S-STEM Scholar Melanie Castaneda's Blog

  Introduction:  This week we continued our work with E. coli pRHAM. Last friday, we completed a plasmid extraction in order to get the pRHAM plasmid out. This week, we focused on confirming that we successfully extracted the plasmid before moving onto using that sample in PCR. If both of these go smoothly, we will be able to begin assembly, using Gibson Assembly, at the beginning of next week.  Methods: 1/29/2024  Using gel electrophoresis was our way to confirm if we had extracted the pRHAM plasmid. We mixed 2 μL of UV blue dye with 10 μL of our plasmid sample in an Eppendorf tube. We spun our tubes down in the pop spinner to make sure it was all one homogenous liquid. Since the plasmid’s size is 947 kb, we added a 1 kb ladder into one of the wells so that we could compare the size of our band to the ladder. We let the gel run and checked it under the UV light.   1/30/2024 Using our plasmid sample, we ran a short-amp PCR. We mixed 8 μL of mastermix ...

  Deinococcus radiodurans Carotenoid Knockout Introduction:  Prior to winter break, I was given the opportunity to work on a new project that was separate from my previous work with minimum inhibitory control on different Deinococcus species. While I am still working on that project, my primary focus has shifted to working with a new group to transform Deinococcus radiodurans from a red color to a white color. We hope to achieve this by knocking out the crtB gene in D. rad . While this is only the tip of the iceberg, if we are able to successfully do this, we can go on to transform Deinococcus caeni.   In order to be able to transform D. rad, we would need to extract separate pieces from different bacteria. We started off by growing E.coli pRAD1 from freezebac and D. rad from a slant that a classmate gave us. After gram staining them to make sure the samples looked good, we went ahead and got started. From E.coli pRAD1, we needed to extract the pRAD 1 plasmid and p...

  Researching Deinococcus Species  After inoculating new species and failing to see growth after multiple days, we were beginning to become frustrated since it was slowing down our project. There seemed to be a lot of waiting around to see when a species would be ready to work with while the current species we were currently testing and growing in the incubator.  This made me question if we were doing something wrong or if there was more information that we could find out about the Deinococcus genus. I decided to go on a search to learn more about Deinococcus in order for us to be more productive and prepare more for the following weeks.  Coming into this project, I was introduced to two species of the Deinococcus genus, Deinococcus radiodurans and Deinococcus aquaticus . Both of these species seemed to have a similar growth time of about 48 hours in a 30°C environment. Our newly inoculated species were placed in the same conditions, for the same amount of time, and...