Week 5

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 Biobrick’d luciferase reporter.

Tomita 2005 Mechanism Review:

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.

Grew liquid cultures and plates from the new PCC 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.

KaiABC extraction Lane 21: ~3kb segment Lane 16, 20, 24: Positive Control Other lanes: experimental (failed)

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

Goal: Verify that our KaiA and KaiBC Biobricks can be expressed in E. coli. Procedure: 1. Tag KaiA/BC (e.g. with a fluorescent subunit). 2. Insert the tagged KaiA/BC into E. coli. 3. Induce promoters for both KaiA and KaiBC. 4. Measure the E. coli fluorescence. We should see some fluorescence, indicating that our promoters are working and the Kai genes are being expressed. KaiAKaiBC 1.

Goal: Verify that KaiA, KaiB and KaiC are interacting and phosphorylating KaiC in E. coli. Procedure: 1. Insert KaiA and KaiBC into E. coli. 2. Induce KaiBC’s promoter and measure the amount of phosphorylated KaiC via Western blot. 3. Induce KaiA’s 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. KaiAKaiBC 1. 2.

Goal: To measure the oscillation of the KaiC phosphorylation cycle given pulsed KaiA and KaiBC production. Procedure: 1. Insert KaiA and KaiBC into E. coli. 2. Induce the promoter for KaiA and KaiBC at the same time. 3. After a short time, turn off the promoters. 4. 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. KaiAKaiBC 1.

Goal: To measure the oscillation of the KaiC phosphorylation cycle given constant KaiA and pulsed KaiBC production. Procedure: 1. Insert KaiA and KaiBC into E. coli. 2. Induce the promoters for KaiA and KaiBC. 3. After a short time, turn off the KaiBC promoter, leaving the KaiA promoter induced. 4. 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 KaiC’s phosphorylation cycle. The constant production of KaiA may even strengthen the oscillation, since KaiA won’t degrade or dilute away. KaiAKaiBC 1.

Goal: To measure the oscillation of the KaiC phosphorylation cycle, given constant KaiA and KaiBC production. Procedure: 1. Insert KaiA and KaiBC into E. coli. 2. Induce the promoters for KaiA and KaiBC. 3. 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. KaiAKaiBC 1.

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.