Preliminary UV Resistance Testing for Deinococcus xinjiangensis

 Introduction

The Deinococcus genus is known for its robust ability to withstand radiation, extreme oxidation, and desiccation. D. radiodurans is the prime example in this genus for this. Previously, we had done biochemical testing on D.xinjiangensis. Now, we are interested in seeing how UV resistant D.xinjiangensis is and if the media the bacteria is grown in may affect these attributes. We will be comparing culture grown in R2B and TGY broth before exposing them to varying degrees of UV radiation in the unit of X100μJ/cm²  using a UV-Crosslinker. 


Methods

To begin, 1 TGY and 2 R2B 20mL cultures of D.xinjiangenis were grown overnight and normalized to an A600 value of 1 in a 1.5mL centrifuge tube. To prepare for dilution, 20 1.5μL centrifuge tubes were filled with 450μL of R2B. 10 1.5μL tubes were filled with 450μl of TGY broth. Next, 50μL of the normalized samples were pipetted onto 3 separate parafilm squares labeled A1 , B1 and C1. Letters represent biological differences and number represents level of energy in hundred thousands of microjoules. The UV Crosslinker was then used to expose the media to constant degree of radiation for 5 minutes. The values we chose were 10 intervals between 100,000-999,999μJ/cm² . After exposure, irradiated cells were carefully pipetted out and into according dilution tubes, to make a total volume of 500μL. Each step past the normalization was repeated for each varying degree. Next, diluted irradiated cells were streaked on 10 different R2A plates. Lastly, these plates were grown for 4 days. 

Results


UV Resistance Tests from 100,000-999,999μJ/cm² 

Discussion

Interestingly, cells grown in TGY were the only ones to grow on plates. There is inconsistency in some of the plates TGY grew on, for instance , there is no growth at 400,000μJ/cm² but there is on all other levels of energy. Both samples grown in R2B did not grow at any energy level. There may be some issues with experimental design. For instance, after normalization, cultures remain in their respective media broth. This may affect the true exposure of the UV, as cells may be protected by the nutrients in their environment. Another theory is that the cells may be better equipped to use DNA repair mechanisms due to the nutrients provided from growing in TGY. Another adjustment could be shortening the duration of exposure to get a better threshold for our R2B samples. Another possible change is using more culture for exposure, as volume is lost due to evaporation post-exposure. Lastly, changing the amount of volume to pre-exposure will keep a constant post-exposure volume and make up for evaporation. 











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