Cyanobacteria Oscillator in E coli Week 9 review

Cyanobacteria Oscillator in E. coli Week 9 review

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

News � Recieved Gene. Art synthetic Kai. A, B, and C on Friday � Received anti-GFP antibodies � Will receive anti-Kai. C antibodies on Tuesday from Prof. Susan Golden

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

Stage I (cont. ) Experiment 3: Interaction of Kai. B and Kai. C Kai. B Kai. C Experiment 4: Interaction of Kai. ABC Kai. A Kai. B Kai. C

Stage II Experiment 1: Pulsed Kai. ABC Kai. A Experiment 2: Synchronized constant Kai. A and pulsed Kai. BC Experiment 3: Synchronized constant Kai. ABC Kai. A Kai. BC

Construct design

Creating Constructs � Currently building on high copy plasmids � Lac + RBS + natural Kai. A � Lac + RBS + synthetic Kai. B � Lac + RBS + synthetic Kai. C � On low copy plasmids � Lac + RBS + GFP

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

To do this week � Practice Western blots with GFP constructs and anti. GFP antibodies � Continue creating Stage I experimental constructs (combinations of Kai. A and Kai. B with Kai. C) � Perform Stage I experiments

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