Week 4: Eureka! Phone conversation with Professor Susan Golden at Texas A&M: • Professor Golden is one of the leading experts in cyanobacteria and has experience working with PCC 7942. • Susan answered a great deal of our questions concerning plasmid choice, cyanobacteria culturing and storage, reporters, and isolating and measuring KaiC. • Received very useful protocol information as well as ideas which helped us to focus our project goals. 1. The PCC 7942 culture we received from Peter Weigele at MIT met with an untimely end. • Left the lid partially open in the incubator to allow for gas exchange: bad idea! • Learned from Professor Golden that sufficient gas exchange will occur even with the lid closed. • Needed to order new PCC 7942, which have arrived. 2. Many cultures from our first batch were contaminated and did not grow any cyanobacteria. • Again, we were worried about gas exchange and so our cultures were not very well protected. • Growing cultures without antibiotic resistance is quite challenging! 3. Synchronizing the KaiC phosphorylation in E. coli. • How do we synchronize phosphorylation initially? • How do we preserve synchronization across multiple generations? Fungus from our first liquid cultures: PCR results from new PCC7942 and liquid culture. SI.com, 2006 1. Create Kai A/KaiBC Biobricks. 2. Knock out wild type Kai A/B/C in PCC 7942 and reinsert with Biobricks to recreate functional oscillation. 3. Transform E. coli with Kai Biobricks to reconstitute KaiC phosphorylation cycle with no reporter attached. 1. Phosphorylation measured by SDS-PAGE/Western blot. 4. Transform E. coli with Kai Briobricks to reconstitute KaiC phosphorylation cycle with Biobrick’d luciferase reporter. 1. Coincidentally, BU’s iGEm project is to create a Lux Biobrick. Reasonably, we can accomplish goals 1-3 by the end of the summer. • Working with newly acquired PCC 7942 strain. • PCR’ing Kai ABC using primers designed last week. • Making new liquid cultures (with calcium thiosulfate and without) • Streaking plates. • Designing and implementing solutions to the “KaiC phosphorylation synchronization” problems. • Designed computer program to model KaiC phosphorylation in multiple cells over time. • Output can be graphed by MatLab. • Model will become more complex as we encode more realistic features. • Hoping to use model to derive a solution. • Considering several possible solutions to pursue in parallel: What we want to do: • 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 must be done on groups of cells, since individual cells don't have enough protein to measure. • This measurement is destructive (we must extract the protein from the cells). • 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 time points. Thus we can only observe group oscillation. The problem: 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 the KaiABC proteins 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, since for every cell at a peak, there is equal probability that another cell is at a trough. The problem: Will our E. coli preserve current cycle phase between mothers and daughters? • Cyanobacteria preserve their clock phase during cell division, so colonies will still be synchronized after several generations through special mechanisms (mostly unkown). E. coli lacks these special mechanisms, so we have no guarantees that daughter cells remains in synch with their parent cells. In that case, even if we solve the initial synchronization problem, our cells will still desynchronize after reproducing. • Unsure if this will actually happen, since the only elements of the clock are the KaiABC proteins, whose interaction in the cytoplasm should not be reset or otherwise phase-shifted by cell division. Put the KaiABC genes under a temperature-sensitive promoter. These genes would be unexpressed in normal conditions, but expressed at high temperature. We could use a pulse of high temperature (a heat shock) to stimulate production of KaiABC for a brief period, then lower the temperature to stop production. Pros: Ideally this would synchronize all the cells by causing them to begin translation at the same time. We could also mitigate the generation problem by starving the cells after heat shocking them, to slow down their rate of reproduction. Cons: The concentration of KaiABC in each cell will grow more and more dilute as cells divide, since no new KaiABC will be produced after the beginning of the experiment. The proteins will also degrade over time. Thus, the oscillator's period will lengthen over time and eventually flatline Use a different promoter that responds to chemicals instead of temperature. We would achieve synchronization by controlling the exogenous chemical. Pros: We would not have the same problems with dilution and degradation of KaiABC as solution #1, since our cells would be producing KaiABC continuously. Cons: The obvious problem with this solution is that constant production of KaiABC may interfere with the clock in unknown ways (in cyanobacteria, KaiA expression remains constant while KaiBC oscillates on a circadian rhythm). Potentially, if KaiABC expression temporarily ceases during cell division, and cell division is unsynchronized, then the KaiABC clock might also become desynchronized after several generations, since production of KaiABC will drop at random intervals for different cells. Use cyanobacteria as an external clock. This would require modifying cyanobacteria to produce a messenger chemical (e.g. AHL) in a circadian rhythm. We would also have to modify E. coli to respond to this chemical. Pros: The latter step has already been done succesfully (Basu et el 2005). Also, there are Biobricks for LuxI and LuxR in the registry. Potential rewards would be great, and probably higher than what we would achieve by reconstituting the KaiABC clock in E. coli, since we already know how to make E. coli react to quorum sensing signals. Cons: We would probably be treading new ground by trying to introduce quorum sensing to cyanobacteria. We would also have to figure out a way to share media between cyanobacteria and E. coli so the messenger chemical can diffuse between them. All of this adds up to a signficant amount of work that may not see results by the end of the summer. Phase α: • Design and order Kai primers done! Phase β 1-2 weeks: • Culture new PCC 7942. • Mutate Biobrick restriction sites onto Professor Golden’s plasmids. • Culture E. coli. • Create plasmids with Kai genes. Phase γ 1-2 weeks: • Insert mutated plasmids into cyanobacteria with wild type Kai genes knocked out. • Transform E. coli with Kai Biobricks to reconstitute KaiC phosphorylation cycle. Phase δ 2-3 weeks: • Measure circadian cycle in Biobrick’d 7942. • Synchronize and measure KaiC phosphorylation in E. coli.
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