IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-6-30: Difference between revisions
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These two points mean that we cannot observe a single cell over a period of time. Instead, we must take aliquots of groups of cells at different timepoints. Thus we can only observe group oscillation. | These two points mean that we cannot observe a single cell over a period of time. Instead, we must take aliquots of groups of cells at different timepoints. Thus we can only observe group oscillation. | ||
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 (and KaiABC do not respond directly to light as far as we know). 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. | 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 (and KaiABC do not respond directly to light as far as we know). 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. The group's level of phosphorylated KaiC would be more or less a flat line. | ||
===Generation problem=== | ===Generation problem=== |
Revision as of 14:42, 30 June 2006
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. Instead, we must take aliquots of groups of cells at different timepoints. Thus we can only observe group oscillation.
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 (and KaiABC do not respond directly to light as far as we know). 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. The group's level of phosphorylated KaiC would be more or less a flat line.
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