BioTech Lab Entry 6

 Introduction

    This week was spent desiging ways to further test the UV resistance of Deinococcus xinjiangensis. 

Methods

   Six flasks of TGY, each containing 25uL, are prepared. Five flasks of 20 uL of R2B and one flask of 20 uL of R2A is prepared. A practice gel is done for electrophoresis. The gel contains 30mL of TAE buffer and 0.3g of agarose. Cryovials contain 2uL of the sample, 8uL of PCR water, and 2uL of the loading dye. The gel is run at 100 amps. The next day, 2 flasks of TGY and one flask of R2B are inoculated with D. xinjinangensis. They are put in the skaing incubator at 30.0 degrees Celsius. I then had a discussion with Dr. Tuohy on further avenues of research (see discussion). Gram stains are done on the flasks inoculated yesterday. However, the results are inconclusive. Aland and I design a new and simplified experimental design for the UV tests that can be easily manipulated to test for specific variables. The following day, Aland and I are introduced to the flourescent microscope. We then discuss our lab work with the other groups in lab. The following day, two plates of TGY and one plate of R2A are inoculated with D. xinjiangensis.

Results

    The experimental design for the UV tests is as follows: two flasks of R2B will be inoculated with D. xinjangensis. Epindorf tubes with 100uL of media will be taken from them and diluted to an OD value of 1. 50uL of the media will be taken from the tubes and exposed to 30,000 uJ/cm^2 and 100,000 uJ/cm^2 of UV radiation respectively. After exposure, 20 uL of each droplet will be diluted in 480 uL of R2B and then streaked onto a plate. Bacteria from the epindorf tubes that have not been exposed to UV will also be streaked onto the R2A plates. 

Discussion

    The UV resistance of D. xinjiangensis offers multiple avenues for further research. Multiple genes are involved in UV resistance, with PprI being a 'master switch' that controls other genes. It is unknown how these genes differ between species and whether or not these genes could be used to build accurate phylogenetic trees. Further questions remain as to how DNA repair in Deinococcus occurs (e.g. homologous vs non-homologous repair). 

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