Team:MSU-Michigan/Experiments

Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

What should this page contain?
  • Protocols
  • Experiments
  • Documentation of the development of your project
Inspiration

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).✓

Title

This procedure is to make LB Broth Media.

Testing the Strains

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.

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

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