DNA Extraction and Visualization of Deinococcus sonorensis

 Introduction:

    Deinococcus sonorensis is a species from the Deinococcus genus that has a history of being particularly difficult to work with, as seen during DNA extractions for sequencing efforts during last summer. It is a gram positive bacteria with a short rod morphology, as well as forming plaque like structures when its grown on agar plate. These plaque like structures, what may be a biolfim formation with further research, prevent these cells to become homogenous in solution even with maximum vortexing and pipette mixing. This issue is very problematic when doing DNA extractions, taking OD values and trying to normalize multiple samples, or most other protocols for any investigative research. This bacteria has only been studied in one paper so far, which is assumed to be because it is so difficult to work with. Previous DNA extractions with bead beating, lysozyme, and boiling as well as multiple combinations of these three techniques, produced very low yields of DNA which was frequently not clean for sequencing standards. For this upcoming poster, we have decided to visualize D. sonorensis with fluorescent microscopy and officially compare three common lysing techniques to demonstrate how effective each one is for bacterial cells that form these types of strange structures that prevent standard lysing methods from working properly. 

Methods: 

    It was decided that sonication, bead beating, and lysozyme were the three methods to be tested for this study using previously adjusted protocols. Each method would be in triplicates, and compared by fluorometer and by agarose gel to compare DNA fragment size and concentration. 

Cells were homogenized for each protocol by scraping colonies from plate into distilled water in an Eppendorf tube and a microbiology mortar and pistil set was used in order to physically break apart plaques for at least two minutes, and then was vortexed at maximum speed for at least thirty seconds. Each tube was then allowed to rest for three minutes so that large plaques can naturally settle to the bottom of the tube, The remaining supernatant is a homogenous solution, which can be Nanodropped with relative consistency, and removed for further use leaving behind the difficult plaques. We normalized to an OD value of approximately 1.00. 

After normalization, it was decided to do an initial test for the sonication method, which has not been done before up until this point. The sonicator was set to 12% intensity, and done for a total of 1 min on 5 seconds on/off intervals. One milliliter of cells was centrifuged and resuspended, once the supernatant was removed, in B1 buffer which is essentially a detergent lysing solution originally used in the QUIAGEN Genomic tip protocol for high molecular weight DNA extraction. The 1 ml solution was sonicated, and immediately put into the Zymo DNA cleanup and concentration kit, using two volumes of DNA binding buffer and a final elution of 25 ul. This samples was measured on the Nanodrop One and seen on the fluorometer, as well as visualized on a 1% gel. 

Fluorescent microscopy was also done using green dye and prepared by Ryan Grosso. Images were captured as seen below, identifying a plaque formation and then individual cells. 

Results: 

The sonicated sample was tested to have 59 ng/ul, and 260/280 value of 1.83, and 260/230 of 1.95, which fits parameters for sequencing. 

The gel showed very low molecular weight with a HINDIII ladder, around 1,000 bp average which is much lower than the high molecular weight seen with the Deinococcus aquaticus isolates, likely due to the fact that sonication time was increased from 30 seconds to 1 minute. 

The images below focus on one plaque formation found, although more were also seen. It is unsure if this bacteria does produce a film in order to be technically considered a biofilm, but more dyes can be used in the future to confirm or deny this suspicion. 

Discussion: 

    This bacteria was visualized with fluorescent microscopy for the very first time, but it is still unclear if it a bioflm or not. From what has been seen I personally do not believe it does form a biofilm, but the plaque like structures is very interesting and should be studied further, even more so with sequencing for any proteins that can be detected that would give insight as to how this bacteria does this and works. The gel did show very low molecular weight, but for the actual data it will likely be decreased to 30 seconds of total time rather than one minute, and hopefully that will resolve the problem enough for the DNA to be acceptable for sequencing, It was very surprising to see sequencing worthy DNA from a crude protocol for sonication and then to DNA cleanup rather than into a specific DNA extraction kit which leaves the possibility of future DNA extractions to be simplified and streamlined for time and resources by doing this routinely, but it needs to be done consistently in order to verify sequence worthy results can be achieved on a regular basis.