Further Investigation of D. sonorensis and Lysis Methods for DNA Sequencing

 Introduction:

As a continuation of last week's project, research into D. sonorensis' optimal lysing method for DNA extraction was further studied, and usable data was necessary in order to create a poster for the upcoming conferences. With this particular goal in mind, it was crucial to maximize time and resources to produce results that could hopefully be  measured and visualized with a coherent story that would be useful for not only this lab, but other labs that are currently dealing with a bacterial species that forms the signature plaque like structures that D. sonorensis likes to make. These plaques have been described in the one paper it is featured in, where it was originally isolated and scarcely characterized due to the "clumping" formations that it makes (Rainey et al 2005). These structures inhibited the work of these individuals, and have historically been difficult for us as well. This is exactly why it is so important that not only can these cells be homogenized in an efficient and relatively easy method, but specifically for sonorensis that it be sequenced in order to evaluate its strange capabilities. 

Methods:

It was decided to adjust a few things from the previous week for the homogenization of the plaques of cells, primarily resuspending the cells in TGY media as opposed to deionized water, to prevent initial and unmeasured osmotic lysis, which could affect the results seen after the protocol of all three lysing techniques. 

One plate of R2A was grown for 24 hours (due to poster time constraints) rather than the typical 48 hour growth time, but enough growth was seen in order to produce nine identically homogenized samples. Three replicates were used for each lysing method, and the nine total samples were normalized to an OD600 value of 0.4 plus or minus 0.1. This was done using the developed technique of scraping numerous plaques from plate and physically disrupting these plaques with the micro mortar tool, and the vortexing these plaques at max speed for about one minute. After allowing to incubate for five minutes at room temperature, the remaining plaque structures will settle to the bottom and the resulting supernatant could be extracted in 500 ul increments for testing. 

The sonication was done at 12% intensity, 30 seconds total with 5 second on/off intervals in 1 mL of B1 buffer from the QIAGEN Genomic tip protocol. 

Bead beating was again a resuspension of the cells in 350 ul of B1 lysis buffer, and done for 10 minutes total with 2 min on/1 min off and on ice intervals. 

Lysozyme was done with 500 ul of 1 mg/mL in a sodium acetate- acetic acid buffer of 4.03 and incubated at 37 degrees Celsius for thirty minutes. 

Measurement was done with the fluorometer and the purity tested with the Nanodrop, after all three different methods and their replicates were immediately put into the ZYMO DNA cleanup and concentrator kit, with a 25 ul total elution in buffer.

A 1% gel was made to visualize the molecular weight of the genomic DNA extracted from the samples, in hoped that the sonicated samples still maintained a relatively high molecular weight. Unfortunately, the 1 kb extended ladder was done incorrectly, as well as the some of the samples not having a high enough concentration to be able to be visualized on a gel. There was also a significant issue loading the gel in the first place, as samples did not seem to "sink" into the wells as they should have. Therefore, no image was taken of this gel because it was useless for the poster and this project in general.  

Results:

Below is the data table of the results, with the aggregate averages at the bottom of the table, indicating the overall performance of each lysis method. 

Discussion:

The results of this week successfully indicated that the sonication method produced not only the highest yield of DNA, which is the most we have ever seen from D. sonorensis, but also acceptably clean for basic 3rd generation sequencing. Given new chemistries these samples may still not be good enough for our current Nanopore technology, but it could be done in a pinch for at least scaffolding data of a species. The high protein ratios seen are indicative of RNA contamination, so next week will also be focused on adapting this protocol to all the other Deinococcus species and including a RNAse A step in order to keep the protein ratio between the goal range of 1.75 and 1.90. This is very narrow, but it may be doable. The other possibility if this protocol cannot be successfully adapted will likely be much more time consuming and expensive, using genomic tips. It is yet to be seen if this will work, or if genomic tips will be necessary to get sufficient data on these other species. 

Source: 

1. Rainey, F. A., Ray, K., Ferreira, M., Gatz, B. Z., Nobre, M. F., Bagaley, D., ... & da Costa, M. S. (2005). Extensive diversity of ionizing-radiation-resistant bacteria recovered from Sonoran Desert soil and description of nine new species of the genus Deinococcus obtained from a single soil sample. Applied and environmental microbiology, 71(9), 5225-5235.