Attempted Transformation of Deinococcus geothermalis

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 enzyme protocol using 1 ul of XbaI, buffer, and the remaining volume of PCR water to 50 ul. The sample was incubated at 37 degrees Celsius for 15 minutes and then inactivated for 20 minutes at 65 degrees Celsius. The entire 50 ul sample was loaded onto a one percent agarose 1x TAE buffer gel to see a fragment approximately 6800 bp in length, measured with the HindIII ladder.  

    To make cells competent for transformation, a broth culture of D. geothermalis was grown over 48 hours at 37 degrees Celsius and 1.5 ml of culture with calcium chloride solution,  was placed in a cryovial tube with 0.5 ml of 60% glycerol and shaken. This tube was frozen at minus eighty degrees Celsius for 24 hours and thawed on ice the next day. One hundred microliters of the 2 ml freezeback sample was placed in one Eppendorf tube with one microgram of plasmid for transformation. Incubation was on ice for ten minutes, and then placed in a 37 degree Celsius incubator for 45 minutes. During the second incubation, the tube was also inverted twice every 15 minutes. 

    The entire sample was transferred into 4 ml of TGY and incubated at 37 degrees Celsius for 16 hours with continuous agitation. The next day, two plates with 3 ug/ml of chloramphenicol and two plates with regular TGY agar were evenly inoculated and allowed to incubate at 37 degrees Celsius for 72 hours to exhibit growth. 


Results:

    The plasmid extraction of Prad1 from E. coli yielded a sample with 190 ng/ul, a 260/280 of 1.90 and a 260/230 of 2.01. The results seen in Figure 1 indicate a 6800 bp fragment that is consistent with the known length of Prad1. 

Figure 1: From left to right- HindIII ladder, and one microgram of Prad1 plasmid post XbaI protocol. The band seen is within markers that indicate 6000 to 8000 bp, correctly identifying the plasmid at roughly 6800 bp in length. 


Discussion:

    Although not much results this week, the preparation has been made to evaluate the two sets of plates after the 72 hour incubation period to visualize the transformation efficiency of D. geothermalis for the first time, if any. There was some concern that the amount of growth visualized after 16 hours overnight incubation for plasmid expression was lower than normal, but this species also holds a higher ideal growing temperature than most Deinoccocus genus species do, which is likely why growth was stunted. That being said, given approximately 72 hours to grown on plates will likely be enough time to see results of the transformation attempt. Regardless if this first time is successful or not, there is plenty of plasmid and competent cells to attempt another transformation, this time with a negative control for the transformation protocol, which would have been better to do the first time around. Thankfully, there is also a greater confidence in the plasmid extraction of Prad1, which can be used in the future, potentially with D. aquaticus as we have come across new findings in its CRISPR-cas system which make it more attractive to transform and evaluate in the future.


References: 

Brim, H., Venkateswaran, A., Kostandarithes, H. M., Fredrickson, J. K., & Daly, M. J. (2003). Engineering deinococcus geothermalis for bioremediation of high-temperature radioactive waste environments. Applied and Environmental Microbiology, 69(8), 4575–4582. https://doi.org/10.1128/aem.69.8.4575-4582.2003

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