Cyanobacteria:

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Cyanobacteria

• Week 6

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Outline

• Background Information
• Update new paper out July 21st
• Experimental Progress
• Current challenges
• Bad template
• Successful colony PCR (try 2)
• Synthesis
• To do

3
Outline

• Background Information
• Update new paper out July 21st
• Experimental Progress
• Current challenges
• Bad template
• Successful colony PCR (try 2)
• Synthesis
• To do

4
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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New Information

• From Kageyama et al. (7/21/06)
• KaiC forms a hexamer
• KaiA phosphorylates the KaiC hexameric subunits
• KaiB traps KaiA when KaiC maximally
  phosphorylated promotes KaiC dephosphorylation
• KaiC subunit suffling
• KaiA FLAG-tagged, KaiB His6-tagged while
  maintaining rhymaticity

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Outline

• Background Information
• Update new paper out July 21st
• Experimental Progress
• Current challenges
• Bad template
• Successful colony PCR (try 2)
• Synthesis
• To do

8
Week in Review

• Received sequencing results, which confirmed our
  digests showing that the 3kb fragment we were
  working with was not KaiABC.
• Began a new extraction of KaiABC fragment from
  cyanobacteria.
• Received primers for sequencing KaiABC and
  extracting coding sequence.
• Sent out our KaiABC synthesis order to Geneart.
• Renovating Wiki!

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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.

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Incorrect Template (

• We found that our template which we extracted two
  weeks ago is not the correct template. Although a
  restriction digest suggested we had the correct
  template, our sequencing primers did not bind and
  site-specific mutagenesis has repeatedly failed.

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Restarting the process )

• From square one, we have successfully extracted
  another 3kb fragment from cyanobacteria DNA. This
  week we will retry cloning it into a Topo Vector,
  site-specific mutagenesis, and sequence the new
  template.

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KaiABC extraction 2

• Lane 4 and Lane 9 Purified liquid cultures.
• 3kb and 400bp fragments
• Lane 5 and Lane 6 Plated PCC7942
• Other lanes purified or raw liquid culture
  (failed)

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Outline

• Background Information
• Update new paper out July 21st
• Experimental Progress
• Current challenges
• Bad template
• Successful colony PCR (try 2)
• Synthesis
• To do

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Synthesis Constructs
kaiB
kaiA
367 bp In standard vector
913 bp In standard vector
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Synthesis Details

• Chose between
• GeneART
• Coda
• Codon Devices
• Blue Heron
• DNA 2.0
• Quote 1.10/bp, 10-15 business days
• Second best offer Coda, at 1/bp but requiring
  assembly (3wks)
• Should arrive between Aug. 7-11

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Outline

• Background Information
• Update new paper out July 21st
• Experimental Progress
• Current challenges
• Bad template
• Successful colony PCR (try 2)
• Synthesis
• To do

17
To Do

• Sequence our new 3kb constructs.
• Extract KaiA and KaiBC coding sequences from
  KaiABC region.
• Select inducible promoters for experiments.
• Create experiment constructs.
• Perform KaiABC synchronization experiments, and
  measure results.