1. Week 9 review Cyanobacteria Oscillator in E. coli

2. 1.Create KaiA, KaiB, and KaiC biobricks. 2.Transform E. coli with Kai Biobricks to reconstitute KaiC phosphorylation cycle (no reporter attached). 3.Distant: Transform E. coli with Kai Biobricks to reconstitute KaiC phosphorylation cycle with Biobrick’d reporter. Project Goal: Reconstitute the cyanobacteria KaiABC oscillator in E. coli.

3. News Recieved GeneArt synthetic KaiA, B, and C on Friday Received anti-GFP antibodies Will receive anti-KaiC antibodies on Tuesday from Prof. Susan Golden

4. Stage I: test for expression and interaction of Kai proteins Stage II: test for oscillation KaiC Experimental overview Stage I Experiment 1: Expression of KaiC Experiment 2: Interaction of KaiA and KaiC KaiA KaiC

5. Stage I (cont.) Experiment 3: Interaction of KaiB and KaiC KaiB KaiC Experiment 4: Interaction of KaiABC KaiB KaiCKaiA

6. KaiBC Stage II Experiment 1: Pulsed KaiABC KaiAKaiBC Experiment 2: Synchronized constant KaiA and pulsed KaiBC KaiAKaiBC Experiment 3: Synchronized constant KaiABC

7. Construct design

8. Creating Constructs Currently building on high copy plasmids Lac + RBS + natural KaiA Lac + RBS + synthetic KaiA Lac + RBS + synthetic KaiB Lac + RBS + synthetic KaiC On low copy plasmids Lac + RBS + GFP

9. Working ligation Attempt to ligate natural KaiA into a high copy backbone Lanes: 1, 8: 1kb+ 2, 4, 6: undigested plasmid 3, 5, 7: digested plasmid 12345678 3000 2000 850

10. To do this week Practice Western blots with GFP constructs and anti- GFP antibodies Continue creating Stage I experimental constructs (combinations of KaiA and KaiB with KaiC) Perform Stage I experiments

11. Questions for faculty For practicing Western blots, is it preferable to use a low-copy or high-copy GFP plasmid? Do our BioBrick parts need terminators?