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    T7PhageLibraryExpansion_GR

    Monday, 11/4/2019Phage_Expansion_Calculator.xlsxExpansion Aliquot Calculator.xlsxTable1
    Monday, 11/4/2019
    Phage_Expansion_Calculator.xlsx
    Expansion Aliquot Calculator.xlsx
    MATERIALS
    8.
    Water (3 L for extraction buffer; molecular biology grade): https://www.sigmaaldrich.com/catalog/product/sigma/w4502?lang=en&region=US
    9.
    DI Water for media to be autoclaved
    13.
    BLT5403 E. Coli (need to check with millipore to see if can purchase separate from packaging kit): https://www.emdmillipore.com/US/en/product/T7Select-Packaging-Kit,EMD_BIO-70014
    18.
    Conical centrifuge tubes 50 and 500 mL (100 or so of 50 and a small pack of about 20 for the 500): https://www.sigmaaldrich.com/labware/labware-products.html?TablePage=17216271
    25.
    Glassware:
    a.
    25 and 50 mL erlenmeyer flasks (2 each)
    b.
    500 mL, 1 L and 2 L bottles (5 each)
    26.
    Micro pipettes and corresponding filtered tips: 20, 200, and 1,000 uL (5 tip boxes of each)
    27.
    Incubator (30°C static and 37°C shaking)
    a.
    Static incubator should be able to accommodate 75x 150 mm petri dishes
    b.
    Shaking incubator should able able to accommodate 2 or more 2 L bottles
    28.
    Centrifuge with adapters suitable for 50 and 500 mL conical tubes
    29.
    Vacuum aspirator
    30.
    Aluminum foil
    31.
    Gloves
    EXPANSION DETAILS
    ●
    Human
    ○
    Human library = 259,364 complexity
    ○
    Want 100x coverage = 2.6E7 PFU
    ○
    Want to add 1E6 PFU per 150 mm plate
    ○
    2.6E7 PFU to cover / 1E6 pfu per plate = 26 plates
    ○
    26 Plates extracted with 12 mL each = 26 * 12 = 312 mL of final library
    ○
    assuming a typical titer of 5E10 pfu/mL, this is sufficient for 312/0.5 (see table 1) = 624 IPs
    ●
    VirScan
    ○
    VirScan library = 96,100 complexity
    ○
    Want 100x coverage = 9.6E6 PFU
    ○
    Want to add 1E6 PFU per 150 mm plate
    ○
    9.6E6 PFU to cover / 1E6 pfu per plate = 10 plates
    ○
    10 Plates extracted with 12 mL each = 10 * 12 = 120 mL of final library
    ○
    assuming a typical titer of 5E10 pfu/mL, this is sufficient for 120/0.19 (see table 1) = 631 IPs
    ●
    ArboScan
    ○
    ArboScan library = 210,976 complexity
    ○
    Want 100x coverage = 2.2E7 PFU
    ○
    Want to add 1E6 PFU per 150 mm plate
    ○
    2.2E7 PFU to cover / 1E6 pfu per plate = 22plates
    ○
    22 Plates extracted with 12 mL each = 10 * 22 = 220 mL of final library
    ○
    assuming a typical titer of 5E10 pfu/mL, this is sufficient for 220/0.42 (see table 1) = 523 IPs
    EXPANSION MEDIA RECIPES (30 Plates)
    500x Carbenicillin
    1.
    Dissolve carbenicillin to 50 mg/mL in sterile water and filter through a 0.22 um filter. Aliquot and store at -20°C .
    2xYTM9 Bottom-Agar
    1.
    Combine the following in 2 L bottle:
    ○
    31 g 2xYT medium
    ○
    11.3 g 5xM9 salts (makes a 1x solution)
    ○
    15 g agar (Cf = 1.5%) or 37 g lb/agar
    ○
    Make up to 1 L with deionized water
    2.
    Add a stir bar to the flask and autoclave.
    3.
    Allow the media to cool with stirring and then add 2 mL of 50 mg/mL carbenicillin (Cf = 100 ug/mL).
    4.
    Keep molten in a 45°C water bath and pour 30 mL per 150 mm plate.
    5.
    Allow the plates to sit undisturbed on a level surface so the agar can solidify.
    2xYTM9 Top-Agarose
    1.
    Combine the following in a 1 L bottle:
    ○
    300 mL water
    ○
    9.3 g 2xYT media
    ○
    3.4 g 5xM9 salts (makes a 1x solution)
    ○
    1.8 g low-melting point agarose (Cf = 0.6%)
    2.
    Autoclave and allow to solidify in the bottle.
    3.
    Use a microwave to melt the 2xYTM9 Top-Agar when ready to use and maintain at 45°C.
    4.
    Once the temperature has equilibrated to 45°C, add 3 mL of 45% glucose (Cf = 0.45 %) and 300 uL of 1M MgSO4 (Cf = 1 mM). Maintain at 45°C throughout.
    2xYTM9 Cell Culture Media (need 600 mL total per 60 plate expansion)
    1.
    Combine the following in a 2 L bottle:
    ○
    1 L water
    ○
    31 g 2xYT medium
    ○
    11.3 g 5xM9 salts (makes a 1x solution)
    2.
    Add a stir bar to the bottle and autoclave.
    3.
    Allow the media to cool with stirring and then add the following:
    ○
    2 mL of 50 mg/mL carbenicillin (Cf = 100 ug/mL)
    ○
    1 mL of 1 M MgSO4 (Cf = 1 mM )
    ○
    10 mL of 40% glucose (Cf = 0.4%)
    Phage Extraction Buffer
    1.
    Combine the following in a 500 mL bottle and store at 4°C:
    ○
    477 mL water (molecular biology grade)
    ○
    10 mL Tris-HCl, pH 8.0 (Cf = 20 mM)
    ○
    10 mL 5 M NaCl (Cf = 100 mM)
    ○
    3 mL 1 M MgSO4 (Cf = 6 mM)
    LIBRARY EXPANSION (30 Plates)
    1.
    In a 50 mL erlenmeyer flask, seed 10 mL LB media containing 100 ug/mL carbenicillin with BLT5403 and shake at 37°C overnight.
    2.
    In separate 2 L flasks, inoculate 300 mL Cell Culture 2xYTM9 media containing 100 ug/mL carbenicillin with 5 mL overnight culture and shake at 37°C until the OD600 reaches 1.0.
    3.
    Meanwhile, melt 300 mL YTM9 top-agar and keep the solution molten by placing the flask in a 45°C water bath. Once the temperature has equilibrated to 45°C, add 3 mL of 40% glucose (Cf = 0.4 %) and 300 uL of 1M MgSO4 (Cf = 1 mM). Maintain at 45°C throughout.
    4.
    Collect and concentrate the cells from step 2 16-fold by resuspending the centrifuged pellet in 37.5 mL YTM9 containing 100 ug/mL carbenicillin. Separate 1 mL for negative infection control.
    5.
    To the remaining 36.5 mL 16x cell suspension, add 36.5E6 pfu phage library. Mix thoroughly and take 30 mL of this mixture immediately onto step 6.*
    6.
    Dilute 30 mL of the phage-infected cell suspension into 300 mL molten YT-M9 top-agar containing 0.4% glucose and 1 mM (held at 45°C in a water bath). Mix thoroughly.*
    7.
    Pipette 10 mL of the phage/cell/agarose mixture onto 150 mm YTM9 agar plates containing 100 ug/mL carbenicillin.*
    8.
    Allow the plates to sit undisturbed on a level surface so the agar can solidify. Incubate until the plaques are nearly confluent (4-6 h at 30°C).
    9.
    To elute the phage, cover each plate with 12 ml of Phage Extraction Buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 6 mM MgSO4) and place on a level surface at 4°C overnight.
    10.
    Harvest the phage by tipping the plates slightly and pipetting the liquid into a single sterile container.
    11.
    Centrifuge to clarify the phage solution and filter the library using a 0.2 micron PES membrane filter.
    12.
    Determine the titer of the amplified library by plaque assay.
    13.
    Add DMSO to a final concentration of 10% and Antibiotic-Antimycotic to 1x (remember to adjust determined titer by the amount of DMSO and AA added).
    14.
    Mix well and aliquot into 50 mL Falcon tubes such that a single tube is sufficient for 110 samples.
    15.
    Store at -80 °C for long term storage
    *Note for step 5-7:
    ●
    36.5E6 pfu in 36.5 mL = 1E6 pfu/mL
    ●
    30 mL of 1E6 pfu/mL in 300 mL top-agar = 0.1E6 pfu/mL
    ●
    Plating 10 mL of 0.1E6 pfu/mL per plate will add 1E6 pfu to each plate.
    PLAQUE ASSAY
    MEDIA
    2xYT Bottom-Agar
    1.
    Combine the following in 1 L bottle:
    ○
    500 mL water
    ○
    15.5 g 2xYT medium (Cf = 31 g/L)
    ○
    7.5 g agar (Cf = 1.5%)
    2.
    Add a stir bar to the flask and autoclave.
    3.
    Allow the media to cool with stirring and then add 1 mL of 50 mg/mL carbenicillin (Cf = 100 ug/mL).
    4.
    Keep molten in a 45°C water bath and pour 10 mL per 100 mm plate.
    5.
    Allow the plates to sit undisturbed on a level surface so the agar can solidify.
    2xYT Top-Agarose
    1.
    Combine the following in a 1 L bottle:
    ○
    300 mL water
    ○
    9.3 g 2xYT media
    ○
    1.8 g low-melting point agarose (Cf = 0.6%)
    2.
    Autoclave and allow to solidify in the bottle.
    3.
    Use a microwave to melt the 2xYT Top-Agar when ready to use and maintain at 45°C.
    Protocol
    1.
    In a 25 mL flask, inoculate 5 mL LB media containing 100 ug/mL carbenicillin with BLT5403 and shake at 37°C overnight.
    2.
    The next day, use 50 uL of the saturated overnight to inoculate 5 mL LB media containing 100 ug/mL carbenicillin.
    3.
    Shake at 37°C until the OD600 = 1.0 (Store the host cells at 4°C until needed. Do not use host cells that have been stored for longer than 1 week)
    4.
    Melt a sufficient amount of YTM9 top-agar to provide 3 ml for each dilution being plated and keep the molten solution in a 45°C water bath.
    5.
    Prepare 10-fold serial dilutions of the phage in using LB as the diluent (100 uL phage + 900 uL LB).
    6.
    For each dilution to be plated, add 250 uL of BLT5403 (OD600 = 1) cells to 5 mL tubes.
    7.
    Starting with the highest dilution, add 100 uL of the phage dilution to each tube.
    8.
    Add 3 ml YTM9 top-agarose to each tube and pour the contents onto a prewarmed 37°C LB plates containing 100 ug/mL carbenicillin. Immediately swirl the plate (gently) to spread the agarose evenly.
    9.
    Allow the plate to sit undisturbed for several min until the top agarose hardens, then incubate for 3–4 h at
    37°C or overnight at room temperature.
    10.
    Count the plaques and calculate the phage titer.
    a.
    The phage titer (expressed as plaque forming units per mL) is the number of plaques on the plate divided by 100 (to account for the 100 uL of dilution plated) then times the dilution factor.
    b.
    Example: if there are 100 plaques on a plate from a 1/108 dilution, then the titer of the sample is (100/100)*108 = 108 pfu/uL.

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