IGEM:Harvard/2008/Lab Notebooks/Meeting notes

5/20: Meeting with Orianna Bretschger

Questions for Orianna Bretschger

General

• Transformation protocol (not via conjugation)
• Plasmids that can be replicated (origins)
• Promoters that work (inducible, etc)
• Request for gifts
• Can mtr genes be regulated (eg lacZ)?

Knocking out EnvZ and/or OmpR (together a bit over 2kbp)

• Homologous recombination- time frame, ease
• Cre-lox- which one is easier?
• effects of removing endogenous envR and ompR

MFC

• suggestions on how to grow Shewie anaerobically
• ask about colorimetric assay used to detect electrical output; differences between different electron acceptors that we could use and what would be an optimal system for us to use (ie. manganese vs iron vs some other detection system, like fumarate?)
• minimum surface area required to detect current over noise
• time to produce current after anaerobic
• suggestions for shewanella sensing electric current(light, heat, magnetism)

Notes from meeting

General

• Transformation protocol (not via conjugation)
  • Diane Newman –ask for protocol
  • Chemical wont work
  • Electroporation probably way to go
• Plasmids that can be replicated (origins)
  • Check paper for the plasmids and get them
• Promoters that work (inducible, etc)
  • LacZ
  • Any promoters that we know DON’T work?
• Can mtr genes be regulated (eg lacZ)?

Knocking out EnvZ and/or OmpR (together a bit over 2kbp)

• Homologous recombination- time frame, ease
• Cre-lox- which one is easier?
  • Get protocol from Orianna
  • Multiple stable knockouts
  • 3 week method
• effects of removing endogenous envR and ompR
• Has had good track record of E. coli and Shewie exchanging plasmids

MFC

• suggestions on how to grow Shewie anaerobically
  • Lactate
  • Fumerate
  • Ioscitrate
  • Manganese oxide
  • Nitrogen co2 headspace
  • LB broth aerobically then transfer to anaerobically
  • GFP wont work anaerobically?
• ask about colorimetric assay used to detect electrical output; differences between different electron acceptors that we could use and what would be an optimal system for us to use (ie. manganese vs iron(slower) vs some other detection system, like fumarate?)
• protocol for colorimetric assay
• minimum surface area required to detect current over noise
  • graphite paper with lower surface area
  • micro mFC’s
  • should be able to pick something up with relatively small surface area
  • cathode size > anode size so cathode isn’t limiting
  • www.fuelcellstore.com
  • air cathode?
    • Great in chemical fuel cell
• time to produce current after anaerobic
  • almost immediate response
• suggestions for shewanella sensing electric current(light, heat, magnetism)
  • Turning electrical signal off?
    • Flush chamber with oxygen
    • Time frame: minutes?
  • Potential:
    • 0.4 volts Cathode: 0.4 V
  • Invert cathode/anode to turn off Shewie? Oxygen output at anode.
    • Colorimetric oxygen assay
    • Promoters that respond to oxygen environment?

Widgetry

• may be easier to make individual wells/MFC’s and arrange them than to create an actual widget
• Keithley multi-model 2700 20-channel
• Trigger mechanisms available
• DigiKey Potentiometer
• Minimize exposure of wire to prevent interference from corrosion
  • Cover with graphite/epoxy?
  • Titanium wire? No insulator but cover with epoxy?

Contact
Orianna Bretschger: bretschg@usc.edu

Diane Newman: dkn@gps.caltech.edu

Things we have

• Silver epoxy
• Graphite sheet
• Nafion
• Titanium wire
• Platinum coated sheet
• Polycarbonate/polypropylene stock
• Silicon rubber sheets for gaskets
• Pierce G-12 mills?