Team:CSU Fort Collins/Protocols

Protocols

Transformation into Thermococcus kodakarensis

  • Grow overnight(12-13 hrs) 100 mL cultures of TK @85C
  • Place culture and centrifuge tube into anaerobic chamber
  • Pour liquid, but not sulfur into the tube
  • Spin with Beckman centrifuge with rotor JLA10.500 at 9,000 RPM for 11 min, at 4 degrees C
  • Flip cultures upside down and bring into chamber with 2-3 ug plasmid aliquot, cold block, and trash bucket
  • Pour out supernatant
  • Resuspend pellet in 3 ml 0.8X asw, then ice for 30 min
  • Add 200uL of cells to plasmid aliquot, flick. Then ice for 1 hour
  • Heat shock at 90C for 45 seconds.
  • Spread onto -trp plates
  • Incubate at 85C, upside down in canister with anaerobic pack for 3-5 days
  • Pick cultures into epi tubes with 10 ul .8X asw (10 cultures per plate)
  • Spot each culture to a discrete portion of a lovastatin, +trp plate
  • Incubate for 3-5 days
  • Remove 20 cultures and put each of them into a 5 mL serum bottles containing +lov, +aug ASW media.
  • ASW Media

    • 20 g Sodium Chloride (NaCl)
    • 6g Magnesium Sulfate (MgSO4*7(H2O))
    • 3 g Magnesium Chloride (MgCl2 *6(H2O))
    • 5g Yeast Extract
    • 5g Tryptone
    • 1g Ammonium Sulfate (NH4(SO4)2)
    • 0.2g Sodium Bicarbonate
    • 0.3g Calcium Chloride (CaCl2 2(H2O)
    • 0.5 g Potassium Chloride (KCl)
    • 0.42g Potassium Phosphate Monobasic (KH2PO4)
    • 50 mg Sodium Bromide (NaBr)
    • 20 mg Strontium Chloride (SrCl2 6(H2O))
    • 10 mg Ferric Ammonium Sulfate (Fe(NH4)2(SO4)2*6(H2O))
    • 5 mL Wolfe Trace Minerals
    • 1L boiled water

    Boil 1.5 L water, then let cool. While heating, mix other ingredients

    Measure 1L water into a 2L plastic bucket and add a stir bar.

    Place bucket, dry ingredients, 10 100mL bottles, stoppers, seals, 100 mL graduated cylinder, and funnel into chamber.

    Place bucket on stir plate and turn it on, then add ingredients.

    A clear, amber liquid will form.

    Fill 100mL bottles with 100mL liquid, then stopper and seal them.

    Autoclave on a liquids cycle for 30 min.

    When inoculating with this media, more additives must be used. Per 100 mL bottle, add

    • 100 ul KOD vitamins (Made in house)
    • 100 uL Agmatine
    • 50 uL (25 mol/L) lovastatin
    • ½ scoop Sulfur

    ASW Additives:

    • ASW liquid media
    • Lovastatin
    • Agmatine
    • Sulfur
    • KOD vitamins

    Place in incubator until grown (supposed to be 12-16 hr)

    SPOT PLATEs (pg6)

    ASW Plates:

    1 recipe makes 3-4 plates

    Acquire 2, 100 mL serum bottles, and fill with desired ingredients.

    Bottle 1:

    • 50 mL 18 Millipore water
    • 1g Gelzan
    • Bottle 2:

      • 50 mL 2X asw, 500 uL Wolfe Trace Minerals, (optional: .5g pyruvate)
        • For minimal plates: add nothing
        • For rich plates: 0.5 g tryptone, 0.5 g yeast extract

      Add stoppers and crimp bottles closed. Place in wire basket in noodle pot with some water. Autoclave using liquid cycle: 30 min.

      Prepare additives:

      • 100 uL KOD vitamins
      • 200 uL polysulfide solution
      • 100 uL agmatine
      • Selective marker: 50 uL Lovastatin(Mevinolin) on Rich plates only
      • For Minimal: 5 mL 19 Amino Acid solution

      When complete, bring bottles into chamber using P2 (for a shorter time than P1)

      Leaving gelzan bottle capped, uncap the other bottle and add premixed additive solution, swil

      Uncap gelzan bottle and pour additive bottle into gelzan bottle

      Pour ¼ mixture into each plate.

      Allow plates to cool, then flip them over for storage.

    gPREP/PCR/gel:

    Transfer 1 mL TK into 1.7 mL eppendorf tube with a syringe (through stopper flange, not center)

    Centrifuge for 5 min at 15,000 RPM

    Snap supernatant into hood sink and pipette excess

    Resuspend in 100 uL 10 mM Tris HCL pH 8.0

    Add 50uL from bottom layer of Phenol/Chloroform/Isoamyl Alcohol 25/24/1 Pipette and put tip in PCI hazardous waste bucket.

    Centrifuge 5-10 min, full speed.

    Add 50 uL 10 mM Tris HCL pH 8.0 to new 1.7 eppes

    Transfer 50 uL from top aqueous layer into new 1.7 mL eppes

    Add 100 uL 100% Isopropanol

    Centrifuge for 30 min high speed. (Centrifuge with hinge facing upwards)

    Pipette extra PCI from first tubes to PCI liquid waste, and put tubes with lids open into solid PCI toxic waste

    When spin is finished, remove all supernatant from pellet into drain.

    Pellet is small, clear, and gelatinous

    Leave caps open in 37 degree incubator for 20 minutes

    Resuspend in 30 uL Tris HCL pH 8.0

    Store in fridge until needed

    PCR

    Mix master mix: enough for the number of reactions plus 2-3 to ensure enough mix

    Components for master mix, per 1 reaction:

    • 3 uL gDNA
    • 0.2 uL Primer 1 (100 uM)
    • 0.2 uL Primer 2 (100 uM)
    • 0.5 uL dNTP (25 mM)
    • 5 uL Home Phusion
    • 10 UL 5X GC
    • 31.1 uL dH2O

    Add 50 uL Master Mix to a .2 uL eppe tube, then add 3 uL gprepped DNA.

    Thermocycler Program (degrees Celsius):

    • 1 min @98
    • 15 sec @98
    • 15 sec @ 60
    • 3 min @ 68
    • Repeat 35X
    • 4 Degree hold

    Gel protocol:

    Use 1% Agarose gel

    • Small tray: .5g Agarose :50 mL 1X TBE
    • Medium tray: 1.2 g Agarose: 120 mL 1X TBE

    Mix and microwave until boiling

    Let cool to touchable temperature and pour into mold. Allow to solidify for 30 min.

    After PCR is finished:

    Add 5 uL 10 X Loading Dye to each sample

    Write out which row corresponds to which sample

    Remember that the DNA starts at negative and flows towards positive. Position in the correct direction

    Load 5uL 1KB plus ladder

    Load 8 uL sample to gel

    Run gel starting at 120V for 50 min

    If dye is not around ¾ of the way down the gel, run for more time.

    Bradford Assay

    Use 2 replicas each of all standards, samples, and blank

    Generate standard curve:

    • Make solutions of 0, 0.1, 0.2, 0.5, 0.8, 1 mg/mL BSA in DIH2O

    Add 15 uL samples and standards to cuvettes, and add 750 uL Bradford reagent to each cuvette.

    Blank spectrophotometer, then use spectrophotometer to read all standards and samples.

    The one used for iGEM experiments could generate standard curves and give concentration data for samples

    Western Blot

    Use overnight incubations in 100 ml bottles- collect biomass by centrifuge to pellet the biomass and collect for western in 15mL falcon tube. OD0.4-0.6. You should have about 1 gram. You can store the pellet in freezer 4◦ degree until ready for western. Beckman centrifuge 1 st floor.

    Western

    Melt pellet at RT then put onto Ice. Add 1 ml Lysing Buffer (Lysing buffer: 25mM Tris-HCl pH7.5/ 200mM NaCl)

    Sonicate on ice 30/50/70 pulse (10” on/ 10” rest), until watery liquid (about 5x) Keep wand in the liquid when power is on. Use earphones.

    Centrifuge 5K 30’ 4◦C

    Replace onto ice. Remove supernatant into 15ul ml falcon tube on ice.

    Prepare 2ul aliquots of 6x SDS Dye in 0.5ul eppes

    Take a 10ul sample of cell pellet and add to SDS aliquot

    Take 4/ 6/ 8/ 12 ul samples of supernatant and add to SDS aliquots

    Prepare 7ul sample of Benchmark ladder and Magic mark ladder in 0.5ul eppes. Do not add SDS.

    Boil all samples controls and ladders for 5 minutes at 98◦ using thermocycler.

    Centrifuge very briefly about 5K 5 Seconds

    Western Blot Buffers:

    • 10x Lamelis Running Buffer: 1L
    • 30.3g Tris
    • 144.2g glycine
    • 10g SDS
    • Adjust pH to 8.3
    • Adjust volume to 1L wH2O
    • No autoclave needed
    • 2.5g BSA
    • 50mL 1x TBST
    • 100mL 10x TBS
    • 1ml Tween 20

    Running the gel:

    Use 1x lamellis Running buffer and Criterion TGX Stain Free gel Biorad precast 4-20%gel

    Run on 200V for 35mins

    Image with gel Doc stain free gel

    Transfer to PVDF paper

    Image PVDF on Gel Doc

    Block with BSA- in glass dish

    Incubate with primary in seal-o- meal bag 4 hours to overnight 4degree rocker (1:10,000 antibody to 1X TBST) anti-rabbit HA antibody

    Wash with 1x TBST 2 x 15 mins- glass dish

    Incubate with secondary 1hour RT rocker seal-o- meal baggie (1:2,000) AP-conjugate to ECF antibody

    Wash 2x15 mins in 1X TBST-glass dish

    Ready to use ECF on Typhoon Fla 9500 for fluorescence image

    Extraction for GC:

    Synechocystis 2 mL aliquot extraction (Bligh & Dyer) 1 Last updated: 10/07/14 YE

    Notes: This protocol was developed for 2 mL aliquots pulled from Synechocystis grown in 250 mL Erlenmeyer flask.

    Procedure:

    • Thaw 2 mL frozen aliquot from -80 o C.
    • Add 200 µL of acetic acid. Vortex 10 sec.
    • Add 5 µL of 4g/L C15:0 FFA. Vortex 10 sec (use RPT plastic pipet tips).
    • Add 7.50mL of 1:2 CHCl 3 :MeOH. Vortex 10 sec.
    • Add 2.50mL of CHCl 3 . Vortex 10 sec.)
    • Add 2.50mL of HPLC H 2 O. Vortex 10 sec.
    • Leave in fume hood at room temperature overnight.
    • Pipet out bottom layer with a Pasteur pipette into 4mL glass tube with septum. (Recover ~80% of it. <5mL)
    • Evaporate all solvents under nitrogen (~1hr). Make sure samples are absolutely dry.
    • Resuspend samples in 300 µL Hexane (HPLC grade).
    • With a glass pipet, aliquot all samples into GC vials. Run sample on GC.
    • Store injected samples in -20 o C. They can be derivatized to FAME for analysis.

    Notes:

    The ideal solvent ratio (chloroform:Methanol:Water) before dilution (at step 4) is 1:2:0.8. The solvent ratio after dilution should be 2:2:1.8 (at step 6, after vortexing). After addition of solvent at step 5, there should be phase separation in the tube (top: organic layer, bottom: solvent layer). If phase separation not visible, add 200uL of CHCl 3 and vortex again. Addition of Acetic Acid acidifies the sample and causes for the conversion of Fatty Acid Soaps to their Free Fatty Acid form.

    Add miniprep protocol

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