Team:USAFA/Experiments

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Experiments

Dehalogenase

  1. Transformed an D. Acidovorans plasmid with Ori.C, CAM (chloramphenicol resistance), Lac Operon and two types of DeHa3 dehalogenases as two separate variables into E.coli
  2. The bacteria was grown overnight in LB broth and transferred to a test solution
    1. The culture required an aqueous solution to facilitate availability for free fluoride ion probe testing
  3. There were a total of four different test solutions, each with varying concentrations of PFAS: 0, 100, 375 and 1500. All test solutions also contained chloramphenicol and Isopropyl β-d-1-thiogalactopyranoside (IPTG). All solutes were dissolved in LB broth. The different tests are shown below.
    1. Negative Control: DE3 690 without IPTG (not a required activator for DE3) expressing nucleocapsid protein from SARSCoV2, which should not affect fluoride ion concentration
    2. Negative Control: DeHa3 with Delta 1 dehalogenase without IPTG, a necessary activator to conduct dehalogenase function
    3. Positive Control: Known fluoride ion solution
    4. DeHa3 with Delta 1 dehalogenase with IPTG
    5. DeHa3 with Delta 2 dehalogenase with IPTG
  4. Cells were sonicated after 24 hours of growth in test solution
  5. At 0, 6 and 24 hours, free fluoride ions were measured using a free fluoride ion probe
    1. If there is an increase in measurements, then a pattern of dehalogenase activity would be present

Fungus Isolate

Winogradsky Sampling

  1. 0.1% PFOA and Agar minimal media plates were made
  2. All winogradsky column samples were streaked on the minimal media plates
  3. Plates were incubated at 30 degrees Celcius for 14 days and observed daily
  4. Upon observing a plate with positive growth, the fungal growth was transferred to a SAB plate
  5. Lastly, once fungus/fungi are grown on a positive media, they are sent out for sequencing


Schriever AFB Contaminated Soil Sampling

  1. Add contaminated soil and DI water into an eppendorf tube
  2. Vortex until fully mixed and let the sediment settle
  3. Pipet 200 μL of the mixed DI on top of the sediment was put over a minimal media plate of PFOA (0.1%) and Agar and was spread
  4. Leave the plate out for 10 days at room temperature
  5. Transfer positive growth to a SAB plate
  6. Send fungus for sequencing


      7. For both the Winogradsky sampling and contaminated soil sampling, slants were created of the same PFOA agar minimal media to test for for growth from SAB grown fungus to minimal media again. (all fungus we grew were able to regrow on the minimal media)

      Winogradsky Community

      1. Three overnight cultures were made for Delftia acidovorans, Pseudomonas flourescens and E. coli in 1/3 conc. TSB
      2. Three mL of bacterial sample transferred into each vial
      3. Centrifuged each vial to form bacterial pellet
      4. Removed supernatant
      5. Added 3mL of either DI water, 1000ppm PFOA, or 500ppm PFOS
        1. Negative controls: just 1000 PPM PFOA, just 500 PPM PFOS, and the bacterial species in DI water
      6. Vortex viles to recombine in solution
      7. Place vials on ice
      8. Half of the samples were sonicated to lyse the cells. Half were left with whole cells.
      9. Once sonication was complete, the fluoride ion level was measured in each vial at 0, 1, 2, and 24 hrs.

      Detection

      1. Searching for constitutive genes within the rhodococcus genome which constitutively/ housekeeping genes = want to show system works/ positive control, chose glyA,16S, recA
      2. See if can activate prmA in rhodococcus has been shown to upregulate in rhodo in presence of PFAS
      3. Evidence of some dehalogenase capability to break down PFOS, wanted to see if promoters would be activated (might degrade, so might be something that promotes degradation pathway)
      4. Flanked promoter with EcoR1 and Not1 cut sites, cut out the Lac promoter region of plasmid pSRKBB using EcoR1 and Pst1 restriction enzymes so only have ORI and kanamyasin resistance gene in order to insert in RFP
      5. Ran gel to check bp length for promoters and pSRKBB
      6. Purify DNA using gel extraction protocol
      7. Ligate RFP sequence into BB plasmid to be used as reporter gene with multi-cloning site immediately upstream of RFP to insert promoters
      8. Transform ligated plasmid (BB with RFP) into E. coli
      9. Grow up colonies
      10. Tested 5 colonies by diagnostic restriction enzyme digest, to cut same fragment out and determine which colonies had fragment inserted using miniprep
      11. 3 out of 5 colonies had correct size fragment for RFP
      12. Once found colony that worked, did miniprep on DNA cut it with different set of restriction enzymes, which allow to ligate promoter sequences immediately upstream of RFP at Not1 site
      13. Grew up on E. coli
      14. Future action:
        1. Check for consecutive promotion
        2. prmA/Dehalogenase to turn red with PFAS transformed

      Basic Lab Prep and Growth Procedures

      Making LB

      1. Read the directions on the bottle . For example, to make LB Agar. The bottle says to add 40 grams off the LB powder per liter of distilled water. (Note: the gram amount may differ for each bottle. See bottle for instructions).
      2. Scale 12.5 grams of LB powder of 500 mL of distilled water with graduated cylinder.
      3. Always heat the mixture to dissolve fully using a heat plate and a flask. Use an autoclaved flask. Make sure to have stir bir to dissolve particles. Black autoclaved tape on the flask means that it has been autoclaved and is sterile. Temperature doesn't matter. Dark urine color.
      4. Autoclaved for 15 minutes in 121 degrees. TIghten media bottle cap . Put foil on top of lid and put autoclave tape. Use double volume bottle for however much liquid mixture being autoclaved. For example, use a liter bottle for a 500 mL mixture.
      5. Tip when labelling with masking tape, flip end of one tape so easy to pull off.
      6. Always triple rinse glassware with DI water after washing with soap and warm water.

      Bacterial Plating

      1. Add 5 mL of LB broth to each test tube (cultured tubes) (3 of them).
      2. From the bacteria plate, take a distinct colony for each test tube and swirl it into the LB broth.
      3. Plate two more plates with the original plate. Streak on one side of the plate, do a quarter turn, and then overlap the streaks at this direction.
      4. Placed the plates in the incubator and ensure that all surfaces are cleaned. The temperature will vary based on different bacteria.

      Making Gel for Electrophoresis

      (For 50ml of 2% agarose gel)

      1. Add 1 gram of iGEM agarose (B/c 2% of 50 is 1)
      2. Add 50 ml of 1x tbe ( a bit more over 50 is okay due to evapoation) and mix
      3. Stuff flask with tissue
      4. Microwave for 30 seconds- stop when mix starts to boil to avoid over flowing, start again when solution settles
      5. OPTIONAL, either mix in GelGreen or GelRed, or dye with EtBr later after Electrophoresis ran.
      6. Continue to microwave until there are no more agarose crystals left. Make sure the ENTIRE solution is clear, Look meticulously.
      7. Once it is clear then it can be poured.
      8. Let it cool in the fridge for about 10mins, or until opaque.

      Gel Preparation(Summer procedure)

      Make a gel buffer, then add agarose.

      1. Obtain a 1 liter container. Add 20 mL of buffer and fill the rest of the container with 18 miliohm water until 1 liter volume is attained.
      2. Pour 50 mL of the solution into a smaller flask. Add 0.4 g of agarose powder. Microwave mixture for about a minute and a half (you can allow it to boil for 20-30 sec).
      3. When the flask can be held comfortably for 5-6 sec, add 5 microliters of ethidium bromide and swirl to mix. Pour mixture into gel container and insert the comb (this makes the wells) onto the side where the DNA and ladders will be inserted. The gel will solidify in about 20 min.
      4. Pull comb out of solidified gel. Plug in power source and hook wires to gel electrophoresis instrument ensuring that the DNA will flow from negative to positive.
      5. Pour buffer into gel electrophoresis instrument so that a thin layer covers the top of the gel.
      6. Add 5 microliters of each sample, keeping track of what well each sample is insterted into.
      7. Set power to 130-140 V for about a half hour or until the DNA has moved about halfway or 3/4 of the way down the plate.

      How to Grow & Preserve Rhodococcus RHA1 in Glycerol for Freezing

      1. Culture bacteria in LB broth in incubating orbital minishaker at 30 degrees C and 250rpm for 48 h.
      2. Under biosafety cabinet (hood), add 500 mL of Rhodococcus RHA1 in broth to Corning cryotube
      3. Under hood, add 500mL 50% sterile glycerol mix (See below if you need to make this)
      4. Place tubes into cryo Freezing Container
      5. Place in -80 Freezer
      6. Next day, take out of -80 freezer and put into a box in the -80 freezer.

      DNA+RNA Prep

      Digestion

      1. For digestion, use 1-5 micrograms of DNA, buffer at 10x concentration
      2. 1 unit of enzyme per microgram of DNA that can be digested in 1 hour
      3. Fill the rest of the desired volume with 18 miliohm water.
      4. Total volume shouldn't exceed 60 microliters.

      Tradition Ligation

      1. 7 ul vector (~10.7 ng/ul)
      2. 10 ul Insert (`10 ng/ul)
      3. 1 ul Enzyme (T4)
      4. 2 ul Buffer (T4)
      5. Mix all four together (in order of descending volume) then let sit at room temp (or 16 degrees celsius) overnight.

      qRT-PCR

      1. For each sample, weigh 25–50 mg acid-washed glass beads (150–600 μm diameter) in a 2 ml Safe-Lock tube (not supplied) for use in step 4.
      2. Harvest bacteria by centrifuging at 5000 x g for 5 min at 4ºC (do not use more than 1 x 109 bacteria). Decant the supernatant, and carefully remove any remaining media by aspiration. If the centrifuge will be used later in this procedure, heat it to 20–25ºC. Note: Incomplete removal of medium will inhibit lysis and dilute the lysate, affecting the conditions for binding of RNA to the RNeasy membrane. Both effects may reduce RNA yield.
      3. Add the appropriate volume of Buffer RLT (see table below). Vortex vigorously for 5– 10 s. 9. Note: Ensure that β-ME is added to Buffer RLT before use (see “Important points before starting”). Ensure that the pellet is thoroughly resuspended in Buffer RLT
      4. Transfer the suspension into the 2 ml Safe-Lock tube containing the acid-washed beads prepared in step 1. Disrupt cells in the TissueLyser for 5 min at maximum speed. Other methods of mechanical disruption can be used instead. We recommend using the TissueLyser
      5. Centrifuge for 10 s at maximum speed. Transfer supernatant into a new tube (not supplied). The volume of the tube must be at least twice that of the Buffer RLT used.
      6. Determine the volume of supernatant. Add an equal volume of ethanol (70%), and mix well by pipetting. Do not centrifuge. After adding ethanol, a precipitate may form. This will not affect the RNeasy procedure.
      7. Transfer up to 700 μl lysate, including any precipitate that may have formed, to an RNeasy spin column placed in a 2 ml collection tube (supplied). Close the lid gently, and centrifuge for 15 s at ≥8000 x g (≥10,000 rpm). Discard the flow-through.* Reuse the collection tube in step 8. If the lysate exceeds 700 μl, centrifuge successive aliquots through the spin column. Discard the flow-through after each centrifugation.*
      8. Add 700 μl Buffer RW1 to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s at ≥8000 x g (≥10,000 rpm) to wash the spin column membrane. Discard the flow-through.* Reuse the collection tube in step 9. Note: After centrifugation, carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow-through. Be sure to empty the collection tube completely. Skip this step if performing optional on-column DNase digestion.
      9. Add 500 μl Buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s at ≥8000 x g (≥10,000 rpm) to wash the spin column membrane. Discard the flowthrough. Reuse the collection tube in step 10. Note: Buffer RPE is supplied as a concentrate. Ensure that ethanol is added to Buffer RPE before use (see “Important points before starting”).
      10. Add 500 μl Buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 2 min at ≥8000 x g (≥10,000 rpm) to wash the spin column membrane. The long centrifugation dries the spin column membrane, ensuring that no ethanol is carried over during RNA elution. Residual ethanol may interfere with downstream reactions. Note: After centrifugation, carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow-through. Otherwise, carryover of ethanol will occur.
      11. Optional: Place the RNeasy spin column in a new 2 ml collection tube (supplied), and discard the old collection tube with the flow-through. Close the lid gently, and centrifuge at full speed for 1 min. Perform this step to eliminate any possible carryover of Buffer RPE, or if residual flow-through remains on the outside of the RNeasy spin column after step 10.
      12. If the expected RNA yield is >30 μg, repeat step 12 using another 30–50 μl RNase-free water, or using the eluate from step 12 (if high RNA concentration is required). Reuse the collection tube from step 12. If using the eluate from step 12, the RNA yield will be 15–30% less than that obtained using a second volume of RNase-free water, but the final RNA concentration will be higher.

      QIAquick PCR Purification

      1. Used for purification of up to 10 ug of PCR product (10 - 100 bp) - (Used for G block)
      2. Add 5 volumes of PB Buffer to 1 volume of PCR product and mix. If color orange or violet add 10 ul of 3 M Sodium Acetate, pH 5.0. Mixture should turn yellow
      3. place a QIAquick column in provided 2 ml collection tube.
      4. To bind DNA apply sample to QUIquick column (centrifuge at 13000 rpm for 30-60 second). Discard flow through and insert column back in the same tube
      5. Wash - add 750 ul PE Buffer to QIAquick column (centrifuge at 13000 rpm for 30-60 second). Discard Flow through. and insert column back in the same tube.
      6. Centrifuge QIAquick column once more in provided 20 ml collection tube to remove residual wash buffer.
      7. Place each column in clean 1.5 ml microcentrifuge tubes. (let dry for a minute due to ethanol in misture).
      8. Elution - add 50 ul EB buffer or water to the center of the QIAquick membrane and centrifuge for 1 minute.
      9. If analyzing Purified DNA on gel add 1 volume loading dye to 5 volumes of purified DNA.

      QIAquick Gel Extraction Kit

      1. Used for purifying of up to 10 ug of DNA from gel.
      2. Cut the DNA from the agarose gel
      3. Weigh gel in colorless tube. Add 3 volumes QC Buffer per 1 volume gel (gel ~ 100 mg = 10 ul).
      4. Incubate tube 50 - 60 degrees for 10 minutes (or until gel completely dissolved). Vortex every 2 - 3 minutes to break down gel. If color orange or violet add 10 ul of 3 M Sodium Acetate, pH 5.0. Mixture should turn yellow
      5. Place sample in 2 ml collection tube. Centifuge for 1 minute or until all sample passes through column. Discard flow through.
      6. Wash - add 750 ul PE Buffer to QIAquick column (centrifuge at 13000 rpm for 30-60 second). Discard Flow through. and insert column back in the same tube.
      7. Repeat step 5
      8. Place each column in clean 1.5 ml microcentrifuge tubes. (let dry for a minute due to ethanol in misture).
      9. Elution - add 50 ul EB buffer or water to the center of the QIAquick membrane and centrifuge for 1 minute.
      10. If analyzing Purified DNA on gel add 1 volume loading dye to 5 volumes of purified DNA.

      MINIPREP

      1. Pellet 5 mL of culture into each Epindorf tube, 6 tubes total. Add 2 mL at a time, only adding 1 mL in the last addition. Spin down tube in centrifuge after each 2 or 1 mL addition.
      2. Resuspend pellet with 250 microliters of Cell Resuspension Solution. Do not vortex after resuspension.
      3. Add 250 microliters of lysis solution to each sample; invert 4 times to mix.
      4. Add 10 microliters of the protease solution to the sample; invert 4 times to mix. Incubate at room temperature for 5 min.
      5. Add 350 microliters of Neutralization Solution; invert 4 times to mix.
      6. Centrifuge at top speed for 10 minutes at room temperature.
      7. Insert spin column into collection tube.
      8. Decant cleared lysate into Spin Column.
      9. Cetrifuge at top speed for 1 minute at room temperature. Discard flowthrough, and reinsert column into collection tube.
      10. Add 750 microliters of Wash Solution. Centrifuge at top speed for 1 minute. Discard flowthrough and reinsert column into collection tube.
      11. Repeat step 10 with 250 microliters of wash solution.
      12. Centrifuge at top speed for 2 minutes at room temperature. Let sit to dry 4-5 min.
      13. Transfer spin column to a 1.5 mL microcentrifuge tube.
      14. Add 100 microliters of nuclease-free water to the spin column. Centrifuge at top speed for 1 minute at room temperature. Let sit for 1 min to allow for better efficiency of elution.
      15. Discard column and store DNA at -20 deg C. If in multiple tubes, combine liquid from all tubes into one.

      MINIPREP (promega)

      1. Pellet 8 mL of culture into 5 Epindorf tube, using an additional counterbalance tube (6 tubes total). Add 2 mL at a time, then spin at 14000 rpm for 1 minute after each. (Add 2 mL, spin, dump, add 2 more.. etc). After last spin, decant lysate and remove any remaining fluid near the pellet with a micropipette.
      2. Thoroughly resuspend pellet with 250 microliters of Cell Resuspension Solution. Do not vortex after resuspension.
      3. Add 250 microliters of lysis solution to each sample; forcibly invert 4 times to mix.
      4. Add 10 microliters of the protease solution to the sample; forcibly invert 4 times to mix. Incubate at room temperature for 5 min.
      5. Add 350 microliters of Neutralization Solution; forcibly invert 4 times to mix.
      6. Centrifuge at top speed (1400 rpm) for 10 minutes at room temperature. Binding of Plasmid DNA
      7. Insert spin column into collection tube.
      8. Decant cleared lysate into Spin Column.
      9. Centrifuge at top speed for 1 minute at room temperature. Discard flowthrough, and reinsert column into collection tube.
        10. Washing
      10. Add 750 microliters of Wash Solution. Centrifuge at top speed for 1 minute. Discard flowthrough and reinsert column into collection tube.
      11. Repeat step 10 with 250 microliters of wash solution.
      12. Centrifuge at top speed for 2 minutes at room temperature. Let sit to dry 4-5 min.
      13. Transfer spin column to a 1.5 mL microcentrifuge tube.
      14. Let column sit 3-5 minutes to allow ethanol to evaporate out.
      15. Add 100 microliters of nuclease-free water to the spin column. Centrifuge at top speed for 1 minute at room temperature. Let sit for 1 min to allow for better efficiency of elution. -Discard columns (keep flowthrough) and digest samples with Eco RI.