S-STEM Scholar Leilani Boren's Blog

  Introduction:                   Last week, two reference genes were confirmed with primer set efficiencies that fit within the desired parameters for experimentation. This week, the two experimental genes need to be evaluated for primer set efficiencies to be able to go forward with the actual experiment of how growth temperature effects the gene expression of Cas2 and Cas10. Each primer set has been verified for hairpins structures and are assumed to be at this point binding to only one site in the D. geothermalis genome. This will be verified using qPCR data, and evidence of any cas2 and cas10 detection would be a first for the lab so far.                 DNA extractions were also carried out for two new Deinococcus species that will be sequenced in the next few weeks. These two species are relatively unknown, D. marmoris...

 Introduction:     The previous week yielded questionable results in terms of a successful Deinococcus geothermalis transformation of pRad1 with chloramphenicol resistance. It seemed like there was some contamination that could have been attributed to the original sources of competent cells or inoculation of transformed/control cultures onto plates. Ultimately, a successful transformation would be a positive stride forward for future experiments challenging the CRISPR-Cas system of D. geothermalis and analyzing changes of gene expression using qPCR. This week, transformation results from the previous week was evaluated and a preliminary qPCR experiemnt with D. geothermalis was begun with the validation of two primer sets, one for each reference gene that will be used going forward. It has been seen in other studies that Cas proteins can be multifunctional within a cell, one paper even detailing Cas2 as heat shock protein as well as a its function within the CRISPR system....

 Introduction:      In the previous week, four plates were inoculated with two different cultures of D. geothermalis in order to evaluate transformation efficiency of Prad. This plasmid from E. coli can be useful for future transformation experiments that would place selective pressures on the CRISPR-cas system of D. geothermalis, which is very detailed and described in comparison to others in the genus such as D. aquaticus . The two cultures were of one control group that underwent the transformation process and but was not exposed to the actual plasmid, whereas the experimental was. The plates that were grown for those 72 hours were evaluated for transformation and possible contamination, and another attempt was made using the same protocol after more precautionary measures were taken. Other plates were also inoculated so that the potential source of contamination in the previous plates could be narrowed down.  Methods:     Th...

Introduction:      Deinococcus geothermalis is a species that has been shown capable of transformation with an ancestor of the Prad1 plasmid, as well as a detailed CRISPR-cas system. These two characteristics can hopefully be used in order to further study the CRISPR system of D. geothermalis with the use of qPCR, but first, transformation must be completed successfully with Prad1 with chloramphenicol resistance. The plasmid was first extracted from E. coli and then verified prior to a transformation attempt. A successful transformation of D. geothermalis can hopefully indicate a potential application for D. aquaticus , in also testing its CRSIPR system.  Methods:     E. coli was grown on a plate and gram stained to assure correct morphology and lack of contamination, and input into the ThermoFisher plasmid protocol kit, yielding 190 ng/ul solution with a total volume of 50 ul. One microgram of plasmid product was verified for identity with restriction e...