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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.

    • Title

    This procedure is to test the current output of modified Shewanella oneidensis MR-1.

    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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