Preparation for Nanopore Sequencing and Competent D. sonorensis

 Introduction:


D. sonorensis has been shown to have competence genes based off of bioinformatic analysis, with potentially even more that have not been identified yet. So far, pil genes associated with competence such as PilN and PilQ are present, as well as ComF, ComEA, and ComEc which are supposed to be directly related to working with PilQ to integrate one strand of foreign DNA into the cell. Other genes have also been identified in the Rec family, that are supposed to aid in DNA recombination and also relate to the integration of foreign nucleic acids. Due to this great amount of preliminary evidence, in addition to some of these genes also being present some of the known Deinococcus that can be transformed (D. radiodurans, D. geothermalis, and D. ficus) it seems prudent to attempt transformation with D. sonorensis. Preliminary work was done to prepare for this attempt next week. 

The other goal of this week was to provide new DNA samples of D. caeni that may be less sheared, or higher in molecular weight, for hopefully more optimal Nanopore long read ligation sequencing. The previous sample used for Illumina seems to be rather fragmented even based off of the data recovered from that run, so avoiding to use this sample is likely beneficial. The bead beating time of the cells will be altered in order in order to produce just enough DNA with hopefully the least amount of fragmentation possible. 


Methods:


D. sonorensis was experimentally grown on 3 ug/ml of chloramphenicol to ensure no significant antibiotic resistance that would skew transformation results with the plasmid Prad1. The cells did not exhibit any significant growth on TGY agar dosed with antibiotic for 72 hours, which concluded this concentration should be sufficient alike other Deinococcus species. R2A plates were made with and without 3 ug.ml of chloramphenicol and D. sonorensis cells were inoculated on plate and then broth to prepare for the chemical competence procedure. 


D. caeni was grown on TGY agar plate for 24 hours prior to being transferred into two tubes of TGY broth and homogenized in solution prior to be used as input for the QUIAGEN DNeasy kit that has been used previously. One tube was bead beated for 30 seconds at 1 m/s, the most minimal speed available on the machine. The other tube was done for 30 seconds at 3 m/s to hopefully ensure at leats one fo these samples would be sufficient and to find the threshold for lysis. These samples were eluted in 25 ul of provided elution buffer after 5 minutes of incubation at room temperature prior to final centrifugation. The results were recorded on the Nanodrop One for approximate concentration and cleanliness. 


D. caeni was taken from TGY plate and inoculated into about 20 ml of TGY broth to attempt again at DNA extraction. The process was similar except the one tube was done at 30 seconds 5 m/s with a final elution of 20 ul. 


Results: 


Sample

Concentration (ng/ul)

A260/A280

A260/A230

1 m/s

6.9

1.41

0.32

3 m/s

15.7

1.71

0.84

5 m/s

169.2

1.94

1.95


Discussion:


The remaining 18 ul of the one D. caeni sample done at 5 m/s will need to undergo DNA cleanup procedure in order to improve the A260/A230 value which absolutely must be above 2.0 to meet parameters for the Nanopore sequencing protocol. The drastic increase of concentration is likely a combination of a few factors; the initial concentration cells to start was probably much higher, the elution volume was smaller, and then finally the increase in bead mill power. It seems evident that Deinococcus species likely need a minimum of the 5 m/s power to have sufficient DNA recovered for sequencing. More work can be done in order to narrow down whether the 4 m/s would maybe also be efficient or if more time at the 1 m/s or 3 m/s powers would actually provide enough DNA and potentially less shearing. The D. sonorensis transformation will continue with carrying out the competence cells procedure next week and an attempt with Prad1 which is already available from older experiments. 





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