Add dH20 to autoclavable bottle with a magnetic stir bar (fill to 1000 mL) and separate into two 1 L glass bottles to prevent overflow in the autoclave process.✓
Mix the solution on a magnetic stir plate until consistent throughout.✓
Ensure the caps of the bottles are loosened to allow steam to enter. Autoclave at a liquid cycle for 30-45 minutes. Tighten the caps after the media has cooled to prevent contamination.✓
Materials
LB powder (Various, currently Miller Acumedic)
Bacto Agar (BD)
dH20
Antibiotics
Petridishes
Media Preperation
Add dH20 to autoclavable bottle (500mL in 1L bottle)✓
Add LB powder (12.5g for 500mL)✓
Add agar (7.5 for 500mL)✓
Mix using magnetic stir bar or shaking✓
Autoclave for 30 minutes on liquid cycle✓
Plate Preperation
Ensure media is cooled to 50-60°C✓
Add antibiotics to final concentratation✓
Mix using stir bar or shaking✓
Pour plates in biosafety hood, ~20mL in each✓
Let cool with lid askew✓
When cool stack with top down and slide back in petri dish bag✓
Tape and Label✓
Materials
0.225 g Potassium Phosphate Dibasic K2HPO4
Magnetic stir bar
0.225 g Potassium Phosphate Monobasic KH2PO4
0.46 g Sodium Chloride NaCl, 0.225 g Ammonium Sulfate NH4SO4
Add the chemical compounds to 880 mL of dH2O and separate into two 1 L autoclavable bottles with magnetic stir bars.✓
Stir the solution on a magnetic stir plate and adjust the pH to 7.2 using 5 M NaOH.✓
Ensure the caps of the bottles are loosened to allow steam to enter. Autoclave at a liquid cycle for 30-45 minutes. Tighten the caps after the media has cooled to prevent contamination.✓
Add 1 x Wolfe’s minerals and 1 x Wolfe’s vitamins (no riboflavin).✓
Colony PCR
This procedure is to run colony PCR on a sample plate.
Materials
2x Master Mix (Promega GoTaq)
Forward Primers
Reverse Primers
Nuclease Free H2O
LB media
Thermocycler
PCR Tubes
Colonies of Interest
Procedure
Select colonies of interest and mark them
Add 50 uL of nuclease-free water to a PCR tube for each colony selected
Touch the colony using a pipette tip and place into water in PCR tube
Prepare the master PCR mix for the samples, control, and a blank. Do not put Template DNA in master mix.
Reagent
Volume
2x Master Mix
5 uL
Forward Primer
0.5 uL (10 uM)
Reverse Primer
0.5 uL (10 uM)
Nuclease-free water
3 uL
Total Master Mix Volume
9 uL
Template (sample DNA)
1 uL
Total Volume
10 uL
Add 9 uL of master mix to clean PCR tubes
Pipette 1 uL of template DNA into each PCR tube and mix gently
Start appropriate thermocycler run
Testing the Strains
IPTG Induction Using the 96 Well Plate
This procedure is to test the fluorescence of modified Shewanella oneidensis MR-1.
Program software (Bio-Tek, Gen5 3.02) to measure OD 600 and Fluorescence
simultaneously in plate reader (Bio-Tek, Synergy HTX Multi-Mode Reader).
Program following conditions: Set plate type to Greiner 96 Black Flat Bottom Fluotrac
and check the use lid box. Run endpoint with orbital shaking (Continuous, Orbital
Frequency= 307 cpm, fast orbital speed) and reading OD 600 (Full plate, normal read
speed, no pathlength correction) and reading GFP (Excitation= 485/20, Emission:
528/20, Optics position= top, Gain= 75, normal read speed, Read Height= 6.00mm).
Run plate with these endpoint settings.
Growth Fluorescence
This procedure is to test the fluorescence of modified Shewanella oneidensis MR-1.
Grow overnight cultures of ∆mtrB, ∆mtrB prL814 and ∆mtrB prL814-mtrB in 5mL LB and
add 5 μL of 1000X Spectinomycin to ∆mtrB prL814 and ∆mtrB prL814-mtrB.
Incubate at 30ºC and shaking at 275 rpm.
Spin down the overnight cultures at 10,000X g for 5 minutes and then discard the
supernatant.
Re-suspend the cells in 1mL of M5 media with 1X Wolfe’s vitamins and minerals.
Check OD 600 on spectrophotometer (Eppendorf, Part No: 2231000288-P17).
Note: Blank spectrophotometer with M5 media.
Dilute the original culture re-suspension to an OD 600 of 0.1 using M5 media.
Note: Use C 1 V 1 =C 2 V 2 to end up with a total OD 600 of 0.1 in a total volume of 5mL for each strain
to have enough diluted culture to add to 96 well plate. Add 5μL of Spectinomycin to ∆mtrB
prL814 and ∆mtrB prL814-mtrB
Pipet 100 μL of each strain and background M5 into 96 well plate (VWR, Part No: 82050-
052) such that 4 technical replicates of each strain and background M5 are obtained for
the following concentrations of IPTG: 0 μM, 50 μM, 100 μM, 150 μM, 200 μM and 250
μM.
Note: 250 μM determined to be beginning of saturation based on preliminary experiments.
Parafilm the plate to avoid condensation buildup.
Running the plate on a reader
Program software (Bio-Tek, Gen5 3.02) to measure OD 600 and Fluorescence simultaneously in plate reader (Bio-Tek, Synergy HTX Multi-Mode Reader).
Program following conditions: Set plate type to Greiner 96 Black Flat Bottom Fluotrac
and check the use lid box. Set temperature to 30ºC and start kinetic (Run= 3:00:00,
Interval= 0:15:00) with orbital shaking (Continuous, Orbital Frequency= 307 cpm, fast
orbital speed) and reading OD 600 (Full plate, normal read speed, no pathlength
correction) and reading GFP (Excitation= 485/20, Emission: 528/20, Optics position= top,
Gain= 75, normal read speed, Read Height= 6.00mm). End this kinetic and insert a
stop/resume pause step. Start a new kinetic (Run= 48:10:00, Interval= 0:15:00) with
orbital shaking and reading OD 600 along with GFP under the same conditions as first
kinetic. End this kinetic step.
Note: Save this experiment as these conditions will be used throughout plate reader
experiments.
Run the plate and once the pause step has been reached, remove the plate and induce
with varying amounts of IPTG.
Resume running the next kinetic after the plate has been induced and run till
completion.
This procedure is to test the current output of modified Shewanella oneidensis MR-1.
Assembling Carbon Electrodes
Cut out a 25 x 25 mm piece of carbon felt (Alfa Aesar, Part No: 43200) with a razor
blade.
Insert 15 cm long titanium wire (Malin Co., .025 Titanium) horizontally through the
felt, and bend in a loop back into the bottom of the electrode.
Apply carbon cement (Fluka Analytical, Part No: 09929-30G) to the wire, where it
contacts the felt. Add Xylene (Jade Scientific, Part No: JS-X0250) and stir to ensure
carbon cement remains in a liquid state.
Allow to dry for at least five hours under a fume hood.
Deoxidize by submerging in a container of dH 2 O and tap against a solid surface to
remove trapped oxygen in the carbon felt. Further remove oxygen by purging the
gas via pressurizing anaerobic chamber to 20psi then depressurizing (2X).
Stoppers
Drill a hole (0.5 in. (1.27 cm) diameter) through the rubber tapered round plug
(McMaster-Carr, trade size: #12) for the reference electrode.
Drill a hole (0.5 in. (1.27 cm) diameter) halfway through for the counter electrode
housing.
Note: Make sure to apply soap/water solution to stopper and inside drill bit to prevent clogging.
Remove the plunger and cut a 3 mL plastic syringe (BD, Part No: 309657) to size for
the counter electrode housing. Add punctures throughout to allow release of
hydrogen gas. Insert securely in the hole drilled halfway through the stopper.
Note: Cut off needle side of syringe housing to allow prevent air pocket formation.
String titanium wire through the plastic housing for the counter electrode.
Note: The end of the titanium wire should be in the middle of the plastic housing.
Insert a sterile 18 gauge needle (BD, Part No: 305196) through the rubber stopper
and into the housing to allow for ventilation of hydrogen gas. Bend to the side and
cover with foil.
Insert second sterile 18 gauge needle through stopper away from electrode
connections for ventilation during autoclaving. Bend to side and cover with
aluminum foil.
String the wire of the carbon felt electrode through the stopper.
Final assembly before autoclaving
Fill each Mason jar (Ball, Part No: 750097) with 125 mL of M5 media. Add stir bar
(McMaster-Carr, Part No: 5678K111).
Place assembled stopper into the jar. Ensure the electrode is completely submerged
in media.
Push a long sterile stainless steel needle (Part No: 5FTZ1) through the stopper for
sampling and ensure it reaches down into the solution. Screw on a male luer cap
(McMaster-Carr, Part No: 51525K371)
Insert a sterile 18 gauge needle that does not interfere with the other hardware for
venting. Cover with foil. This needle will be used to inject various additives.
Note: [Place Figure 9 here]
Autoclave bioreactors in a liquid cycle for 35 minutes. Allow to cool before adding
vitamin/mineral solution and antibiotic.
Use the inserted 18 gauge needle to add up to total volume of 140 mL: 1.25 mL
Wolfe's minerals, 1.25 mL of Wolfe’s vitamins (no riboflavin) and 12.5 mL of 200 mM
lactate (carbon source).
Add 140 µL of Spectinomycin antibiotic to reactors with antibiotic resistant strains.
Note: Antibiotics are prepared at 50mg/mL concentrations.
Oxidation the silver electrode
Cut a 6 cm long piece of silver wire (Sigma Aldrich, Part No: 265608) and insert
through a rubber tapered round plug (McMaster-Carr, trade size: #C).
Add 1 g of KCl Crystal (J.T. Baker, Part No: 3040-05) to 50mL of dH 2 O in a small
beaker.
Insert a carbon rod (Electron Microscopy Sciences, Part No: 70230), Ag/AgCl
reference electrode (BASi, Part No: MF- 2052) and silver electrode into dilute KCl
solution.
Connect white wire (REF 2) from potentiostat (Bio-Logic Science Instruments, Part
No: VMP3) to Ag/AgCl reference electrode, connect blue wire (REF 3) to a carbon
rod and connect red wire (REF 1) to silver electrode.
Note: Connections should not touch inside beaker and alligator clips from potentiostat wires
should not contact the solution.
Program EC-Lab software to the following settings: Set electrical potential (E we ) to -
0.200V (E i ) vs Ref. (Ag-AgCl), hold for 0.0 min (E i for t i ) and record every 1.00 sec (dt i ).
Scan electrical potential (E we ) over time (dE/dt= 20.0mV/s) to vertex potential (E 1 =
0.400V vs Ref) then hold for 10.0 min (E 1 for t 1 ) and record every 1.00 sec (dt 1 ).
Reverse scan electrical potential (E we ) to vertex potential (E 2 = 0.300V vs Ref) then
hold for 0.0 min (E 2 for t 2 ) and record every 0.10 sec (dt 2 ). Measure current (<I>) over
the last 50% of the step duration, record current (<I>) averaged over one voltage
steps (N= 1), repeat cycle zero times (n c = 0) and record the first and every cycle for 2
cycles (n r = 2). Set sensitivity to specified settings (E Range= -10.0V; 10.0V). Leave
current and bandwidth at default settings (I Range=Auto, Bandwidth= 5-medium).
Note: Save these settings as they will be used for every experiment.
Run these preprogrammed settings.
Note: Wire should change to a brown/purple color when reduction is complete (~ 10 min.).
Note: Placing the silver electrodes in concentrated household bleach for 30 min. also oxidizes
them
To prepare the reference electrode
Combine 50 mL of dH 2 O and 0.75 g Bacto Agar (BD, Part No: 214010) in a 100 mL
glass beaker, containing a small stir bar (McMaster-Carr, Part No: 5678K111).
On a heat/stir plate, stir (650 rpm) and heat (~300 ◦ C) until Agar dissolves and
solution appears clear
Note: May have a slightly yellow tint.
Add KCl Crystal until saturated. Look for particles to start settling at the bottom (~ 23
g).
Pour the hot solution into a glass cylinder reference electrode (Custom made,
dimensions: [Place Figure 10 here]), stoppered by 60 mm long Magnesia stick (Sigma
Aldrich, Part No: 31408)) , filling it halfway. Insert a syringe with stainless steel
needle (see 1.19) into the bottom and draw up any air before filling the cylinder.
Insert the reduced silver wire that is connected into the rubber stopper and create a
secure seal.
Wipe off any excess salt around the reference with water and Kim wipes.
Programming potentiostat:
Connect potentiostat to EC-Lab software.
Program EC-Lab to the following settings: Apply 0.200V (E i ) vs Ref. (Ag-AgCl) for the
amount of run time desired (t i ). Leave limits at default settings (I max , I min = “pass” mA,
(|ΔQ|> ΔQ M )= 0.0 mAh). Record data every second (dt a =1.00 s). Leave sensitivity at
default settings (E Range= -2.5V; 2.5V). Leave current and bandwidth at default
settings (I Range=Auto, Bandwidth= 5-medium).
Note: Save these setting because they will be used for every reactor over all experiments.
Running the bioreactors
Place bioreactors on a low profile magnetic stirrer (VWR, Part No: 10153-690),
careful to position stir bar to avoid damaging hardware.
Connect to potentiostat by attaching white wire (REF 2) to reference electrode,
attach blue wire (REF 3) to counter electrode and attach red wire (REF 1) to carbon
felt electrode.
Inspect the units for anything out of place or for bubbles in the counter electrode
housing. This will cause errors in potentiostat readings.
Turn on the stir plate and ensure the stir bars are stable. Run the reactors without
bacteria for at least 2 hours to obtain baseline values.
Inoculate with bacteria, dilute to an OD 600 of 0.01 () in a final volume of 140 mL.
Note: C i , V i refer to initial concentration and initial volume, respectively. C f , V f refer to final
concentration and final volume respectively.
Bacterial strains grown in the reactors include: ΔmtrB (negative control), ΔmtrB
prL814 (positive control) and ΔmtrB prL814-mtrB.
Allow growth in the bioreactor overnight. Use a syringe to remove 2 mL for sampling
to measure OD 600 and GFP fluorescence.
Induce with 200 µM concentration of 0.1M IPTG once current begins to decrease.
Note: The 200 µM was determined to be optimal concentration based on previous preliminary
experiments.
Disposal
Disconnect all leads from potentiostat.
Soak all parts in a diluted bleach solution for 30 minutes, wash with soap, and rinse
with dH2O.
Note: When removing glass reference housing from stopper use soap to avoid breaking
housing.
Dispose of used carbon felt electrodes as biohazardous waste.
Allow all parts to dry for a minimum of 5 hours in a drying cabinet.
Building Measurement Devices
Purpose:
To create a large-scale liquid biosensor that uses a single chamber to conduct current when inoculated with modified strains of Shewanella Oneidensis and induced by IPTG
Materials:
For each bioreactor:
250 mL mason jars
Rubber stoppers (2.5 cm tapering to 2 1/8 cm)
Titanium wire (~15cm per unit)
Carbon felt
Glass reference housing
Oxidized nickel wire + Small ~3 mm rubber stoppers
Large metal needles for sampling
3 mL plastic syringe (to be cut to act as housing for a counter-electrode)
Create ultra-low cost MFCs to be innoculated with Shewanella Oneidensis MR-1 and then induced with a selected compound.
Materials:
Whatman Paper
Razor
Scissors
Aluminum foil
8B graphite pencils
Carbon cement
Crayon
Superglue (cyanoacrylate)
Parchment paper
Procedure:
Cut out 30mm x 30mm squares of whatman paper (6 per reactor) and one piece of parchment paper of the same size.✓
Colour the sides of five pieces of whatman paper (5mm in from edges) with crayon (both sides). One piece can be set aside, this will be the blank.
✓
Fully Colour one piece of whatman paper. This will be the cover.
✓
Draw on the center of two pieces of paper with 8B graphite pencil for at least five repetitions. These will be the anode and cathode.
✓
Cut out 2cm long 1cm wide strips of aluminum foil.
Lightly superglue aluminum foil to wax part of both anode and cathode. Allow foil to extend 1cm onto anode/cathode.
✓
Paint cement glue onto the anode and cathode. Ensure proper ventilation and safety. Allow to dry a minimum of five hours.
✓
Cut the center out of two pieces of coloured whatman paper. These will be the chamber pieces.
✓
Superglue the whatman paper together with small dabs of glue on only the corners of the paper in the order of cover, chamber, chamber, cathode (with foil facing up), blank, parchment paper, anode (with foil facing down).✓
