IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-6-30
Two problems
Today we realized that there are two major obstacles to our project:
Synchronization problem
After inserting the KaiABC genes into E. coli, we will measure oscillation by doing Western blots of our colonies and observing the relative amounts of phosphorylated versus unphosphorylated KaiC (recall that the phosphorylation state of KaiC is what oscillates when KaiA, KaiB, and KaiC are mixed in vitro).
- This measurement is destructive (we must extract the protein from the cells)
- This measurement must be done on groups of cells, since individual cells don't have enough protein to measure
These two points mean that we cannot observe a single cell over a period of time.
Nick raised a big problem with our project: how will we synchronize our E. coli clocks? E. coli don't have the same light-sensing apparatus as cyanobacteria, so light/dark entrainment is unlikely to work. If our cells are out of phase with each other, we won't be able to detect any group oscillation in KaiC phosophorylation, even if the oscillator works perfectly in individual cells.
Generation problem
There is a second related problem: will our E. coli preserve clock phases between mothers and daughters? Cyanobacteria preserve their clock phase during cell division, so that colonies which were entrained at the same time will still be synchronized after several generations. E. coli doesn't have any special mechanisms to preserve phase, so daughters might end up at different phases from their mothers after division. In that case, even if we solve the synchronization problem, our cells will still desynchronize after reproducing.
Reading
Read these three papers:
- Circadian rhythms in rapidly dividing cyanobacteria, Kondo et al., 1997
- Independence of Circadian Timing from Cell Division in Cyanobacteria, Mori and Johnson, 2000
- Article on synchronization
