Protocols

P. denitrificans Protocols

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

Thaw and mix 50 uL portions cells with 5 uL DNA
Transfer to ice cold electroporation cuvette
Electroporate with single pulse set at 25 uF and 2.0 kV for 4.5 to 5 ms
The electroporator as a Gene-Pulser by BioRad connected to a 200-(-) resistor also by biorad
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
Incubate cells at 30 C for 2 hours in a shacking incubator.
Take 100 uL portions and plate.

P. denitrificans Nutrient Agar

Combine 23g 3 Nutrient Agar/Broth Agar Medium Nutrient Agar with 1000 mL deionized water, then autoclave

P. denitrificans Nutrient Broth

Combine 8g Medium Nutrient Broth with 1000 mL deionized water, pH to 6.8 +/- 0.2, then autoclave

Isolate Single colonies

Select glycerol stock cultures from -80 C storage to begin
Thaw on ice for 15 minutes and observe when the cell stock begins to melt
Take a plastic p-loop or other spreading instrument and spread on a selective media agar plate containing the appropriate antibiotics
Incubate both P. denitrificans and E. coli overnight at 37 C
Isolate single colonies after overnight incubation and resuspend in 60 mL volumes of liquid selective LB media with appropriate antibiotics

Preculture for Device Testing

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
Obtain a 1 mL sample and measure the OD600. If at a satisfactory level, then proceed with device testing
Divide the 60 mL pre-cultures into 3.5 mL aliquots in 14 mL falcon RB tubes or comparable sterile culture tube
Start incubation in a shaking incubator at 37 C and 220 rpm

Device Measurements

Starting with the unsealed tube, use a vernier Dissolved Oxygen (DO) membrane probe or comparable measurement system to measure the do levels
Immediately take three 150 uL samples of the culture and dispense into appropriate wells in an optical PCR well plate
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.
Once all tubes have been unsealed and measured for DO content, seal the PCR optical well plate with an optical grade adhesive cover
After all samples for optical fluorescence measurement have been taken, remove cuvettes from storage to measure and record OD600 absorbance values

PCR Fluorescence Measurement

Immediately after all samples have been collected and the well plate covered, run a pre-created protocol for fluorescence measurement
The protocol should have three measurement cycles set at 30 seconds and 37 C
After measurements are made by the optical reader, export the data for analysis

Disposal

Disinfect all Cuvettes with bleach or ethanol before washing with tap/sink water. Then rinse with deionized water and wipe dry
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
All PCR well plates should be sterilized disposes of similarly

Minimal Media Preparation

Experimental Procedure

Prepare 1L minimal media
Dissolve 6.68g NH4Cl in 50 mL deionized water (spike solution)
Preculture experimental bacteria overnight in minimal medium at 30oC in an erlenmeyer flask
Culture experimental bacteria overnight in minimal media
Remove three aliquots of 294 mL when culture reaches OD=0.3 suspension. Use three separate flasks labeled Flask 1, 2, and 3
Put the experimental flasks on hot plates with magnetic stirrer and stabilize the temperature at 30oC; stirrers function as aerators
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)
Use 1mL of each sample to measure the OD
Get initial thermometer readings; adjust plates as necessary
After the min 6 sample, quickly spike with 6 mL of spike solution to create 50mM ammonium cultures (culture volume is now 300ml)
At T=6min measure [ammonium], [nitrate], [DO] after spike solution added
Continue to rotate probes between flasks 1, 2, and 3 at two minute intervals for 90 minutes
Measure OD at 30 minute intervals at times t=0, t=30, t=60, t=90

Step 1

Step 2

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

Step 3

Growing N. europaea

Add 1 x 100 ml aliquot of solution prepared in Step 2 to the flask prepared in Step 1
Now add 8 ml of the solution prepared in Step 3 to the flask prepared in Step 1
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
The bacterium should reach approximately OD600nm~0.1 in three to four days