Cyanobacteria:

1
Cyanobacteria

• Week 5

2
Project Goals

• Reconstitute the KaiC phosphorylation cycle in E.
  coli.

1. Create KaiA and KaiBC biobricks.
2. Transform E. coli with Kai Biobricks to
   reconstitute KaiC phosphorylation cycle with no
   reporter attached.
3. Distant Transform E. coli with Kai Biobricks to
   reconstitute KaiC phosphorylation cycle with
   Biobrickd luciferase reporter.

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Mechanism Review
Tomita 2005
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(No Transcript)
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Current Challenges

1. Using PCR to create and extract the correct
   constructs.
2. Synchronizing the Kai cycle within one E. coli
   cell.
3. Pick inducible promoters for KaiA and KaiBC.
4. Performing Western blots to detect KaiC within E.
   coli.

6
Week in Review

• Grew liquid cultures and plates from the new PCC
  7942.
• Our PCR produced a 3kb fragment which we hope is
  KaiABC (sequencing pending) .
• Began crossover PCR for site-specific
  mutagenesis.
• Planned future experiments with E. coli.
• Decided to eliminate the branch of our project
  dealing with cyanobacteria.
• Designed primers for sequencing KaiABC and
  extracting coding sequence.

7
KaiABC extraction

• Lane 21 3kb segment
• Lane 16, 20, 24 Positive Control
• Other lanes experimental (failed)

8
Failed site-specific mutagenesis PCR

• Lanes 1-3 2402 bp expected
• Lanes 4-6 209 bp expected
• Lanes 7-8 80 bp expected
• Lanes 9-10 370 bp expected
• Lanes 11-13 370 bp expected

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Experiment 1

• Goal
• Verify that our KaiA and KaiBC Biobricks can be
  expressed in E. coli.
• Procedure
• Tag KaiA/BC (e.g. with a fluorescent subunit).
• Insert the tagged KaiA/BC into E. coli.
• Induce promoters for both KaiA and KaiBC.
• Measure the E. coli fluorescence.
• We should see some fluorescence, indicating that
  our promoters are working and the Kai genes are
  being expressed.

KaiA
KaiBC
1.
10
Experiment 2

• Goal
• Verify that KaiA, KaiB and KaiC are interacting
  and phosphorylating KaiC in E. coli.
• Procedure
• Insert KaiA and KaiBC into E. coli.
• Induce KaiBCs promoter and measure the amount of
  phosphorylated KaiC via Western blot.
• Induce KaiAs promoter and measure the amount of
  phosphorylated KaiC via Western blot.
• Ideally, we would see an increase in the amount
  of phosphorylated KaiC between steps 2 and 3.
  This would indicate that the Kai proteins are
  interacting as they do in vivo. However, some
  care needs to be taken in deciding when exactly
  to measure the phosphorylated KaiC, to determine
  when/if KaiC is phosphorylated in the absence of
  KaiA. In that case, we may not see as much of a
  difference between steps 2 and 3.

KaiA
KaiBC
1.
2.
11
Experiment 3

• Goal
• To measure the oscillation of the KaiC
  phosphorylation cycle given pulsed KaiA and KaiBC
  production.
• Procedure
• Insert KaiA and KaiBC into E. coli.
• Induce the promoter for KaiA and KaiBC at the
  same time.
• After a short time, turn off the promoters.
• Measure the amount of phosphorylated KaiC at
  regular intervals (e.g. every hour) via Western
  blot.
• We hope to initially see strong oscillation, with
  the amplitude decreasing over time. Since no
  KaiA/B/C should be produced after the initial
  pulse, we expect that the intracellular
  concentrations of the Kai proteins would decrease
  as the E. coli divide.

KaiA
KaiBC
1.
12
Experiment 4

• Goal
• To measure the oscillation of the KaiC
  phosphorylation cycle given constant KaiA and
  pulsed KaiBC production.
• Procedure
• Insert KaiA and KaiBC into E. coli.
• Induce the promoters for KaiA and KaiBC.
• After a short time, turn off the KaiBC promoter,
  leaving the KaiA promoter induced.
• Measure the amount of phosphorylated KaiC at
  regular intervals.
• We expect that, since KaiA is produced at a
  constant (non-oscillating) rate in cyanobacteria,
  its constant production in E. coli should not
  interfere with KaiCs phosphorylation cycle. The
  constant production of KaiA may even strengthen
  the oscillation, since KaiA wont degrade or
  dilute away.

KaiA
KaiBC
1.
13
Experiment 5

• Goal
• To measure the oscillation of the KaiC
  phosphorylation cycle, given constant KaiA and
  KaiBC production.
• Procedure
• Insert KaiA and KaiBC into E. coli.
• Induce the promoters for KaiA and KaiBC.
• Measure the amount of phosphorylated KaiC at
  regular intervals.
• We do not know how constant KaiBC transcription
  will interfere with the oscillation of KaiC (in
  cyanobacteria, KaiBC transcription oscillates on
  a circadian rhythm). This experiment should help
  us figure it out.

KaiA
KaiBC
1.
14
To Do

• Sequence our 3kb construct.
• Extract KaiA and KaiBC coding sequences from
  KaiABC region.
• Select inducible promoters for experiments.
• Create experiment constructs.
• Learn Western blot protocol.
• Perform experiments and measure results.
• Design new experiments as the need arises.