S-STEM TRAIN Scholar Emerson Turensky

 Introduction:  The previous week in lab involved a series of plasmid extractions to collect the bank of plasmid we will need to complete mutagenesis. From there, we started preparing the materials called for in the protocol we plan on following for cloning in the pgRNA plasmid vector. We started building our primers for the kanamycin gene we plan to target with PCR to clone into our vector. We reattempted transformation of pwtCas9 as well. This blog will go through how we went about the week and our results that follow.  Methods 1. Grow E. coli with pRHAM on LB plate with kanamycin concentration of 30ug/mL. 2, Grow E. coli with pgRNA on LB plate with Ampicillin concentration of 50ug/mL. 3. Grow E.coli with pwtCas9 on LB plate with Ampicillin concentration of 50ug/mL. 4. After 24-48 hour incubation period, gram stain each plate to confirm bacterial species. 5. Refer to Zyppy Plasmid Miniprep Kit to extract plasmid from each culture after overnight growth. 6. Run plasmid e...

 Introduction: This past summer, the time in lab has been focused around the successful mutagenesis of D. sonorensis. Our preliminary goal is to target the Carotenoid pathway to "turn off" the production of the classic pink pigment, which will result in white D. sonorensis.  We began with a proof of concept trial using pRad1, which went smoothly and showed us transformation can be done with D. sonorensis. Since then, our transformation attempts with the plasmids needed for mutagenesis have posed challenges. We have been unable to confirm any successful transformation with the plasmid responsible for cutting the target gene, pwtCas-9. To confirm transformation in the cell, any particular plasmid will contain antibiotic biomarkers that will gift the cell a new resistance to the specific antibiotic on the given plasmid. The biomarkers found on pwtCas-9 do not cooperate well with the bacteria chosen to transform. The TetR gene has been a poor biomarker, since D. sonorensis itsel...