IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-6-28

Phone conversation with Susan Golden

• Hybrid plasmids
  • Hybrid plasmids exist, there are a few of them out there. They'd probably be fine for what we want to do. Golden doesn't use them because they caused minor changes in expressions. Using non-replicating vectors to insert into the chromosome is more stable.
  • Golden says it probably won't make a difference for our purposes whether we use endogenous or exogenous plasmids.
  • Endogenous plasmid is 8Kb; fairly large.
• Light sources
  • Golden uses cool white flourescent lights; says we can throw in grow lights for more red.
  • Be aware that conversions between lux and µE aren't perfect. Grow lights have more energy in the 400-700 nm range.
  • We're not in danger of giving cyanobacteria too much light
• Air
  • Use flasks that give (?) negative air space, shake them a little to exchange
  • Take cheesecloth, put cotton in it, tie it up and autoclave it in the flask; use as a plug with aluminum foil fit lightly over.
  • Tried various commercial caps, but Golden is worried that they may release toxins over time.
• Plate storage
  • Plates can be stored in a drawer at room temperature
• Reporters
  • Doesn't work in cyanobacteria
  • YFP and CFP work (in van Oodenaarden's lab), but they haven't done anything circadian with it
  • GFP is too stable (doesn't decay in the troughs of expression)
  • Luciferase doesn't require you to illuminate the cells with high energy light (which damages them)
  • You can passively measure bioluminescence in real time
  • Don't need a great CCD if you're not trying to measure single colonies
• Isolating and measuring KaiC
  • Tagging KaiC might not work (oscillation ceases)
  • Kondo lab has a new paper in press where they succesfully tagged some of the Kai proteins
  • RNA polymerase is very similar between cyano and E. coli; but the other factors may cause problems
• What we can reasonably
  • Make a BioBrick, try to get the oscillation going in E. coli. Might as well try a reporter gene, but the easiest thing is to directly measure the KaiC phosphorylation.
  • There's skill to running the SD-PAGE.

Questions for Susan Golden

• Are plant lights viable for growing cyanobacteria?
• Can we use GFP as a reporter in cyanobacteria instead of luciferase? (We don't have the right equipment for automatically measuring bioluminescence).
• Do we need to aerate our cultures with CO2? If so, how do we prevent contamination and dry out?
• How do we store plates for a long time?
• For isolating and measuring the phosphorylation of KaiC, what are the pros and cons of using KaiC antibody assay versus purifying with HisTag?
• Why not use endogenous autoreplicating plasmids for transformation, instead of non-replicating vectors?

Agar longevity

We put clean agar plates on the shaking floor of the incubator to test how quickly they dry out. One plate is open while the other plate is closed but not sealed.

Green growth

Some of our PCC 6803 flasks showed growth since yesterday (single 2006-6-26 and streak 2006-6-26). Nick vortexed the streak 2006-6-26 to disperse the fungus poof.

Email from Susan Golden

We received an email from Susan Golden with lots of information.

• Susan's expertise lies with PCC 7942. She warned that growth conditions and the circadian oscillator may be very different in other strains.
• We shouldn't be worried about giving our cyanobacteria too much light. The more light the better. We can even keep them in constant light conditions instead of the 16h light / 8h dark cycle we've been using.
• Susan is willing to send us plasmids from her lab.
• Getting KaiC phosphorylation to oscillate in E. coli would be a major achievement by itself. We can still try to transplate RNAP and sigma factors from cyanobacteria into E. coli, but we'd be heading into unknown territory. We should make sure that KaiC will phosophorylate on a circadian rhythm in E. coli.