - Constant loading of
centrifuges and transferring of liquids
Uses disposable materials - Consumable plastics and
chemicals cost about $60,000 per genome screen
Disposables used for one run of 96
samples
Design GoalsWalk-Away Automation
- Streamline the current process to minimize human interactionRedirects focus from preparation to research More research can be performed at a lower cost to the
lab
- Increase throughput to purify 10,000 plasmid DNA samples per week
Lower costs - Reduce disposable materials
Reduce cost per sample by at least 50%
Improve purity of samples- No cell debris in purified sample: only
plasmid
Constraints
Continue to use 1-2-3 alkaline lysis process- Doesn’t use proprietary methods or
expensive chemicals
Use standard dimensions of 96-well plate - Easily integrated with common
laboratory robotics
Maintain consistent yield96-well plateBase is 5” x 3
3/8”
Plate picture: www.hamptonresearch.com/products/productdetails.aspx?cid=10&sid=158&pid=453
Streamlining the Process
Replace CentrifugationCentrifuge is difficult to automate
- Vacuum filtration Limited to 14.7 PSIG (atmospheric) Uses proprietary filter plates (plates
with filters built in)- Centrifuge filters
Uses filter plates Same difficulties as centrifugation
Positive pressure filtrationAttributes of filtration with no
pressure limitationsVery fastFiltration does not need lysate
clearing step
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
Filter(First separation
step)
Filter(First separation
step)
Filter(Second
separation step)
Filter(Second
separation step)
Filter RequirementsEfficiently remove E. coli bacteria from growth
mediaEfficiently remove cellular debris allowing
passage of plasmid DNALow protein bindingWithstand 30 PSIG without damage (assuming
proper support)Chemically compatible with alkaline lysis
solutionsRestrict lateral flow through membraneLow costAvailable in sheet form
Polyether Sulfone Polycarbonate Track Etched Polyester Track Etched
Filter pictures: http://www.sterlitech.com/products.htm
Filters Tested
Polyether Sulfone
Precise fiber pore structure
Low protein binding
High lateral flow rate
Track Etched Vertically etched pores
eliminate lateral flow cross-contamination
Extremely accurate pore sizing
Lowest protein binding Different material
options
Filter Testing
Multiple test columns have been manufacturedBacteria is added and pressurized gas is applied
Test Column
Progression of Filtration Testing
Filtration of bacteria from growth media- Initial testing used PES membrane- Clogging of filter was overcome using Celpure a filtration
aid- Testing of PCTE membrane cut time down to 90 sec- Pressure and time trends enabled selection of
parametersFiltration of cellular components from
plasmid DNA- Track etched membranes were tested - Time and pressure were varied to select parameters- Analysis of yield and purity proved comparable
Start to finish filtration- The two filtration steps were run successfully in series - The use of one filter to accomplish both filtrations
succeeded- Filtration was run against centrifugation and analyzed
Filter clogging was prevalent- Hindered data collection
Introduced to Celpure®
- Powdered filtration aid (diatomaceous earth)
- Acts as a pre-filter
P300 Celpure® - Filtering 0.4 – 0.6 µm particles
Celpure picture: http://www.advancedminerals.com/celpure.htm
Celpure®
Celpure® Added to Eliminate Clogging
Progression of Filtration Testing
Filtration of bacteria from growth media- Initial testing used PES membrane- Clogging of filter was overcome using Celpure a filtration
aid- Testing of PCTE membrane cut time down to 90 sec- Pressure and time trends enabled selection of
parametersFiltration of cellular components from
plasmid DNA- Track etched membranes were tested - Time and pressure were varied to select parameters- Analysis of yield and purity proved comparable
Start to finish filtration- The two filtration steps were run successfully in series - The use of one filter to accomplish both filtrations
succeeded- Filtration was run against centrifugation and analyzed
Yield Comparison
Filtration (ng) Centrifugation (ng) Mean 5394.82 7975.38
Standard deviation 835.64 1117.39Coefficient of variation 6.46 7.14
DNA Yield Analysis
0
2000
4000
6000
8000
10000
12000
0 5 10 15 20 25
Test Number
DN
A Y
ield
(ng)
Centrifugation (Control)Filtration
DNA Yield (ng)Centrifu-
gation
5961 8895 6543 9700 10921 8068 7798 8540 8443 7869 6867 6891
8384 6894 8517 8648 7903 9011 8690 7905 7471 6285 7441 7764
Filtration 5867 6466 6540 5648 5781 4666 4959 3643 5496 5418 4859 -
Kilobase Pairs
239.46.64.4
2.22.0
Filtering Centrifugation
Purity
Genomic
nicked
linear
supercoiled
DNA Classification
Supercoiled DNA represents plasmid DNAGenomic and Chromosomal DNA trace is
negligibly small
First Filtration: Extract bacteria from growth media- 0.2µm Polycarbonate track etched membrane- 10mg Celpure per well- 30 PSIG- 90 seconds
Second Filtration: Remove cellular debris - 0.2µm Polycarbonate track etched membrane- 10mg Celpure per well- 30 PSIG - 30 seconds
Uses same filter for both stepsTrack Etched Membrane
http://www.2spi.com/catalog/spec_prep/grease-coated-membrane-filters.shtml
Filtration Parameters
Filtration EfficiencyApproximately 70% decrease in
process time- Down from 64 minutes to 20
minutes20 minutes per plate equates to 24
plates per day, or 2,304 samples per 8-hour day
- Surpassed our goal of 2,000
0
10
20
30
40
50
60
70
Process Time (m
inutes)
OldNew
Centrifuge 16
Add & mix Solution 1 6
Add & mix Solution 2 6
Add & mix Solution 3 2
Centrifuge 16
Transfer to lysate plate 2
Centrifuge 16
TOTAL 64
Transfer to custom plate 4
Filter 1.5
Add & mix Solution 1 4
Add & mix Solution 2 6
Add & mix Solution 3 2
Filter 2.5
TOTAL 20
Current process Proposed process
Breakthroughs in Filtration Testing
Testing has proved that :
Two different filtration processes are possible with the use of one filter
Celpure, a cheap and simple to add solution, is capable of preventing clogging at all stages of filtration
Filtration has decreased the purification process time by 70%, or 44 minutes
Filtration is equally comparable to centrifugation in both DNA yield and purity
Designing a Reusable Plate
Design a custom reusable 96-well plate to allow for more efficient fluid handling and to reduce the cost of consumables
Similar to a filter plate, but with replaceable filters
Need to seal each well properly to prevent cross-contamination
96 Piston Design
Single Piston Design
Pressurized Air Supply
Pressurized air inlet
Component Assembly
Component Assembly
Five elastomeric materials- Isobutylene-isoprene rubber (IIR)- Chloroprene rubber (CR)- Ethylene-propylene diene monomer rubber (EPDM)
Important material properties- Chemical compatibility
The chemicals used in alkaline lysis can be very harmful to elastomers
- Erosion resistanceThe repeated usage during automation degrades material
properties over time
- Compression set- Gas impermeability
- Styrene-butadiene rubber (SBR)- Nitrile rubber (NR)
EPDM has been chosen as the gasket material
Material Selection: Gaskets
Compression Requirement
Fb = (π/4)G2P + 2b πGmP
Fb ≈ 400 lbs
Fb = total load for operating conditions
Using a pressure of 30 PSIGTakes into account the compression
needed to seal the interface as well as containing the hydrostatic end force
Four securing points on assembly - 100 lbs force on each securing point
Integrity of design analyzed with 200 lbs (SF=2)
Max Displacement: 0.159 e-7 in316L Stainless Steel
ANSYS Analysis of Clamped Tabs
Leak Rate AnalysisFinish of custom well plate would be made
with tolerance of +/- 0.001 in Low amount of leak due to
- Open area of filter 99.982% of total flow area
- Distance to be filteredAcross filter is = 0.0127mm Along leak path = 0.991 mm
For the 1.5 ml filtered- Flow across filter = 1.4997 ml- Flow through leak path = 0.263 µl
Cost Analysis
Consumable Current way Proposed process
Qty. Price Qty. Price
Deep-well block for cell growth 1 $2.00 1 $2.00
Lysate clearing microplate (800 ml) 1 $12.40 (none)
Box LTS 1000 pipet tips 0.5 $2.25 0.1 $0.45
Solution I (RNAse) 28.8mL $1.50 28.8mL $1.50
Solution II 28.8mL (negligible) 28.8mL (negligible)
Solution III 28.8mL $0.30 28.8mL $0.30
Celpure (none) 20 mg $0.22
0.2 µm filter (none) 1 $4.77
Total (approximate, before volume discounts, w/tips) $18.45 $9.25
Automated process would cut consumables cost of primary separation in half
Cost of consumables for 96-well plate Primary separation only: isolating bacteria, alkaline lysis, and
capturing plasmid
Future Work
Final, toleranced prototypes will be manufactured/machined to specifications
Testing will be performed on the 96 through hole filtration assembly
- Consistency among wells- Cross-contamination- Leaks
A thorough integration into an automated sequence
AccomplishmentsTesting showed that filtration is very
plausible for an automated process- The same 0.2 µm filter can be used in both
separation steps- Track etched membranes speed up flow and
lessen the chance for cross-contamination- Celpure® was found to nearly eliminate
cloggingProcess time was reduced by 70 %,
providing the potential for throughput of at least 2,304 samples per 8-hour day
- Goal was 2,000 per dayConsumables costs can be cut in half
- Labor dramatically reduced, alsoDesigned an assembly which is
automatable- Calculations showed gaskets will seal
properly