Difference between revisions of "Team:Virginia/Experiments"

 
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<li>Grow P. denitrificans cultures in liquid media<br></li>
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<li>Culture P. denitrificans in SGM17 medium until OD600=0.6-0.8.<br></li>
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<li>Dilute 500mL of P. denitrificans culture in 500mL SGM17 medium<br></li>
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<li>***The following procedure should be done in a cold room at 0-4˚C.***</li>
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<li>***Cold Room from here on***</li>
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<li>Harvest by centrifuge 250mL samples each in four centrifuge bottles at 5000xg at 4 C. Decant supernatant<br></li>
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<li>Harvest by centrifuge 250mL samples each in four centrifuge bottles at 5000xg at 4 C. carefully dump out supernatant<br></li>
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<li>Completely resuspend the pellets in 50mL of 0.5 M sucrose solution with 10% glycerol, for each bottle<br></li>
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<li>Completely resuspend the pellets(shake side to side to avoid liquid loss) in all centrifuge bottles each with 50mL of 0.5 M sucrose solution with 10% glycerol<br></li>
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<li>Combine the resuspended cells into one centrifuge bottle,ncounterbalance with water in another centrifuge bottle<br></li>
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<li>Combine the liquid(containing resuspended pellets) into one centrifuge bottle, add appropriate amount of water to the other empty centrifuge bottle for counterbalance<br></li>
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<li>Centrifuge at 5000xg at 4 C<br></li>
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<li>Centrifuge the two bottles at 5000xg at 4 C<br></li>
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<li>Decant supernatant and resuspend the pellets with 100mL of 0.5 M sucrose solution with 10% glycerol. Counter-balance bottle should have 100mL of water</li>
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<li>Dump out supernatant and resuspend the pellets with 100mL of 0.5 M sucrose solution with 10% glycerol. Counter-balance bottle should have 100mL of water</li>
+
 
<li>Centrifuge the two bottles at 5000xg at 4 C</li>
 
<li>Centrifuge the two bottles at 5000xg at 4 C</li>
−
<li>Dump out supernatant and resuspend the resulting pellets in 10mL of 0.5 M sucrose solution with 10% glycerol</li>
+
<li>Decant out supernatant and resuspend the resulting pellets in 10mL of 0.5 M sucrose solution with 10% glycerol</li>
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<li>Aliquot the final 10mL of liquid into 0.25mL(250uL) with 40 microcentrifuge tubes</li>
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<li>Aliquot the cells into microcentrifuge tubes</li>
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<li>Flash freeze with liquid nitrogen and store at -80 C </li>
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<li>Flash freeze the aliquoted cells with liquid nitrogen and store at -80 C </li>
  
 
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<li>Electroporate with single pulse set at 25 uF and 2.0 kV for 4.5 to 5 ms  
 
<li>Electroporate with single pulse set at 25 uF and 2.0 kV for 4.5 to 5 ms  
 
<li>The electroporator as a Gene-Pulser by BioRad connected to a 200-(-) resistor also by biorad  
 
<li>The electroporator as a Gene-Pulser by BioRad connected to a 200-(-) resistor also by biorad  
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<li>IMMEDIATELY following electroporation: carefully mix suspension with 0.95 mL ice cold SGM17 containing 20 mM MgCl2, 2 mM CaCl2 and incubate on ice for 5 minutes  
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<li>IMMEDIATELY following electroporation: GENTLY resuspend the cells with 0.95 mL ice cold SGM17 containing 20 mM MgCl2, 2 mM CaCl2 and incubate on ice for 5 minutes  
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<li>Dilutions were made in the SGM17 media
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<li>Incubate cells at 30 C for 2 hours in a shacking incubator.
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<li>Incubate cells at 30 C for 2 hours  
+
 
<li>Take 100 uL portions and plate.
 
<li>Take 100 uL portions and plate.
 
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Latest revision as of 23:49, 1 December 2017



Protocols


P. denitrificans Protocols


  1. Culture P. denitrificans in SGM17 medium until OD600=0.6-0.8.
  2. ***The following procedure should be done in a cold room at 0-4˚C.***
  3. Harvest by centrifuge 250mL samples each in four centrifuge bottles at 5000xg at 4 C. Decant supernatant
  4. Completely resuspend the pellets in 50mL of 0.5 M sucrose solution with 10% glycerol, for each bottle
  5. Combine the resuspended cells into one centrifuge bottle,ncounterbalance with water in another centrifuge bottle
  6. Centrifuge at 5000xg at 4 C
  7. Decant supernatant and resuspend the pellets with 100mL of 0.5 M sucrose solution with 10% glycerol. Counter-balance bottle should have 100mL of water
  8. Centrifuge the two bottles at 5000xg at 4 C
  9. Decant out supernatant and resuspend the resulting pellets in 10mL of 0.5 M sucrose solution with 10% glycerol
  10. Aliquot the cells into microcentrifuge tubes
  11. Flash freeze the aliquoted cells with liquid nitrogen and store at -80 C


  1. Thaw and mix 50 uL portions cells with 5 uL DNA
  2. Transfer to ice cold electroporation cuvette
  3. Electroporate with single pulse set at 25 uF and 2.0 kV for 4.5 to 5 ms
  4. The electroporator as a Gene-Pulser by BioRad connected to a 200-(-) resistor also by biorad
  5. IMMEDIATELY following electroporation: GENTLY resuspend the cells with 0.95 mL ice cold SGM17 containing 20 mM MgCl2, 2 mM CaCl2 and incubate on ice for 5 minutes
  6. Incubate cells at 30 C for 2 hours in a shacking incubator.
  7. Take 100 uL portions and plate.


    P. denitrificans Nutrient Agar
  1. Combine 23g 3 Nutrient Agar/Broth Agar Medium Nutrient Agar with 1000 mL deionized water, then autoclave
      3g Nutrient Agar Composition Beef Extract, 5g peptone, 15g agar


    P. denitrificans Nutrient Broth
  1. Combine 8g Medium Nutrient Broth with 1000 mL deionized water, pH to 6.8 +/- 0.2, then autoclave
      3g Nutrient Agar Composition Beef Extract, 5g peptone

Measurement Protocols


    Isolate Single colonies
  1. Select glycerol stock cultures from -80 C storage to begin
  2. Thaw on ice for 15 minutes and observe when the cell stock begins to melt
  3. Take a plastic p-loop or other spreading instrument and spread on a selective media agar plate containing the appropriate antibiotics
      If the culture is P. denitrificans, culture on PD media or standard LB
      If the culture is E. coli use LB media
      If using untransformed bacteria, equivalent non-selective media should be used
  4. Incubate both P. denitrificans and E. coli overnight at 37 C
  5. Isolate single colonies after overnight incubation and resuspend in 60 mL volumes of liquid selective LB media with appropriate antibiotics


    Preculture for Device Testing
  1. Allow the resuspended liquid culture to incubate for 4 to 6 hours in a shaking incubator at 37 C and 220 rpm to reach late- to mid-exponential phase of growth, look for an OD 600 value of ~0.6
  2. Obtain a 1 mL sample and measure the OD600. If at a satisfactory level, then proceed with device testing
  3. Divide the 60 mL pre-cultures into 3.5 mL aliquots in 14 mL falcon RB tubes or comparable sterile culture tube
      Prepare one tube for each hour that the planned test calls for, mark each tube with the number of hours it will remain unsealed and permeable to oxygen
      At the start of testing, cover the tubes marked “0 hours” with parafilm and leave sealed for the duration of testing
  4. Start incubation in a shaking incubator at 37 C and 220 rpm
      Every hour stop the shaking incubator and cover the next tube with parafilm. Repeat this step until all tubes except the last ones are covered
      Allow the final set of tubes to incubate unsealed for an hour and stop the incubation


    Device Measurements
  1. Starting with the unsealed tube, use a vernier Dissolved Oxygen (DO) membrane probe or comparable measurement system to measure the do levels
      Do this for all samples that were left open for a given time interval before moving to the next
      In between DO measurements, clean the probe with ethanol and then rinse with deionized water to minimize cross-contamination between samples
  2. Immediately take three 150 uL samples of the culture and dispense into appropriate wells in an optical PCR well plate
  3. Take a 500 uL sample and dilute with 500 uL deionized water for a total volume of 1 mL in a spectrophotometer cuvette to make a final 1:2 dilution. Store on ice if possible.
  4. Once all tubes have been unsealed and measured for DO content, seal the PCR optical well plate with an optical grade adhesive cover
  5. After all samples for optical fluorescence measurement have been taken, remove cuvettes from storage to measure and record OD600 absorbance values


    PCR Fluorescence Measurement
  1. Immediately after all samples have been collected and the well plate covered, run a pre-created protocol for fluorescence measurement
  2. The protocol should have three measurement cycles set at 30 seconds and 37 C
  3. After measurements are made by the optical reader, export the data for analysis
      f the Bio Rad MyiQ Optical Reader is used, copy and paste the relative fluorescence unit table presented by the output and export to either a .txt or .excel format


    Disposal
  1. Disinfect all Cuvettes with bleach or ethanol before washing with tap/sink water. Then rinse with deionized water and wipe dry
  2. All remaining tubes should be sterilized with bleach or ethanol and drained down an appropriate lab sink. All tubes are to be disposed of in a lab waste receptacle
  3. All PCR well plates should be sterilized disposes of similarly

    Minimal Media Preparation
  1. Combine:
      1L deionized water
      0.2g MgSO4
      0.8g NH4Cl
      1.0g NH4SO4
      0.5g NaCl
      5.2g K2HPO4
      2.0g KH2PO4
  2. Autoclave solution
  3. Add 5.0g glucose


    Experimental Procedure
  1. Prepare 1L minimal media
  2. Dissolve 6.68g NH4Cl in 50 mL deionized water (spike solution)
  3. Preculture experimental bacteria overnight in minimal medium at 30oC in an erlenmeyer flask
  4. Culture experimental bacteria overnight in minimal media
      When conducting experiment with pBAD promoter, add 0.15g arabinose 3-4 hours before experimentation begins
  5. Remove three aliquots of 294 mL when culture reaches OD=0.3 suspension. Use three separate flasks labeled Flask 1, 2, and 3
  6. Put the experimental flasks on hot plates with magnetic stirrer and stabilize the temperature at 30oC; stirrers function as aerators
  7. Measure initial [ammonium], [nitrate], [DO] concentrations in three beakers using ammonium, nitrate, and dissolved oxygen probe at T=0 (THIS WILL REQUIRE A FULL ROTATION OR 6 MINUTES)
  8. Use 1mL of each sample to measure the OD
  9. Get initial thermometer readings; adjust plates as necessary
  10. After the min 6 sample, quickly spike with 6 mL of spike solution to create 50mM ammonium cultures (culture volume is now 300ml)
  11. At T=6min measure [ammonium], [nitrate], [DO] after spike solution added
  12. Continue to rotate probes between flasks 1, 2, and 3 at two minute intervals for 90 minutes
  13. Measure OD at 30 minute intervals at times t=0, t=30, t=60, t=90

N. europaea Protocols

    Step 1
  1. Put 900 ml of deionized water in a 2-liter Erlenmeyer flask
  2. Add 3.3 g (NH4)2SO4 (50mM)
  3. Add 0.41 g KH2PO4
  4. Add 0.75 ml 1 M MgSO4 stock solution
  5. Add 0.2 ml 1 M CaCl2 stock solution
  6. Add 0.33 ml 30 mM FeSO4 /50 mM EDTA stock solution
  7. Add 0.01 ml 50mM CuSO4 stock solution
  8. Autoclave solution


    Step 2
  1. Put 400 ml of deionized water in a beaker. Add: 27.22 g KH2PO4, 2.4 g NaH2PO4
  2. Adjust pH to 8.0 with 10 N NaOH, and bring it to a final volume of 500 ml with deionized water
  3. Autoclave in 100 ml fractions in 250-500 ml Erlenmeyer flasks


    Step 3
  1. Prepare 500 ml of 5% (w/v) Na2CO3 (anhydrous)
  2. Sterilize it in an autoclave


    Growing N. europaea
  1. Add 1 x 100 ml aliquot of solution prepared in Step 2 to the flask prepared in Step 1
  2. Now add 8 ml of the solution prepared in Step 3 to the flask prepared in Step 1
  3. Finally add 10 ml of 3-day old culture to the flask prepared in Step 1, and incubate it on a rotary shaker (100-150 RPM) at 30°C in darkness
  4. The bacterium should reach approximately OD600nm~0.1 in three to four days