S-STEM Scholar Leilani Boren's Blog

 Introduction: This week there was a continuation of sequencing, as three isolates were successfully completed given the current data analysis. The two isolates that were not in sufficient mass to reach a 1 nM concentration for loading, P34 and P71, were reprocessed again with the adjustments made to the protocol including an additional PCR cycle and not diluting the adaptor solution added to the samples. A DNA extraction had to be completed for D. sonorensis due to contamination suspicions the last time it was attempted, and this new sample was processed making up the third specie included in this run. Samples of D. metalli and D. xinjiangensis that were DNA extracted approximately a year and a half ago were cleaned up and accounted for samples four and five for the pooling and sequencing attempt. Finally, SecA and Gap3 reference genes were primer validated and results were calculated for efficiency.  Methods: D. sonorensis was DNA extracted by growing cells on R2A plates for...

 Introduction: This week mostly consisted of prepping DNA samples of Deinococcus aquaticus isolate species that would be used for the first multiplex sequencing run on Illumina technology. These isolates were selected based on environment and previous data collected by another lab, in order to complete the genomes and potentially move forward with a study of the chemotaxis and motility pathways of D. aquaticus. In addition to this DNA prepping, another set of primers for the D. aquaticus qPCR experiment were also validated using identical methodology from the previous run with CSP. By the end of this week, a sequencing attempt was made with three of the five isolates originally planned, and HSP primers were validated.  Methods: Five high molecular weight DNA samples, P21, P17, P34, P71, and FR100, were removed from storage in the minus *0 freezer, and allowed to thaw for initial testing of the concentration and purity values to confirm eligibility to be used in DNA prep protoc...

 Introduction:  This week included an RNA extraction from D. aquaticus with the intent of using this as template for qPCR primer validations, and another attempt at sequencing of D. sonorensis. The experiment with D. aquaticus has been founded upon the sequencing data produced from this lab, identifying the Type III-A CRISPR-Cas system that is fairly unique within the genus, and especially being the only system in the species without interaction or even the presence of another type. The original project was actually utilizing D. geothermalis, but its multisystem components were a potential source of major error or a level of interaction we were not prepared to investigate yet. By studying D. aquaticus, we have effectively decreased the level of complexity for the time being and likely have decreased the range in temperature that will be used for the final experiment. The genes of interest in this species is Cas1 and Cas10, both major factors in bacterial immunity. Cas1 is prom...

 Introduction:  This week included three major advancements in two projects. The majority of the week went towards efforts in sequencing, in DNA extraction of D. sonorensis and then DNA sampling prep for Illumina NEBNext sequencing for both the D. sonorensis and the D. caeni sample extracted the previous week. Both preps were attempted to be sequenced, with one run of D. caeni successfully completed with a genome coverage of 99.999% and the run of D. sonorensis unable to be completed due to technical issues. The latter part of the week was dedicated to trying to identify the problem in the long amp PCR protocol of the 7kb plasmid from D. aquaticus , which is set to be amplified with Gibson primers to attempt Gibson assembly with the already attained Cmr gene. The week ended in mixed results, but with directions for future attempts.  Methods:  D. sonorensis DNA extraction was completed with the DNeasy QUIAGEN kit, with slight alterations to the original protocol. Cell...