Week 8

OutlineProject GoalExtracting and biobricking KaiA and KaiBSynthesis updateWestern Blotting updateSite-Specific Mutagenesis updatePromoter ChoiceFuture Plans

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 GoalProject GoalReconstitute the cyanobacteria KaiABC oscillator in E. coli.

Clarification:We now have 3 different methods for getting our KaiA,

B, and C in a biobrick

1. Extraction of Kai A, B, and C separately w/ BB ends• Attempted this week

2. Synthesis of Kai A, B, and C separately w/ BB ends3. A cassette of Kai A, B, and C w/ BB ends

• Attempted past month

We have KaiA, and KaiB ; we will be getting KaiC at the end of the week.

Performed a PCR to extract kaiA, kaiB, and kaiC sequences individually (with BioBrick ends) We successfully extracted

kaiA and kaiB, but not kaiC Performed a digestion of

KaiA, KaiB, and a digestion of RFP-containing plasmid (Bba_J04550)

Performed a ligation/transformation of the RFP-containing backbone with kaiA and kaiB

Currently growing up the transformants

Kai BioBricksKai BioBricks

kaiB

367 bpIn standard vector

kaiA

913 bpIn standard vector

Susan Golden expressed willingness to send us antibodies for KaiC

“Let me know how many westerns you think you want to run, and we'll send you some antiserum. I am pretty sure the anti-KaiC was made in chickens-- an important point for ordering secondary antibodies for visualization. “

We received sequencing results for our “templates” cloned into BLUNT TOPO which we had been using for the past month… Of 6 clones, 4 were “background” for the blunt TOPO kit Other two had bad transformants

Decided to pursue PCR off of the template directly instead of after cloning

Site Specific Mutagenesis Lanes 1, 6, 11: 1kb+

Lane 2 and 4: expected 210bp segment

Lane 3 and 5: expected 80bp segment

Lane 8 and 10: expected 3kb segment

1 2 3 4 5 6 7 8 9 10

11

Site-Specific Mutagenesis Site-Specific Mutagenesis Future PlansFuture Plans

We will not pursue the mutagenesis of the Kai ABC cassette further unless one feels the need to create a “cyanobacteria” BioBrick.

We are experimenting with the Lac promoter Lac + RFP, high copy LacIQ + RFP, high copy Lac + RFP, low copy LacIQ + RFP, low copy

Promoter ChoicePromoter ChoiceLacIQ + RFP, high copy

Kai BioBricks Ligate KaiA/B into BB vector for amplification We’ve decided to wait for synthesis of KaiC

Promoter search Test the tightness of the Lac promoter on a low-copy

plasmid with/without LacIq

Test a second promoter Experimental constructs

Ligate promoters, Kai BBs Decide which operons will go on which plasmids

Tradeoff between doing multiple ligations and multiple transformations

Western blots Reply to Prof. Golden regarding antibodies and prepare

experiments

Future GoalsFuture Goals