Week 7 review

Cyanobacteria Oscillatorin E. coli

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.

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

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

Mutagenesis PCR #6, Gel 21. 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 master22. 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

Digest recap1. 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 TP9. control 10.1 kb+ ladder

• 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

• 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

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

To-doContinuing 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

To-doIn preparation for experiments:

Finalize promoter choice, acquire promotersContinue investigating Western blot and

possibly mass spectrometry as measurement techniques

Learn more about tagging KaiABC proteins