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

Paramaters 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

Results

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