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!

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=

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

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











=Thursday: July 3, 2008=

Current Test with mtrA Shewie
Results: Current production on the same order of magnitude as wt Shewie
 * Ran with the same parameters as July 3, but with mtrA 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?)