Attempted Transformation of Deinococcus sonorensis

 Introduction

Deinococcus. sonorensis has recently been hypothesized to be transformable. This understudied species of Deinococcus was retrieved from the Sonoran desert and a nonarid soil from a Louisiana forest. It's unique in that it forms a plaque-like biofilm which makes the cells extremely difficult to lyse. A solution we've found to this is using R2A as a selective media which affects the morphology of this species. The changes after growth on R2A make the cells easier to work with. Due to being believed to have no plasmid, and no attempts at transformation, it may be an interesting objective to try and transform D.sonorensis. Our transformation will be using the cmr gene from pRAD1 found in E.coli. 


Methods

To begin, competent cells of D.sonorenis  were made with R2B. Afterwards, competent cells were thawed on ice and 100μL of cells were from each tube were transferred into three 1.5mL centrifuge tubes. Next, 9μL (~647ng) of cmr PCR product was introduced to the two of the tubes. For our negative control, plasmid was replaced with elution buffer. The tubes were immediately placed on ice for 15 minutes then placed in a 30 degrees Celsius still incubator for 45 minutes. During this time, cells were agitated via inverting every 15 minutes. Next, these tubes were taken out, and 109μL of the solutions were pipetted separately into 3 15mL tubes that contained 4mL of R2B media each. These tubes were placed in a shaking incubator overnight ( >16 hours) at 30 degrees Celsius. The next day, cells showed growth and selective plates were pre-warmed in the still 30 degrees Celsius incubator for 40 minutes. Next 100μL of the transformed cells were plated on 2 plates each. Experimental controls were plated on 2 R2B plates containing 3μg/mL of chloramphenicol. Our negative control was plated on an R2A plate without antibiotic, and an R2A plate with the same concentration of antibiotic. These were then grown over the weekend at 30 degrees Celsius. 


Results

One colony grew one of the Transformation #1 plates. The colony was transferred into R2B broth containing 3μg/mL chloramphenicol and observed. No growth was detected in the broth after 3 days of growth. 


Discussion

Transformation needs to be reattempted. Additionally, colonies from transformation should be transferred to antibiotic plates to observe growth rather than broth. Transformed Deinococcus cells have previously been observed to not grow in broth regardless of media. Therefore, it is imperative that colonies are allowed to grow over generations prior to usage. The next steps after transforming would be to perform plasmid extraction and verify via electrophoresis. 





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