Plasmid DNA Purification Project by EfV8D7Bc

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									     Plasmid DNA Purification
               Capstone Design Project


    Sponsored by:                         Advised by:
      Ed Harlow                      Professor Jeff Ruberti
Harvard Medical School              Northeastern University


                      DESIGN TEAM:
                    Forrest Harrington
                     Alexandre Lessis
                     Ashley Mattison
                    Jason McDermott
                      Michael Zabbo
             Problem Statement

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.
   Harlow Lab and DNA Research
 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)
                     Why Plasmid DNA?
 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


Gene to be studied                         Chromosomal DNA


   Plasmid DNA
                                               E. coli bacterium
Current Purification Process -
                                                                  Start with bacteria grown
                                                                       in 96-well plates

        A “Mini-prep”                                                     Centrifuge              16
                                                                       (First separation step)    min
 Separate out bacteria in centrifuge
   - Draw off supernatant (throw away 96 pipette tips)                    Add & mix                6
 3 Step alkaline lysis                                                   Solution 1              min

  - Add Solution #1 (throw away 96 pipette tips)
                                                                          Add & mix                6
       Resuspends bacteria                                               Solution 2              min
       Prevent degradation
   - Add Solution #2 (throw away 96 pipette tips)                         Add & mix                2
       Breaks open cell wall to release plasmid                          Solution 3              min
       Very alkaline
   - Add Solution #3 (throw away 96 pipette tips)                         Centrifuge              16
                                                                      (Second separation step)    min
       Neutralizes mixture
       Precipitates out everything but plasmid
       Very acidic
                                                                     Transfer to lysate-
 Separate out plasmid in centrifuge
                                                                                                  18
                                                                     clearing plate and           min
   - Draw off plasmid in solution (throw away 96 pipette tips)           centrifuge
   - Transfer to lysate-clearing plate (throw away first plate)
 Centrifuge to clean plasmid                                         Capture plasmid            TOTAL
   - Collect plasmid in standard well plate (throw away lysate-            DNA                    over
      clearing plate)                                                                            1 hour
                Current Problems
 Time Consuming
  - 8 hours to do 800 purifications by
    mini-prep
  - Cuts into research time
 Heavily 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 Goals
 Walk-Away Automation
  - Streamline the current process to minimize human
    interaction
      Redirects 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 yield

                                                                     96-well plate
                                                                    Base is 5” x 3 3/8”


                 Plate picture: www.hamptonresearch.com/products/productdetails.aspx?cid=10&sid=158&pid=453
                                                                         Start with bacteria grown

Streamlining the Process
                                                                              in 96-well plates


                                                                                 Centrifuge
                                                                              (First separation step)
                                                      Filter
 Replace Centrifugation                       (First separation step)

     Centrifuge is difficult to automate                                        Add & mix
                                                                                 Solution 1

  - Vacuum filtration                                                            Add & mix
     Limited to 14.7 PSIG (atmospheric)                                         Solution 2
     Uses proprietary filter plates
        (plates with filters built in)                                           Add & mix
                                                                                 Solution 3
  - Centrifuge filters
     Uses filter plates                                                         Centrifuge
     Same difficulties as centrifugation                                    (Second separation step)
                                                     Filter
                                             (Second separation step)

  Positive pressure filtration                                             Transfer to lysate-
                                                                            clearing plate and
    Attributes of filtration with                                              centrifuge
      no pressure limitations
    Very fast
                                                                             Capture plasmid
    Filtration does not need lysate clearing step
                                                                                  DNA
              Filter Requirements
 Efficiently remove E. coli bacteria from growth media
 Efficiently remove cellular debris allowing passage of
    plasmid DNA
   Low protein binding
   Withstand 30 PSIG without damage (assuming proper
    support)
   Chemically compatible with alkaline lysis solutions
   Restrict lateral flow through membrane
   Low cost
   Available in sheet form
                           Filters Tested
Polyether Sulfone                      Track Etched
 Precise fiber pore                    Vertically etched pores eliminate
  structure                              lateral flow cross-contamination
 Low protein binding                   Extremely accurate pore sizing
 High lateral flow rate                Lowest protein binding
                                        Different material options



Polyether Sulfone      Polycarbonate Track Etched Polyester Track Etched




                                              Filter pictures: http://www.sterlitech.com/products.htm
                     Filter Testing




                      Test Column

 Multiple test columns have been manufactured
 Bacteria is added and pressurized gas is applied
   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 parameters
 Filtration 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
Celpure® Added to Eliminate Clogging
 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®


                                Celpure picture: http://www.advancedminerals.com/celpure.htm
   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 parameters
 Filtration 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
                                                     DNA Yield Analysis
                          12000

                          10000
         DNA Yield (ng)
                           8000

                           6000

                           4000
                                                                                        Centrifugation (Control)
                           2000
                                                                                        Filtration
                              0
                                  0              5           10                 15             20              25

                                                              Test Number

                                                       DNA Yield (ng)
Centrifu-           5961 8895         6543    9700   10921   8068        7798        8540   8443     7869    6867   6891
 gation             8384 6894         8517    8648   7903    9011        8690        7905   7471     6285    7441   7764
Filtration          5867 6466         6540    5648   5781    4666        4959        3643   5496     5418    4859     -

                                                       Filtration (ng)           Centrifugation (ng)
                                   Mean                    5394.82                    7975.38
                           Standard deviation              835.64                      1117.39
                          Coefficient of variation           6.46                        7.14
                              Purity
                  Filtering      Centrifugation

                                                  DNA
                                                  Classification
Kilobase
    Pairs
      23                                             Genomic
     9.4
     6.6
     4.4                                             nicked
     2.2                                             linear
     2.0
                                                     supercoiled




  Supercoiled DNA represents plasmid DNA
  Genomic and Chromosomal DNA trace is negligibly small
           Filtration Parameters
 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 steps
                                                    Track Etched Membrane

                       http://www.2spi.com/catalog/spec_prep/grease-coated-membrane-filters.shtml
                         Filtration Efficiency
 Approximately 70% decrease in process time                             0

     - Down from 64 minutes to 20 minutes
                                                                         10
 20 minutes per plate equates to 24 plates per day,
  or 2,304 samples per 8-hour day                                        20




                                                                              Process Time (minutes)
     - Surpassed our goal of 2,000
                                                                         30

      Current process                 Proposed process                   40
  Centrifuge                 16   Transfer to custom plate    4
  Add & mix Solution 1        6   Filter                     1.5
                                                                         50
  Add & mix Solution 2        6   Add & mix Solution 1        4
  Add & mix Solution 3        2   Add & mix Solution 2        6
  Centrifuge                 16   Add & mix Solution 3        2          60
  Transfer to lysate plate    2   Filter                     2.5
  Centrifuge                 16                                          70
                                                                   Old
  TOTAL                      64   TOTAL                      20    New
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
       Material Selection: Gaskets
 Five elastomeric materials
                                                    - Styrene-butadiene rubber (SBR)
  - Isobutylene-isoprene rubber (IIR)               - Nitrile rubber (NR)
  - 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 resistance
      The repeated usage during automation degrades material properties over time
  - Compression set
  - Gas impermeability


 EPDM has been chosen as the gasket material
       Compression Requirement
                 Fb = (π/4)G2P + 2b πGmP
                        Fb ≈ 400 lbs

                Fb = total load for operating conditions


 Using a pressure of 30 PSIG
 Takes 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
 ANSYS Analysis of Clamped Tabs




                       316L Stainless Steel
                  Max Displacement: 0.159 e-7 in

 Integrity of design analyzed with 200 lbs (SF=2)
             Leak Rate Analysis
 Finish 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 filtered
        Across 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
                        Cost of consumables for 96-well plate
       Primary separation only: isolating bacteria, alkaline lysis, and capturing plasmid
                      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
                 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
                  Accomplishments
 Testing 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 clogging
 Process 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 day
 Consumables costs can be cut in half
   - Labor dramatically reduced, also
 Designed an assembly which is automatable
   - Calculations showed gaskets will seal properly

								
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