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

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

PHOTO WILL GO HERE

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

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

Photos?

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
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