Week 6

1. Week 6

2. 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. Create KaiA and KaiBC biobricks. Transform E. coli with Kai Biobricks to reconstitute KaiC phosphorylation cycle with no reporter attached. Distant: Transform E. coli with Kai Biobricks to reconstitute KaiC phosphorylation cycle with Biobrick’d luciferase reporter.

5. 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

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

8. 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!

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

11. 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.

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

13. 1 2 3 4 5 7 10 11 6 8 9 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)

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

15. NotI NotI KpnI XhoI EcoRI XbaI SpeI PstI kaiC mutation mutation Bba_00000 21bp Bba_00001 22bp 1618 bp In standard vector kaiB kaiA 367 bp In standard vector 913 bp In standard vector

16. 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

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

18. 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.