S-STEM Scholar Madi Cook's Blog

 We are going to do cloning for the first time, and this includes inserting kanamycin resistance into our vector (pgRNA). This section has three parts take place simultaneously, this includes starting with prahm which has the promoter first on the plasmid, and then kanamycin resistance to be read, at which we will insert this into pgRNA which is our guide plasmid, the guide plasmid will have Amp resistance, and the restriction sits xbam and xbai, with which will be read from a counter clockwise direction. Xbai and Xbam in which will cut a certain piece of DNA with which we will stick it via engineered primers onto the guide RNA plasmid. The PCR product will be 919bp long, this includes the promoter, and kanamycin resistance. 

 The values the nanodrop had give for pgRNA were of the following: 78.0 (ng/ul),  1.87 (A260/A280), 2.22 (A260/A230). PWTCas9 58.4 (ng/ul), 1.85 (A260/A280), 2.09 (A260/A230). Prahm- 46.1 (ng/ul), 1.92 (A260/A280), and 2.09 (A260/A230). Performed plasmid extraction on Cas9  and gram stained plain Sonora. 250ml of LB broth were made as well as a gel. Several passages were also performed, on PWTCas9, D. sonora, and pgRNA. We had to reattempt PWT transformation as it could not be confirmed. This could be due to contamination, or not enough cells were present within the transformation protocol. Both a broth and a plate of R2A were inoculated with Sonora, we thought it would be best if we could successfully have transformed Sonora growing within a broth. 

 Returning back to lab on Wednesday, we had decided to explore the parameters of which Sonora was Tetracycline resistant as it had managed to grow on 150ug/ml plates. It was discovered that Tetracycline and Ampicillin would not be a good biomarker for D. sonora, as it was observed to have resistance to both innately. Thus, it was decided that we would move forward with prahm using Kanamycin as a biomarker, and Chloramphenicol resistance via dCas9. In order to test if Sonora had Kanamycin resistance, it was plated on 9ug/ml plates in order to observe the growth rate of such. It was observed to have no growth on Kanamycin, in which we decided to move forward with Kanamycin resistance for transforming Sonora. It was also confirmed that dCas9 with Chloramphenicol would be easier to work with as opposed to Tetracycline and Ampicillin as Sonora does not have innate Chloramphenicol resistance. First, we had to grow our plasmids on E.coli ( pgRNA, Prahm, PwtCas9/dCas9).