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

Title

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

Title

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

Title

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

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).✓