Cyanobacteria

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Cyanobacteria

• Week 4 Eureka!

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Weeks in ReviewThe Good

• Phone conversation with Professor Susan Golden at
  Texas AM
• 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.

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Weeks in ReviewThe Bad

• 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.
• 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!
• Synchronizing the KaiC phosphorylation in E.
  coli.
• How do we synchronize phosphorylation initially?
• How do we preserve synchronization across
  multiple generations?

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Weeks in ReviewThe Ugly

• Fungus from our first liquid cultures

PCR results from new PCC7942 and liquid culture.
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Project Goals
SI.com, 2006

• Create Kai A/KaiBC Biobricks.
• Knock out wild type Kai A/B/C in PCC 7942 and
  reinsert with Biobricks to recreate functional
  oscillation.
• Transform E. coli with Kai Biobricks to
  reconstitute KaiC phosphorylation cycle with no
  reporter attached.
• Phosphorylation measured by SDS-PAGE/Western
  blot.
• Transform E. coli with Kai Briobricks to
  reconstitute KaiC phosphorylation cycle with
  Biobrickd luciferase reporter.
• Coincidentally, BUs iGEm project is to create a
  Lux Biobrick.
• Reasonably, we can accomplish goals 1-3 by the
  end of the summer.

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Current Status

• Working with newly acquired PCC 7942 strain.
• PCRing 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

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KaiC Phosphorylation in E. coli

• 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.

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KaiC Phosphorylation in E. coli, Problem I

• 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.

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KaiC Phosphorylation in E. coli, Problem II

• 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.

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Proposed Solution 1

• 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

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Proposed Solution 2
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.
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Proposed Solution 3
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.
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Project Timeline

• Phase a
• Design and order Kai primers ? done!
• Phase ß ? 1-2 weeks
• Culture new PCC 7942.
• Mutate Biobrick restriction sites onto Professor
  Goldens plasmids.
• Culture E. coli.
• Create plasmids with Kai genes.
• Phase ? ? 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 d ? 2-3 weeks
• Measure circadian cycle in Biobrickd 7942.
• Synchronize and measure KaiC phosphorylation in
  E. coli.