S-STEM Scholar Leilani Boren's Blog

 Introduction: D. sonorensis has been shown to have competence genes based off of bioinformatic analysis, with potentially even more that have not been identified yet. So far, pil genes associated with competence such as PilN and PilQ are present, as well as ComF, ComEA, and ComEc which are supposed to be directly related to working with PilQ to integrate one strand of foreign DNA into the cell. Other genes have also been identified in the Rec family, that are supposed to aid in DNA recombination and also relate to the integration of foreign nucleic acids. Due to this great amount of preliminary evidence, in addition to some of these genes also being present some of the known Deinococcus that can be transformed (D. radiodurans, D. geothermalis, and D. ficus) it seems prudent to attempt transformation with D. sonorensis. Preliminary work was done to prepare for this attempt next week.  The other goal of this week was to provide new DNA samples of D. caeni that may be less shear...

This week the top priority of the lab was to make sure that everyone had presentable posters for the ANAS and ASM conferences. My role in this week was to finalize my poster and then help other groups to finish theirs as well in terms of the writing, graphics, and the design. Although a very busy week, all of the posters were completed as well as another attempt at sequencing of four samples on Illumina. The first flow cell malfunctioned somehow, so this time before leaving for Las Vegas we pooled and diluted B81, D, caeni, D. pimenis, and D. grandis leaving out P65. These samples were prepared similarly to the last attempt with the exception of pooling 5 ul of each sample rather than 4 ul to make up the total 20 ul of volume necessary for library loading on the sequencer. Next week work will begin to analyze the electron microscopy images of D. sonorensis that were received, as well as preparing to potentially transform this specie for gene editing purposes.   Below is a copy...

Introduction: Last week the preparation was done to be able to sequence five samples using Illumina short read technology. The list of samples includes three species, two of which have not been sequenced in the lab before, and the two isolates of Deinococcus aquaticus. This week, D. pimensis, D. grandis, D. caeni, P81, and P65 were all prepared and sequenced. These sequences are so critical because they are provide new insight into each of these samples that can be utilized for potential experiments.   Methods: The samples were thawed and centrifuged, with approximately 85 ng of total mass used as input for the Illumina protocol. The samples were diluted with 1X TE provided by the kit to meet the volume requirement for the fragmentation step, which had zero modifications to it. Post fragmentation, adaptors were ligated with zero adaptor dilution as per experimentally validated protocol. The samples were cleaned using the magnetic beads, and eluted in 15 ul of 0.1X TE Buffer so...