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...

 Introduction: In previous weeks Illumina sequencing has been well underway, with multiple species and some isolates have newly developed de novo sequences that has provided a plethora of information and insight. This week was a continuation and preparation for the following week in which more isolates and species will be sequenced, including a replication of D. caeni due to incomplete data acquired the first run on Illumina. The two isolates chosen are considered to be 16s outliers in comparison to other isolates, and suspected of being the most genetically variant from type strain of D. aquaticus, PB314. The other two species, D. pimensis and D. grandis were chosen due to only having limited sequencing data available. D. pimensis has only scaffolding information and D. grandis has contig files is listed as having high levels of contamination that can and should be improved upon for clarity. This week included ensuring the quality of the stored DNA samples, cleaning up a few of th...

Introduction: Deinococcus sonorensis is a species of the genus that has been intensively studied in this lab previously due to its abnormal and unique characteristics when it is grown on TGY media. A poster presentation to develop an optimal DNA extraction method even when it forms its plaque structure was completed about a year ago, and now it is time to revisit the advancements that have been made in the past six months. Fortunately Illumina sequencing became available and the mysteries of D. sonorensis has started to unravel, thanks to being able to bioinformatically process this species and the addition of biochemical testing that has shed light on some new ideas. Not only are we now able to grow D. sonorensis on media and prevent plaque formations, we can extract its DNA better than ever before, and have findings that allow us to infer what proteins or systems may be responsible for its odd behaviors that make this species so interesting in the genus.  Methods: D. sonorensis w...

  Introduction: The 7 kb plasmid of Deinococcus aquaticus has been a long term goal for the lab to not only isolate effectively, but to genetically engineer in a way that it may potentially be useful for other Deinococcus applications in the future. The roadblocks with this plasmid began in just extraction methods from D. aquaticus, with those efforts not resulting in any significant clarity in how to most efficiently isolate this plasmid from living cells in high enough quantities to work with. Thus the idea to amplify the plasmid in a long amp procedure came to be, in which primers were made by Jonathan to not only produce considerable amounts of the plasmid but equip it with long overhangs of nucleotides that would be compatible for the Gibson assembly. So far there has been considerable difficulty validating this procedure in order to put chloramphenicol resistance (Cmr) onto the 7 kb so that this new plasmid could be tested in an array of species in the genus. This week, progr...