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Team:Harvard/Dailybook/Week2/Widgetry
From 2008.igem.org
Contents |
Goals for this week
- Talk to Helen, Pete, etc. =)
- Work on growing anaerobically, the electric current, and everything together?
- Check that Shewie is growing by putting it with media, bubbling N2 gas.
Monday: June 30, 2008
Goals
- autopsy/cleaning
- LML current test
- anaerobic burnicite test
- research/buy O2 test kit
- 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
|
| 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 |
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?)
- physical arrangement of parts
