IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week2/Widgetry

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Goals for this week

  1. Talk to Helen, Pete, etc. =)
  2. Work on growing anaerobically, the electric current, and everything together?
  3. Check that Shewie is growing by putting it with media, bubbling N2 gas.

Monday: June 30, 2008

Goals

  1. autopsy/cleaning
  2. LML current test
  3. anaerobic burnicite test
  4. research/buy O2 test kit
  5. Questions to Peter/Helen/Orianna

Results from autopsy

  • membrane is pink but looks perfectly intact
  • regular epoxy on cathode looks different
    • dull grey color rather than silver black
    • feels like rubber, starting to rub off
  • fluffy white stuff in the cathode chamber where no bacteria was present
  • white/clear gluey stuff growing in cathode chamber
    • looked at under microscope -> looks like bacteria!

Lab

MFC Design changes

  • remade anode and cathode without using epoxy:
    • anode: titanium wire is wrapped around carbon felt
    • cathode: titanium wire weaves around Pt covered carbon cloth

Anaerobic burnicide test

  • see Tuesday for results

LML Current test

  • baseline current of 1 microampere
  • injected Shewie into anode side
    • did not observe any noticeable change in current
    • will try again with a higher density of Shewie

**Note: There was an initial hook-up error in cables**

Tuesday: July 1, 2008

Lab

Results from Anaerobic Burnicide test

Done at pH 7.3

Mfc burnicide.JPG

  • LEFT: Anaerobic chamber with Shewie: went from brown to light yellow
  • RIGHT: Aerobic chamber with Shewie: turned slightly lighter but noticeably darker than anaerobic chamber with Shewie
  • BOTTLE: Anaerobic chamber without Shewie: stayed a clear brown


Conclusion: LML media should be fine for anaerobic growth in Shewie.

Experiment: Plated anaerobic Shewie and aerobic Shewie from chambers to grow colonies.

Resazurin test

4 vials

  • LML, open, 1 mL of 0.8 mM resazurin
  • LML, open, 1 mL of 0.8 mM resazurin, 1 mL Shewie
  • LML, closed, 1 mL of 0.8 mM resazurin, 1 mL Shewie
  • LB, closed, 1 mL of 0.8 mM resazurin, 1 mL Shewie

Results

  • Within a minute, vial with LB changed color
  • Half an hour to an hour later, the closed vial with LML and Shewie also changed color

Mfc resazurin.JPG

Conclusions

  • Resazurin works as an indicator of O2
  • Worth trying LB and lactate (neutralized with NaOH) as the media since it appears to work much faster

Note: Resazurin will turn from clear to pink when exposed to oxygen again, but not to blue (first reduction step is irreversible)

Current Test

Parameters

  • Media: LB + 20 mM lactate at a pH of 6.96 (neutralized with NaOH), in both chambers
  • Shewie: 10 mL of Shewie spun down, resuspended in 1 mL of LML
  • Resistance: 400 ohms
  • No bubbling of oxygen on cathode side initially

Results

  • Observed current increase approximately 7 minutes after Shewie were inserted
  • Leveled off at approximately 34 microamps

ShewieCurrentProduction1.jpg

  • Bubbled oxygen on cathode side at approximately 1700 s
  • Injected 1 mL of LB/lactate (same as media) at 2350 s

ShewieCurrentProduction2.jpg

Wednesday: July 2, 2008

Current Test

Parameters

  • Media: LB + 20 mM lactate at a pH of 6.96 (neutralized with NaOH), in both chambers
    • 1.5 mL of 0.8mM resazurin
  • Shewie: 10 mL of Shewie spun down, resuspended in 1 mL of LB/lactate
  • Resistance: 400 ohms
  • Differences from the previous day:
    • Used 2-inch needles: bubbling right on the membrane (better exchange of ions?)
    • Cathode partially in the air (5 mm above media)
    • Shewie was a day older (three days old) -> stationary phase possible
    • Shewie was from a different plate, but same strain
    • Different cathode/anode of the same material used with about the same resistance (25-30 ohms for the entire setup)

Results

  • Observed much greater current, but the increase was significantly slower
  • Leveled off for a while at approximately 110 microamps, but then continued to increase
  • Addition of O2 in the anode corresponded with a drop in current
  • Removal of O2 from the anode corresponded with a steep increase in current

Shewie/Resazurin Test

  • Ran test parallel to current test because it was taking much longer than the previous day
  • Added 1 mL of resazurin, 1 mL of Shewie used in current test, and filled vial with LB+lactate

Results: took nearly 29 minutes for the resazurin to turn purple (took less than 1 minute in previous day's test)


Time Color


0s Midnight blue
1735s Starting to turn dark crimson purple
2300s Partly magenta pink
2885s Nearly all magenta pink
2925s Part clear
3755s All clear

Mfc current1.JPG

Mfc current2.JPG

Mfc current3.JPG

Mfc current4.JPG


ShewieCurrentTest2.jpg

Thursday: July 3, 2008

Current Test with mtrA Shewie

  • Ran with the same parameters as July 3, but with mtrA Shewie

Results: Current production on the same order of magnitude as wt Shewie

MtrA.jpg

Goals for the Future

  • LabVIEW vs. Excel?
  • Running multiple MFCs concurrently
    • How to set up Keithley for multiple inputs
  • Make more MFCs
  • Tic-Tac-Toe design
    • physical arrangement of parts
      • connections to Keithley
    • computer interface
      • selecting squares on computer -> output that Shewie will detect (checmical? light?)

Mfc happykids.JPG