Primer Efficiency of Cas2 and Cas10 in D. geothermalis and PCR of Prad1

 Introduction:

    Last week there was a failed attempt at calculating the primer efficiencies of the Cas2 and Cas10 genes from D. geothermalis needed for the impending experiment to determine whether or not there is any change in gene expression at different growth temperatures over a 72 hour period. This week another attempt was made and failed at Cas2 and Cas10 due to over loaded cDNA from high yield RNA extractions, and finally the third attempt was successful in attaining favorable primer efficiencies for both Cas2 and Cas10 that indicates the experiment can move forward. As well as that, confirming the transformation of D. geothermalis with Prad1 was done with Prad1 AMP primers that would hopefully amplify a fragment 861 bp in length that could be visualized on a gel. The Prad1 primers were also verified to make sure they were working properly, and D. geothermalis tested in two ways. 


Methods:

    RNA was extracted from a culture of D. geothermalis grown for 72 hours at 45 degrees Celsius, using the standard Zymo kit with an elution volume of 50 ul in PCR water. These samples were run on an RNA gel to verify identity and confirm a lack of DNA contamination. Each of the two samples had about 1 ug/ul and 14 ul was input to cDNA synthesis, which included a Dnase incubation period and then a reverse transcriptase incubation period. These two cDNA samples from RNA extraction sample 1 was frozen overnight and used in 1:5 serial dilutions with 5 points of data for qPCR validation of the Cas2 primer set 2 and Cas10 primer set 1 with 4 ul of template DNA at the different dilutions and 6 ul of SYBR green and primer solution in each well. Each well was pipetted up and down three times for mixing and the plate was centrifuged for 5 minutes at max speed prior to analysis. The first set of unknowns with higher concentrations for both Cas2 and Cas10 had erratic amplification results, but lesser dilutions appeared to be close in standard deviation and curves that we would expect. It was determined that too much RNA was input into cDNA synthesis, which should have a maximum of 1 ug RNA input. New cDNA was made after diluting the original RNA sample, and qPCR was attempted again and results recorded and used to calculate primer efficiency values. 

D. geothermalis that was presumed to be transformed with Prad1 underwent a plasmid extraction and a DNA extraction in order to verify transformation. The plasmid extraction was found to be 44 ng/ul using the Thermo fisher kit, but on a gel it could not be visualized. AMP primers were used during pCR with one control (PCR water) and D. geothermalis DNA extraction on one gel, and then another gel the same primers and PCR conditions with the same control in addition to a positive control with Prad1 extracted from E. coli and plasmid extraction sample from D. geothermalis, which previously wasn't seen on the gel to confirm identity. 

D. geothermalis was inoculated from a single colony on a plate and will be allowed to grow at 40 degrees Celsius for 72 hours until temperature differentiation on Monday morning. 

Results:

    The primer efficiencies of Cas2 and Cas10 resulted in 104.4% and 107.7% respectively.

Figure 1: RNA gel from D. geothermalis used for qPCR

Figure 2: qPCR results and primer efficiency calculations for Cas2

Figure 3: Gel 1 with AMP primers, also including Jonathan's potential aquaticus transformation with Prad1 and 7kb plasmid verification from aquaticus. The only band seen in the 7kb verification related to a different project group. 

Figure 4: Gel 2 with Prad1 from E. coli for positive control and D. geothermalis plamsid as template DNA from plasmid extraction. Only bands visible are from Prad1 E. coli control, the AMP fragment being at 861 bp in length and most prominent band. 

Figure 5: Melt curves from Cas2 and Cas10 qPCR verifying that no secondary binding sites are present. 










Discussion:


The results are positive for the D. geothermalis Cas2 and Cas10 experiment, although the very beginning, having primer efficiencies that are well within acceptable range is encouraging for moving forward with the actual experiment of seeing a change at 30 and 50 degrees Celsius in comparison to 40 degrees. It was realized the utmost importance to dilute the RNA for cDNA synthesis, and knowing thus going forward will be crucial for the impending RNA extractions and qPCR in the near future. As well as that, it is hopeful that only adjusting temperature is a relatively easy challenge that will allow for a swift path to tangible results that we can analyze for CRISPR information. The D. geothermalis transformation results is worrisome, but transformed cells were passaged onto a new chloramphenicol plate since the old plate was likely grown so long the antibiotic degraded and cells may have expelled the plasmid we had originally transformed. Hopefully new colonies will grow and verification of transformation will be undeniably successful next week. 

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