Week of 8/21/24 Plasmid Extraction and D. sonorensis Transformation

 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). 

Comments

Post a Comment

Popular posts from this blog

Week of 11/22/24 pH Experiment on Sonora's Biofilm Breakdown

Image
 After incubating the pH range of 6,7, and 8 the cells were gram stained after 24 hrs along with a plain TGY broth containing Sonora for a comparison and all of the flasks were homogenized strangely enough. The morphology was still irregular as if the cells were still dying. The gram stains albeit were not completely viable, as there was a variable gram negative to gram positive ratio. The OD values were taken as well, and the results were also varied as pictured below.
Read more

Week of 11/18/24 Breakdown of sonora's Biofilm via pH Experiment

 In order to move forward with determining how to breakdown Sonora's persistent biofilm, a trial run of using varying pH values (pH 6, 7, 8) with 20mMolar MOPS buffers in TGY broth were made. Upon inoculating Sonora and incubating in the -30 degree shaking incubator for 72hrs, all of the flasks of varying pH had homogenized Sonora. The gram stains had revealed that it was in fact Sonora, yet the morphology was irregular as if the cells were dying. Going forward the trial will be adjusted to a incubation time of 24hrs rather than 72hrs in order to determine if it is the buffers that are effecting the cells or simply the incubation time. 
Read more

Week of 11/4/24

 A gram stain on pdCas9 e.coli was performed in which it came out to be contaminated we think this may be due to the parent plate having been contaminated. So we are retransforming e.coli with pdCas9 and then confirming that contamination did not take place. In the event that we had e.coli with pdCas9 confirmed, we did a freezeback of it so as to have a source. 
Read more