1. Cyanobacteria Oscillator
in E. coli
Week 10
review

2. 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.

3. 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

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

5. Construct design
Necessary for Stage 1.1

6. 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

7. Anti-GFP Western Blot 1 2 3 4 5 6 GFP expected: 27kDa
1: SeeBlue Plus 2
2: 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

8. Digestion Assays

9. 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 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

11. 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