Amburn Brian (Talk | contribs) |
Amburn Brian (Talk | contribs) |
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<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"> | <div class="w3-ul"> | ||
− | <li> | + | <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. | ||
+ | </div> | ||
</div> | </div> | ||
<button onclick="myFunction('openclose2')" class="w3-btn w3-block w3-black w3-left-align">Growth Fluorescence</button> | <button onclick="myFunction('openclose2')" class="w3-btn w3-block w3-black w3-left-align">Growth Fluorescence</button> | ||
<div id="openclose2" class="w3-container w3-hide"> | <div id="openclose2" class="w3-container w3-hide"> | ||
− | <h4> | + | <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. | ||
+ | |||
+ | </div> | ||
</div> | </div> | ||
Revision as of 01:01, 2 November 2017
Experiments
General
Materials
- LB Broth, Miller (acumedia)
- Magnetic stir bar
- dH20
- Autoclave
Media Preperation
- Add LB broth (25 g for 1000 mL)
- 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
- 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
- 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
Procedure
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 |
Testing the Strains
IPTG Induction Using the 96 Well Plate
This procedure is to test the fluorescence of modified Shewanella oneidensis MR-1.
Growth Fluorescence
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 IPTGMaterials:
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
- Shewanella Oneidensis Δmtrb_GFP_mtrb (spec resistance)
- Shewanella Oneidensis Δmtrb_GFP (spec resistance)
- Shewanella Oneidensis Δmtrb
- 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:
- 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).
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