qPCR of CRISPR Cas Genes and DNA Isolations of D. marmoris and D. saxicola

 

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 and D. Saxicola, with very little information about them besides growth media and temperature conditions. They originate from the center landmass of Antarctica and grow in PYGV media at 10-15 degrees Celsius. They will be valuable contributions to the overall species sequencing data. Another species. D. fridgens was also attempted, but unsuccessful due t possible contamination but will also be included in sequencing.

Methods:

                Cas2 and Cas10 primer sets, primer set 2 for Cas2 and primer set 1 for Cas10, were verified by conducting a 1:2 serial dilution with 5 data points. 40 ul of cDNA was needed for the original dilution with 40 ul of PCR water, and mixing was done by pipette, flicking, and centrifugation. Again, 4 ul of diluted cDNA was used in each well which was carefully pipette mixed with a 6 ul combination of SYBR green and the necessary primer set for each gene. Three control wells with PCR water was also done for each gene. Settings were identical to the reference gene runs, with 36 cycles with an amplification temperature of 60 degrees Celsius. No melt curve was available at the end of the run because a setting was not correctly adjusted, which did hinder proper evaluation of the results.

                The two Antarctic species were grown on PYGV plate for 1 week and inoculated into PYGV broth and allowed to grow for 5 days. An OD600 value was taken of each culture and gram stains were done to verify identity and lack of contamination. Cells were packed 6 times due to the low OD600 value, and sonication was done for 30 seconds total with 5 seconds on and 5 seconds off. This solution was then integrated into the DNEASY extraction kit, but yielded little to no results that could not be used for sequencing. It was then decided to directly use just the DNEASY kit with bead beating although potentially sacrificing genomic fragment size, and bead beating was done for 2 minutes total, 1 minute at a time with 2 minutes on ice in between and after bead beating cycles. The samples were eluted in 30 ul of buffer and measured using the Nanodrop One.

Results:

      

Figure 1: Gram stain of D. saxicola, gram positive cocci

Figure 2: Gram stain of D, marmoris, gram positive cocci

Figure 3: qPCR results of Cas2 and Cas10 graphically and plate layout

Figure 4: Cq data for qPCR primer efficiency of Cas2 and Cas10

Discussion:

                The two species, D. marmoris and D. Saxicola were verified with DNEASY extraction with plenty of DNA needed for sequencing, and well within cleanliness parameters. The attempt at sonication shows that future attempts with D. fridgens should just be done with bead beating from the beginning. The blending of the sonication protocol with the DNEASY kit is something that can probably be worked out, but for purposes of sequencing and just needing one more sample, it is not worth investigating the protocol combination at this time.

                The Cas2 and Cas10 results show a lot of promise for being able to use these genes experimentally in the future. We did 1:2 dilutions of the cDNA assuming very little expression was going to be detected in a standard cell culture with no challenges. This was a gross underestimation of what was seen, but without the melt curve it cannot be officially determined that there is not another unexpected binding site of the primers. But based upon what was seen in amplification, next week we will do 1:5 dilutions for both Cas2 and Cas10, be sure to include melt curves, and hopefully get more consistent data for proper calculation of primer efficiencies. It is assumed to look so messy this time around because of the 1:2 dilution, and hopefully doing a 1:5 would correct that issue. It is also a possibility that there could be a bacteriophage in the culture that was grown for RNA extraction and cDNA synthesis, although a very small chance, which would be why Cas2 and Cas10 were so evident during qPCR amplification. The qPCR that will be conducted next week will be with cDNA from newly inoculated culture, so gene expression will be compared and could potentially be different if the original culture was coincidentally challenged and cas2 and cas10 were being unknowingly upregulated.