Team:Virginia/Experiments



Protocols


Pc. denitrificans Protocols


  1. Grow Pc. denitrificans cultures in liquid media
  2. Dilute 500mL of Pc. denitrificans culture in 500mL SGM17 medium
  3. ***Cold Room from here on***
  4. Harvest by centrifuge 250mL samples each in four centrifuge bottles at 5000xg at 4 C. carefully dump out supernatant
  5. 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
  6. Combine the liquid(containing resuspended pellets) into one centrifuge bottle, add appropriate amount of water to the other empty centrifuge bottle for counterbalance
  7. Centrifuge the two bottles at 5000xg at 4 C
  8. 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
  9. Centrifuge the two bottles at 5000xg at 4 C
  10. Dump out supernatant and resuspend the resulting pellets in 10mL of 0.5 M sucrose solution with 10% glycerol
  11. Aliquot the final 10mL of liquid into 0.25mL(250uL) with 40 microcentrifuge tubes
  12. Flash freeze 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: carefully mix suspension with 0.95 mL ice cold SGM17 containing 20 mM MgCl2, 2 mM CaCl2 and incubate on ice for 5 minutes
  6. Dilutions were made in the SGM17 media
  7. Incubate cells at 30 C for 2 hours
  8. Take 100 uL portions and plate.


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


    Pc. denitrificans Nutrient Broth
  1. Combine 8g Medium Nutrient Broth with 1000 mL deionized water
      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 Pc. 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 Pc. 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

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