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). 

Returning to lab on Monday, we found that another lab mate's competent cells we had utilized for our project was contaminated so we had to scrap all of our bacterial specimens that was used with those cells. This set us back quite a bit, however we were able to bounce back. In the future, we intend on using our own supply of any cells we may need in order to reduce the risk of contamination again due to technical errors. It was established that we will be making R2A media every Monday, preferably 250ml of such in order to comfortably have a steady line of media. On Tuesday, we took the OD value of Sonora using R2A as our blank, with which we received an OD value of 1.63. On Wednesday we inoculated 600 micro liters of our bacterial culture with colony PCR in order to verify that our transformation of Sonora was successful. E. Coli was our positive control, while Sonora would be our negative control. This is because E. Coli contains the plasmid PRAD1 already, which is what we are try...

We are currently working with Deinococcus Sonora in the hopes of transforming the bacteria with PRAD1 plasmid, PRAD1 being the general plasmid that is utilized the most due to it's ability to be accepted easier compared to that of other plasmids. Returning to lab on Tuesday, we curated 250ml of R2B media and 250ml of R2A media. Within the R2A media there were two additional flasks, one containing the antibiotic Chloramphenicol with a concentration of 3mg/ml. The other flask contains R2A media without any antibiotic present. On Wednesday we made competent cells and inoculated those in order to engage in the transformation attempt of Sonora. We also poured 20 plates, and made more R2A media (250ml) to autoclave and be ready for use when we return to lab on Monday. Gram stains were performed on the transformed cells of Sonora to ensure no contamination was present, with which we garnered gram positive results, shape being that of purple spherical-short rods, that confirmed our use of ...

 We made Urease solution for several plates, 2 plates were inoculated with Staph and Subtillus. This was also a facet through which we could observe mechanotaxis and chemotaxis motility phenomena. On Tuesday we inoculated two plates of R2A with Sonorensis in order to redo plasmid extraction. On Wednesday, we performed the plasmid extraction finally, using eight plates and increase the radiation by 25,000 mj/cm squared per plate. One plate was our control, which was not exposed to any radiation. We also needed to fix our dilution factor for non-iradiated bacteria, it was too high compared to what we established for our experiment. On Friday we listened to a presentation about epigenetics and methylation. Methylation occurs near regions with hemimethylation. We also observed hypomethylation. The process for epigenetics includes starting with initial growth of bacteria, then performed DNA isolation. From there, we do DNA fragmentation, and continue forth with Bisulfide conversion. Wit...

 Upon returning to lab, we observed that our previous media containing R2B had reached a timeout due to not being autoclaved properly. In the future, more emphasis will be accrued to ensuring media and respecting specimens are to be autoclaved. Therefore, we had to once again remake R2B media and TGY media. We curated three flasks of R2B with Deinococcus Sonorensis in order to perform plasmid extraction, if there was a plasmid to extract in the first place. We did this via the zippy kit. We also poured urease plates, and inoculated four flasks of R2B with Xinjiangensis to continue forward with our UV testing. Sonorensis was observed to have formed white plaque within R2B, possibly due to the TGY component therein. We then took 1ml each in eight epindorph tubes. Centrifuged that for 1 minute at 13,000 rpm. Thereafter discarding the super natent, ensuring OD values of 1, and repeating the process of centrifuging if needed to enlarge the bacterial pellet. We made it a point to inocula...