Week 8

Outline Project Goal Extracting and biobricking KaiA and KaiB Synthesis update Western Blotting update Site-Specific Mutagenesis update Promoter Choice Future 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 Goal Reconstitute 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 ends 3. 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 BioBricks

kaiB 367 bp In standard vector kaiA 913 bp In 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

Site-Specific Mutagenesis Future 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 Choice LacIQ + 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 LacI q 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 Goals