Week 9 review

Cyanobacteria Oscillatorin E. coli

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.

NewsRecieved GeneArt synthetic KaiA, B, and C

on FridayReceived anti-GFP antibodiesWill receive anti-KaiC antibodies on Tuesday

from Prof. Susan Golden

Stage I: test for expression and interaction of Kai proteins

Stage II: test for oscillation

KaiC

Experimental overview

Stage IExperiment 1:Expression of KaiC

Experiment 2:Interaction of KaiA and KaiC

KaiA KaiC

Stage I (cont.)Experiment 3:Interaction of KaiB and KaiC

KaiB KaiC

Experiment 4:Interaction of KaiABC

KaiB KaiCKaiA

KaiA KaiBC

Stage IIExperiment 1:Pulsed KaiABC

KaiA KaiBC

Experiment 2:Synchronized constant KaiA and pulsed KaiBC

KaiA KaiBC

Experiment 3:Synchronized constant KaiABC

Construct design

Creating ConstructsCurrently building on high copy plasmids

Lac + RBS + natural KaiALac + RBS + synthetic KaiALac + RBS + synthetic KaiBLac + RBS + synthetic KaiC

On low copy plasmidsLac + RBS + GFP

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

1 2 3 4 5 6 7 8

30002000

850

To do this weekPractice Western blots with GFP constructs

and anti-GFP antibodiesContinue creating Stage I experimental

constructs (combinations of KaiA and KaiB with KaiC)

Perform Stage I experiments

Questions for facultyFor practicing Western blots, is it preferable

to use a low-copy or high-copy GFP plasmid? Do our BioBrick parts need terminators?