Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

1. Make modifications to the plasmids
a. Use PCR to amplify the plasmids
b. Point mutagenesis with specific primers to introduce digestion sites (to S.pyCas9 Plasmid)
c. Transform in DH5α and plate to isolate colonies
i. Check to ensure that our point mutation is correct either by Sanger Sequencing or colony PCR with the mutated primers
d. Digest at the newly introduced sites to cut out the portion of S.pyCas9 plasmid that we need
e. Ligate this insert into one of the empty backbones from iGEM
f. Clone the SaCas9 that was order into the other empty backbone from iGEM
2. Make point mutations to the WT-Cas9 plasmids to disable nuclease activity and make them dCas9
a. Use primers introduce these point mutations in each of the plasmids
b. Confirm the mutations by transformation into DH5α to separate colonies and submit to purified sample to Sanger Sequencing
3. Transform the testing plasmid into BL21 Competent E. coli and use corresponding selection antibiotic to confirm
4. Establish Controls
All Controls need to be performed on triple selection plates with triple transformation
a. Control 1 - Average Colony count with Testing Plasmid and the other empty plasmids
b. Control 2 - Average Colony count with WT-SpyCas9, testing plasmid, and the other empty plasmid
c. Control 3 - Average Colony count with WT-SaCas9, testing plasmid, and the other empty plasmid
d. Control 4 - Average Colony count with d-SpyCas9, testing plasmid, and the other empty plasmid
e. Control 5 - Average Colony count with d-SaCas9, testing plasmid, and the other empty plasmid
5. Perform the final competition assay again with triple transformation

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.