Difference between revisions of "Team:MSU-Michigan/Experiments"

 
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                     <button onclick="myFunction('openclose7')" class="w3-btn w3-block w3-black w3-left-align">LB Agar Plates</button>
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                     <button onclick="myFunction('openclose7')" class="w3-btn w3-block w3-flat-green-sea w3-left-align">LB Agar Plates</button>
 
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                         <h5>Materials</h5>
 
                         <h5>Materials</h5>
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                     <button onclick="myFunction('openclose9')" class="w3-btn w3-block w3-black w3-left-align">Colony PCR</button>
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                     <button onclick="myFunction('openclose9')" class="w3-btn w3-block w3-flat-green-sea w3-left-align">Colony PCR</button>
 
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                         <h4>Colony  PCR</h4>
 
                         <h4>Colony  PCR</h4>
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   <td>Nuclease-free water</td>
 
   <td>Nuclease-free water</td>
 
   <td>3 uL</td>
 
   <td>3 uL</td>
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</tr>
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<tr>
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  <td>Total Master Mix Volume</td>
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  <td>9 uL</td>
 
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                     <button onclick="myFunction('openclose')" class="w3-btn w3-block w3-black w3-left-align">End Point Fluorescence</button>
 
                     <button onclick="myFunction('openclose')" class="w3-btn w3-block w3-black w3-left-align">End Point Fluorescence</button>
 
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                         <h4>Title</h4>
+
                         <h4>IPTG Induction Using the 96 Well Plate</h4>
 
                         <p>This procedure is to test the fluorescence of modified <em>Shewanella oneidensis MR-1</em>.</p>
 
                         <p>This procedure is to test the fluorescence of modified <em>Shewanella oneidensis MR-1</em>.</p>
 +
<div class="w3-ul">
 +
<li>Program software (Bio-Tek, Gen5 3.02) to measure OD 600 and Fluorescence
 +
simultaneously in plate reader (Bio-Tek, Synergy HTX Multi-Mode Reader).
 +
<li>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).
 +
<li>Run plate with these endpoint settings.
 +
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                     </div>
  
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                     <button onclick="myFunction('openclose2')" class="w3-btn w3-block w3-black w3-left-align">Growth Fluorescence</button>
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                     <button onclick="myFunction('openclose2')" class="w3-btn w3-block w3-flat-green-sea w3-left-align">Growth Fluorescence</button>
 
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                         <h4>Title</h4>
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                         <h4>Growth Fluorescence</h4>
 
                         <p>This procedure is to test the fluorescence of modified <em>Shewanella oneidensis MR-1</em>.</p>
 
                         <p>This procedure is to test the fluorescence of modified <em>Shewanella oneidensis MR-1</em>.</p>
 +
<div class="w3-ul">
 +
<li>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.
 +
<li> Incubate at 30ºC and shaking at 275 rpm.
 +
<li>Spin down the overnight cultures at 10,000X g for 5 minutes and then discard the
 +
supernatant.
 +
<li> Re-suspend the cells in 1mL of M5 media with 1X Wolfe’s vitamins and minerals.
 +
<li> Check OD 600 on spectrophotometer (Eppendorf, Part No: 2231000288-P17).
 +
Note: Blank spectrophotometer with M5 media.
 +
<li> 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
 +
<li>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.
 +
<li>Parafilm the plate to avoid condensation buildup.
 +
<h4>Running the plate on a reader</h4>
 +
<li>Program software (Bio-Tek, Gen5 3.02) to measure OD 600 and Fluorescence simultaneously in plate reader (Bio-Tek, Synergy HTX Multi-Mode Reader).
 +
<li>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.
 +
<li> Run the plate and once the pause step has been reached, remove the plate and induce
 +
with varying amounts of IPTG.
 +
<li> Resume running the next kinetic after the plate has been induced and run till
 +
completion.
 +
 +
</div>
 
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                     <button onclick="myFunction('openclose3')" class="w3-btn w3-block w3-black w3-left-align">Bioreactors Current Measurement</button>
 
                     <button onclick="myFunction('openclose3')" class="w3-btn w3-block w3-black w3-left-align">Bioreactors Current Measurement</button>
 
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                         <h4>Title</h4>
+
                          
 
                         <p>This procedure is to test the current output of modified <em>Shewanella oneidensis MR-1</em>.</p>
 
                         <p>This procedure is to test the current output of modified <em>Shewanella oneidensis MR-1</em>.</p>
 +
 +
<div class="w3-ul">
 +
<h5>Assembling Carbon Electrodes</h5>
 +
 +
<li> Cut out a 25 x 25 mm piece of carbon felt (Alfa Aesar, Part No: 43200) with a razor
 +
 +
blade.
 +
<li>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.
 +
 +
 +
<li>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.
 +
 +
<li> Allow to dry for at least five hours under a fume hood.
 +
<li>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).
 +
 +
<h5>Stoppers</h5>
 +
<li> 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.
 +
 +
<li> 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.
 +
<li>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.
 +
<li> 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.
 +
<li> 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.
 +
 +
<li>Insert second sterile 18 gauge needle through stopper away from electrode
 +
connections for ventilation during autoclaving. Bend to side and cover with
 +
aluminum foil.
 +
 +
<li>String the wire of the carbon felt electrode through the stopper.
 +
 +
<h5> Final assembly before autoclaving</h5>
 +
 +
<li>Fill each Mason jar (Ball, Part No: 750097) with 125 mL of M5 media. Add stir bar
 +
 +
 +
(McMaster-Carr, Part No: 5678K111).
 +
 +
<li> Place assembled stopper into the jar. Ensure the electrode is completely submerged
 +
 +
in media.
 +
 +
<li> 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)
 +
 +
<li> 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]
 +
<li> Autoclave bioreactors in a liquid cycle for 35 minutes. Allow to cool before adding
 +
 +
vitamin/mineral solution and antibiotic.
 +
 +
<li>Use the inserted 18 gauge needle to add up to total volume of 140 mL: 1.25 mL
 +
Wolfe&#39;s minerals, 1.25 mL of Wolfe’s vitamins (no riboflavin) and 12.5 mL of 200 mM
 +
lactate (carbon source).
 +
 +
<li> Add 140 µL of Spectinomycin antibiotic to reactors with antibiotic resistant strains.
 +
Note: Antibiotics are prepared at 50mg/mL concentrations.
 +
 +
<h5>Oxidation the silver electrode</h5>
 +
<li>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).
 +
 +
<li>Add 1 g of KCl Crystal (J.T. Baker, Part No: 3040-05) to 50mL of dH 2 O in a small
 +
beaker.
 +
 +
<li> 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.
 +
 +
<li> 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.
 +
<li> 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 (&lt;I&gt;) over
 +
the last 50% of the step duration, record current (&lt;I&gt;) 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.
 +
<li>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
 +
 +
<h5> To prepare the reference electrode</h5>
 +
 +
<li> 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).
 +
<li> 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.
 +
<li> Add KCl Crystal until saturated. Look for particles to start settling at the bottom (~ 23
 +
 +
g).
 +
 +
<li> 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.
 +
<li> Insert the reduced silver wire that is connected into the rubber stopper and create a
 +
 +
secure seal.
 +
 +
<li> Wipe off any excess salt around the reference with water and Kim wipes.
 +
 +
<h5> Programming potentiostat:</h5>
 +
<li> Connect potentiostat to EC-Lab software.
 +
<li>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|&gt; 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.
 +
<h5> Running the bioreactors</h5>
 +
<li>Place bioreactors on a low profile magnetic stirrer (VWR, Part No: 10153-690),
 +
 +
careful to position stir bar to avoid damaging hardware.
 +
 +
<li> 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.
 +
 +
<li> Inspect the units for anything out of place or for bubbles in the counter electrode
 +
 +
housing. This will cause errors in potentiostat readings.
 +
 +
<li> 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.
 +
 +
<li>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.
 +
<li> Bacterial strains grown in the reactors include: ΔmtrB (negative control), ΔmtrB
 +
 +
prL814 (positive control) and ΔmtrB prL814-mtrB.
 +
 +
<li> Allow growth in the bioreactor overnight. Use a syringe to remove 2 mL for sampling
 +
 +
to measure OD 600 and GFP fluorescence.
 +
 +
<li> 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.
 +
 +
<h5> Disposal</h5>
 +
 +
<li> Disconnect all leads from potentiostat.
 +
 +
<li> 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.
 +
<li> Dispose of used carbon felt electrodes as biohazardous waste.
 +
<li> Allow all parts to dry for a minimum of 5 hours in a drying cabinet.
 +
 +
 +
</div>
 
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                     </div>
  
  
 
                     <h2>Building Measurement Devices</h2>
 
                     <h2>Building Measurement Devices</h2>
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                     <button onclick="myFunction('openclose4')" class="w3-btn w3-block w3-black w3-left-align">Building Bioreactors</button>
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                     <button onclick="myFunction('openclose4')" class="w3-btn w3-block w3-flat-green-sea w3-left-align">Building Bioreactors</button>
 
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                         <h5>Purpose: </h5>
 
                         <h5>Purpose: </h5>
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{{MSU-Michigan-Footer}}

Latest revision as of 02:39, 2 November 2017

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Parts
Applied Design Hardware Measurement Model

Experiments

General

Materials
  • LB Broth, Miller (acumedia)
  • Magnetic stir bar
  • dH20
  • Autoclave

Media Preperation
  1. Add LB broth (25 g for 1000 mL)✓
  2. 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.✓
  3. Mix the solution on a magnetic stir plate until consistent throughout.✓
  4. 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
  1. Add dH20 to autoclavable bottle (500mL in 1L bottle)✓
  2. Add LB powder (12.5g for 500mL)✓
  3. Add agar (7.5 for 500mL)✓
  4. Mix using magnetic stir bar or shaking✓
  5. Autoclave for 30 minutes on liquid cycle✓

Plate Preperation
  1. Ensure media is cooled to 50-60°C✓
  2. Add antibiotics to final concentratation✓
  3. Mix using stir bar or shaking✓
  4. Pour plates in biosafety hood, ~20mL in each✓
  5. Let cool with lid askew✓
  6. When cool stack with top down and slide back in petri dish bag✓
  7. 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
  • 0.117 g, Magnesium Sulfate Heptahydrate MgSO4 * 7H2O
  • 23.8 g HEPES Free Acid
  • 0.1 g Casamino Acid
  • dH2O
  • 5 M NaOH
  • Autoclave

Media Preperation
  1. Add the chemical compounds to 880 mL of dH2O and separate into two 1 L autoclavable bottles with magnetic stir bars.✓
  2. Stir the solution on a magnetic stir plate and adjust the pH to 7.2 using 5 M NaOH.✓
  3. 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.✓
  4. 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)
    • Small magnetic stir bars
    • Needles and syringes of various size (sterile)
    Chemicals:
    • Carbon paste suspended in Xylene
    • M5 Minimal Media (100 Mm Hepes)
    • KCl, crystal
    • dH2O
    • Bacto Agar
    • Vitamins/Minerals
    • 200 mM Lactate
    • Spectinomycin Antibiotic
    • IPTG Inducer Stock
    Bacterial strains:
    • Shewanella Oneidensis Δmtrb_GFP_mtrb (spec resistance)
    • Shewanella Oneidensis Δmtrb_GFP (spec resistance)
    • Shewanella Oneidensis Δmtrb
    Other equipment:
    • Hot/stir plate
    • Multiple stir plate
    • Potentiostat
    • Autoclave

    Creating Paper Microbial Fuel Cells

    Purpose:
    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:
    1. Cut out 30mm x 30mm squares of whatman paper (6 per reactor) and one piece of parchment paper of the same size.✓
    2. 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. ✓
    3. Fully Colour one piece of whatman paper. This will be the cover. ✓
    4. 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. ✓
    5. 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. ✓
    6. Paint cement glue onto the anode and cathode. Ensure proper ventilation and safety. Allow to dry a minimum of five hours. ✓
    7. Cut the center out of two pieces of coloured whatman paper. These will be the chamber pieces. ✓
    8. 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).✓

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