Cyanobacteria Oscillator in E. coli Week 10 review

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

These past weeks… Received synthetic KaiA, B, and C from GeneArt Received anti-KaiC antibodies from Prof. Susan Golden Revised experimental plan, with focus on construct building for Western Blot experiments

Experimental Design By August 30th, we want to accomplish Stage 1.1: Test for phosphorylated KaiC.

Construct design Necessary for Stage 1.1

Completed this week XbaI-PstI and SpeI-PstI digest optimization Ligation attempts of 9 products on high copy plasmids Lac + RBS + KaiA / KaiB / Kai C 3 attempts KaiA / KaiB / Kai C 2 attempts RBS + KaiA / KaiB / Kaic Successful anti-GFP Western Blot

Anti-GFP Western Blot 1 2 3 4 5 6 GFP expected: 27kDa 1: SeeBlue Plus 2 2: GFP @0.4 OD 3: X:X dilution of lane 2 4-6: Fill in Jeff… and is the ladder right? 148kDa 98kDa 64kDa 50kDa 36kDa 22kDa 16kDa 1 2 3 4 5 6 GFP expected: 27kDa

Digestion Assays

Ligation Protocol Using Roche 5min DNA Ligase Kit 1:3 molar ratio of vector-insert, 50ng vector target If nanodrop values are low, we use 1:3 volume ratio 30 minute incubation @ RT Transformation into Top10 cells 25uL cells / ligation product (using all 21uL) Incubate on ice for 30 minutes Heat shock at 42C for 30 seconds SOC recovery for 1 hour

This week: Continue ligation attempts, especially of Lac + RBS + KaiC Perform Western blot against KaiC on E. coli transformed with above construct Begin working on final presentation