Deinococcus geothermalis Transformation and the beginnings of qPCR

 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. That being said, it is much more infrequent in current literature to evaluate gene expression of the CRISPR genes using qPCR, its baseline expression as well as expression under certain environmental stressors. We intend to test the expression of Cas2 and Cas10 genes found in D. geothermalis by growing three cultures separately at different temperatures, 30, 40, and 50 degrees Celsius. In doing this, it may be evaluated that there are different expression levels at different growth temperatures.  

Methods:

    Plates that had been grown for over 48 hours (over the weekend) were evaluated from growth after being incubated at 37 degrees Celsius.  The plates inoculated from the freezeback competent cells tubes and the culture prior to those tubes were all visualized and gram stained to confirm identity. The plate with supposed transformed cells showed preliminary growth with a few colonies, which were gram stained and then taken and inoculated onto another set of plates, one with standard TGY and the other on TGY with 3 ug/ml of chloramphenicol. These plates were allowed to grow again at 37 degrees Celsius for 48 hours and evaluated for growth and gram stained again. Before freezeback tubes of transformed cells were taken, the plate with chloramphenicol was allowed to grow longer so colony PCR could be done and potentially a plasmid extraction for molecular verification. These primers for colony PCR had not arrived this week to accomplish this, and the incubator was accidentally shut off at some point which inhibited the growth. Official validation of transformation will be conducted in the following week. 

    Primers were constructed using IDT with an ideal Tm of 60 degrees Celsius, and verified for multiple binding sites and possible hairpin structures, The reference genes to be used for qPCR will be SecA and Gap3, each believed to be consistent even in a change in temperature in the environment. RNA isolation was done with D. geothermalis that was grown in a broth culture for 86 hours at 45 degrees Celsius, with the standard Zymo kit with a final eltuion of 50 ul and two separate samples. cDNA synthesis was immediately accomplished with a BIO RAD kit using undiluted 14 ul of each RNA sample, undergoing DNase incubation and subsequent cDNA synthesis with a total 20 ul volume, which was stored overnight for primer validation. It was discovered the following day that one tube contained a volume that was wildly incorrect, and was discarded for lack of confidence. 

    Primer validation of secA was done first by 1:10 dilutions, but results indicated a lesser dilution was necessary to get data on all points. Five data points were made using a 1:5 serial dilution using the cDNA previously synthesized. Primer set 2 was tested with 4 ul of template cDNA (at the different dilution factors) and 6 ul of Mastermix and primer solution. Each well of the plate was mixed by pipetting, and three control wells with PCR water used as a substitute for the template DNA  were placed distinctly away from the sample wells. The plate was carefully covered and centrifuged for five minutes before undergoing qPCR with 98 degrees for 3 minutes, 95 degrees for 15 seconds, 60 degrees for 30 seconds (steps 2 and 3 repeated for 36 cycles) and finally 65 degrees for 5 seconds and 95 again for 5 more seconds. Ct values from qPCR were used to determine primer efficiency percentages. This process was identically done with Gap3, with the exception of using a 1:10 serial dilution and only four data points rather than five. 

Results:

    There seems to be cells that were transformed on the plates with chloramphenicol in Figure 1 and gram staining shows consistent results with D. geothermalis. None of the plates in Figure 3 showed any signs of contamination as seen in Figure 4 as well. 

    The RNA samples, one with 185 ng/ul and 160 ng/ul were used for the cDNA synthesis and resulted in two successful qPCR runs. SecA primer set two was validated with an overall primer efficiency of 102.5% and Gap3 set two was also validated with a primer efficiency of 100.25%. 

Figure1: Transformed D. geothermalis grown after 72 hours on a chloramphenicol plate 3 ug/ml. These colonies were taken and inoculated onto secondary plates.  

Figure2: Gram stain of transformed D. geothermalis from plate in Figure 1. Gram positive spherical cells seen. 

Figure 3: Plates inoculated and grown for 72 hours to verify source of contamination seen from the plates of the previous week. Two of the plates are from competent cell freezeback tubes and the third and fourth from original culture used to make the freezeback tubes. 

Figure 4: One gram strain from the four plates plates pictured in Figure 3, spherical gram positive tetrads seen in abundance. 

Figure 5: Visual curve of SecA primer set 2 qPCR results with five data points used to calculate primer efficiency. 

Figure 6: The calculations done for Gap3 with four data points, using given equations to verify that primer set 2 has an efficiency value of approximately 100.25%.

Discussion:

    This week was successful on multiple levels, likely confirming that D. geothermalis was transformed and SecA and Gap3 primer sets both being validated well within the range needed for reference genes going forward with qPCR. It was also verified almost definitely that the freezeback tubes with competent cells are not contaminated, and the source of contamination seen the previous week was solely due to poor aseptic technique during plating of the cultures. This was likely successful since plating this time was done in a sterile hood as opposed to open air. The primers in order to do colony PCR will hopefully arrive next week and the plate will also ideally exhibit more growth in order to officially verify transformation both in PCR and plasmid extraction to be seen on a gel, so that it can be deemed successful and freezeback samples can be taken. This is very exciting as a possible tool for CRISPR research and a potential indicator that pRad1 can be transformed into other deinococcus species such as aquaticus. The primer sets verified for the reference genes was also successful, but efficiencies well within the 90-110% efficiency range needed for experimentation. That being said, Cas2 and Cas10 primer sets will need to be verified next week, which will likely be much more difficult due to a presumed lack of baseline expression. It is also yet to be seen how exactly the experiment will be run on a single plate, and it may be needed to calibrate multiple plates with a reference gene with even tighter parameters, which would be very difficult to do but may be necessary for statistically sound results. 

References:

 

Campbell, J. A., & Cianciotto, N. P. (2022). Legionella pneumophila CAS2 promotes the expression of small heat shock protein C2 that is required for thermal tolerance and optimal intracellular infection. Infection and Immunity, 90(10). https://doi.org/10.1128/iai.00369-22