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

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Contents

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

  • 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

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

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

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


0sMidnight blue
1735sStarting to turn dark crimson purple
2300sPartly magenta pink
2885sNearly all magenta pink
2925sPart clear
3755sAll clear


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

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?)

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