1. Week 7 review Cyanobacteria Oscillator in E. coli

2. 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.

3. Week in Review Performed a splitting PCR to extract KaiA, KaiB, and KaiC individually from previous genomic extraction Failure (no bands showed) Performed mutagenic PCR #5 on HH5 and HH6 First mutagenic PCR with corrected primers Streaking Didn’t find bands of expected size Ran a gel testing for contamination Primer diagnostic (negative) Performed mutagenic PCR #6 on LC1 and LC2 (newly extracted template) Still streaking in some experimental lanes Primer controls (primer w/o template) show faint streaking

4. 1. 1kb+ ladder 2. 10 ng LC1 template, 3-10 primers 3. 10 ng LC1 template, 9-8 primers 4. 10 ng LC1 template, 6-7 primers 5. 10 ng LC1 template, 5-4 primers 6. 5 ng LC1 template, 3-10 primers 7. 5 ng LC1 template, 9-8 primers 8. 5 ng LC1 template, 6-7 primers 9. 5 ng LC1 template, 5-4 primers 10. 1 ng LC1 template, 3-10 primers 11. 1 ng LC1 template, 9-8 primers 12. 1 ng LC1 template, 6-7 primers Mutagenesis PCR #6 Gel #1

5. Mutagenesis PCR #6, Gel 2 1. 1kb+ ladder 2. 1 ng LC1 template, 5-4 primers 3. 10 ng LC2 template, 3-10 primers 4. 10 ng LC2 template, 9-8 primers 5. 10 ng LC2 template, 6-7 primers 6. 10 ng LC2 template, 5-4 primers 7. 5 ng LC2 template, 3-10 primers 8. 5 ng LC2 template, 9-8 primers 9. 5 ng LC2 template, 6-7 primers 10. 5 ng LC2 template, 5-4 primers 11. 1 ng LC2 template, 3-10 primers 12. 1 ng LC2 template, 9-8 primers 13. 1kb+ ladder 14. 1 ng LC2 template, 6-7 primers 15. 1 ng LC2 template, 5-4 primers 16. no template, 3-10 primers-- negative control for primers 17. no template, 9-8 primers-- negative control for primers 18. no template, 6-7 primers-- negative control for primers 19. no template, 5-4 primers-- negative control for primers 20. Cat template, Cat primers-- positive control for LC2 master 21. Cat template, Cat primers-- positive control for primer control master 22. no template, no primers-- negative control for LC1 master 23. no template, no primers-- negative control for LC2 master 24. no template, no primers-- negative control for primer control master Mutagenesis PCR #6, Gel 3

6. Digest recap 1. 1 kb+ ladder 2. kaiabc+topo HH1 3. kaiabc+topo HH2 4. kaiabc+topo HH3 5. kaiabc+topo HH4 6. kaiabc+topo HH5 7. kaiabc+topo HH6 8. kaiabc+topo TP 9. control 10. 1 kb+ ladder

7. Sequencing results from one clone: M13F and M13R from TopoII(blunt) only primers which bound BLAST results: M13F; 758bp identity (99%) to E. coli K12! (out of 822bp) Position(64,822) to (4267776, 4267018) M13R: 744bp identity (99%) to E. coli K12! (out of 832bp) Position (85, 828) to (4266486,4267230) AlignX finds no homology of M13R/F sequence to Topo vector Could not find BB prefix/suffix in M13R/F sequence Where did the e. coli fragment come from? Sequencing results: HH1 template

8. We used this template for most of our PCR experiments (mutagenesis, splitting) because it looked promising in our digest BLAST results: M13F; did not look like it binded well M13R: 759bp identity (98%) to Haemophilus somnus ! (out of 859bp) Position (84, 849) to (1632, 864) Also 98% to Streptococcus agalactiae No similarity with PCC 7942 cyanobacteria Seems to be a shared transposase Sequencing results: HH6 template

9. Strategy We’ve spent a lot of time performing and debugging PCRs in the last couple weeks (5 genomic extraction PCRs, 6 mutagenesis PCRs, 1 splitting PCR). Our diagnostic digest suggests that we have template, but our sequencing results and failed mutageneses suggest we don’t Our primer stocks may also be contaminated We need to be ready to run experiments as soon as our synthesized KaiABC constructs arrive on 8/8 Therefore we want to shift our emphasis from beating up cyanobacteria for its lunch money to preparing for our experiments

10. To-do Continuing work with cyanobacteria: Try a splitting PCR straight off the cyanobacteria genome. Saves a PCR step Mutagenesis and crossover will probably be easier Immediate feedback: if we get 850bp, 300bp, and 1600bp bands, we can be almost positive that they’re KaiA, KaiB, and KaiC Order new mutagenesis primers? Further diagnostic digests on new template if necessary

11. To-do In preparation for experiments: Finalize promoter choice, acquire promoters Continue investigating Western blot and possibly mass spectrometry as measurement techniques Learn more about tagging KaiABC proteins