Experimental qPCR of Deinococcus geothermalis with Cas2 and Cas10 Genes

 Introduction: 

    Deinococcus geothermalis, the thermophilic bacterial species that has not one but two CRISPR systems according to genetic evidence, has been confirmed to have primers that are within range for acceptable efficiency levels so experimentation can begin. The original experiment included growing Deinococcus geothermalis and then exposing cultures to three different temperatures for a period of time to evaluate a change in gene expression of Cas2 and Cas10. This would potentially indicate that a change in environment can upregulate or downregulate CRISPR genes, which can be useful in understanding how these systems operate in life. It had been previously seen during primer efficiency validation runs that the baseline gene expression of D. geothermalis grown at a constant 45 degrees Celsius has a Cas2 and Cas10 transcription level that is similar to genes considered to be crucial for survival like our reference genes, Gap3 and SecA. This week, D. geothermalis was grown and exposed to three different temperatures, 30, 40 and 50, degrees Celsius and then RNA extractions were carried out and verified prior to going into qPCR, first testing 40 and 50 degrees. 


Methods: 

    D. geothermalis was grown for 72 hours from a single colony on a plate at 40 degrees Celsius at 100 RPM continuously in a 250 mL flask with a cell culture total volume of about 15 mL in TGY. This culture was evenly divided between three autoclaved flasks which approximately 5mL of the grown culture a piece. These aliquots were exposed to their various temperatures, one flask at 30 degrees, one still at 40 degrees, and the third at 50 degrees Celsius with no rotation for 4 hours. After 4 hours of exposure time, three biological RNA samples were extracted from each flask, for a total of nine samples processed simultaneously. They were eluted in PCR water with a total volume of 50 ul and verified for cleanliness and concentration with the Nanodrop One and then visualized on an RNA gel with Midori Green dye for identity. cDNA synthesis was conducted with a 20 ul total volume using only 1 ug of RNA for each sample of cDNA. 

    The plate was loaded according to the diagram below, with 6 ul of the designated mastermix with each gene's primers, and 4 ul of all of the cDNA samples, diluted to a 1:5 solution. 36 cycles were done at data was evaluated. 

Cas2

50°C

Cas2

50°C

Cas2

50°C

Cas10

50°C

Cas10

50°C

Cas10

50°C

SecA

50°C

SecA

50°C

SecA

50°C

Gap3

50°C

Gap3

50°C

Gap3

50°C

Cas2

50°C

Cas2

50°C

Cas2

50°C

Cas10

50°C

Cas10

50°C

Cas10

50°C

SecA

50°C

SecA

50°C

SecA

50°C

Gap3

50°C

Gap3

50°C

Gap3

50°C

Cas2

50°C

Cas2

50°C

Cas2

50°C

Cas10

50°C

Cas10

50°C

Cas10

50°C

SecA

50°C

SecA

50°C

SecA

50°C

Gap3

50°C

Gap3

50°C

Gap3

50°C

Cas2

40°C

Cas2

40°C

Cas2

40°C

Cas10

40°C

Cas10

40°C

Cas10

40°C

SecA

40°C

SecA

40°C

SecA

40°C

Gap3

40°C

Gap3

40°C

Gap3

40°C

Cas2

40°C

Cas2

40°C

Cas2

40°C

Cas10

40°C

Cas10

40°C

Cas10

40°C

SecA

40°C

SecA

40°C

SecA

40°C

Gap3

40°C

Gap3

40°C

Gap3

40°C

Cas2

40°C

Cas2

40°C

Cas2

40°C

Cas10

40°C

Cas10

40°C

Cas10

40°C

SecA

40°C

SecA

40°C

SecA

40°C

Gap3

40°C

Gap3

40°C

Gap3

40°C

 

 

 

 

 

 

 

 

 

 

 

 

Cas2

NTC

Cas2

NTC

Cas2

NTC

 

 

 

SecA

NTC

SecA

NTC

SecA

NTC

 

 

SecA

40°C

Results: 

The results were disappointing in that the standard deviations between identical biological samples, across all genes and temperatures, were too large to evaluate the data. It seemed off of initial analysis that the approximate values of Cas2 and Cas10 were not very different but it is not possible to say so at this point without a statistically successful run. 

The RNA samples that will be used going forward:

 

Ng/ul

260/280

260/230

30A

806

2.07

1.48

30B

767.3

2.11

2.07

30C

1102

2.13

2.14

40A

1008

2.00

1.14

40B

1107

2.10

2.09

40C

600.9

2.13

1.79

50A

933

2.08

2.04

50B

636

2.09

1.83

50C

854

2.11

2.08


Discussion: 

    In the future, these same RNA samples will be used to make new cDNA samples that can be used for qPCR. The problem that was discovered as to why this qPCR run did not work with wild standard deviations, was because the cDNA for reach biological sample and temperature was not normalized, therefore different amounts of cDNA was input for each temperature, which doomed the run. That being said, it is hopeful that correcting this issue will solve this problem in the next run and useful data will be attained for proper analyses. That being said, we are no expecting a higher likelihood a change in gene expression from 40 to 30 degrees Celsius, or a greater difference than will ever been seen between 40 and 50. It is also a possibility that the cultures need to be exposed to different temperatures for a longer amount of time than 4 hours to appropriately assess a change in transcription.  










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