BioTech Lab Entry 8

 Introduction

    We have tested the UV resistance in Deinococcus xinjiangensis twice before this week. In the most recent iteration of the experiment, the dilution factor of the non-irradiated bacteria (our controls) was higher than it was for the bacteria exposed to UV. This week, we repeated the UV test while correctin this error and lowering the exposure amounts for the bacteria. 

    The enzyme urease catabolizes the hydrolysis of urea into carbon dioxide and ammonia. The presence of this enzyme in a given bacteria strain can be tested for. According to the paper in which D. xinjiangensis was first described, D. xinjiangensis is urease positive (Peng et al. 2009). However, when we repeated the experiment, we found D. xinjiangensis to be urease negative. We repeated the experiment this week to clarify our results.

    D. sonorensis is an unusual member of the genus Deinococcus. One strange feature of this bacteria is its ability to form "plaques" in solution. These plaques make the cells very difficult to lyse and thus pose a problem to attempts to detect the presence of plasmids under 25k bp. Cells of D. sonorensis were grown on plates and then lysed to account for this challenge. 


Methods

     Two plates are inoculated with Staphylococcus epidermis (the positive control for the urease test) and two with Bacillus subtilis (the negative control for the urease test) and left to incubate at 37 degrees Celsius for 24 hours. 450 mL of agar is heated at 360 degrees Celsius and cooled to 55 degrees Celsius. 50 mL of ureas base is poured and mixed into the solution in the chemical hood. The solution is titrated with hydrochloric acid until it is an organe-yellow color. The solution is then poured into six plates which are left to cool. Two of the urease plates are inoculated with D. xinjiangensis, two with S. epidermisis, and two with B. subtilis. The plates with S. epidermis and B. subtilis are incubated at 37 degrees Celsius for 48 hours. The plates with D. xinjiangensis are incubated at 30 degrees Celsius for 48 hours. Results are examined visually.

    Four 25 mL flasks of R2B are inocluated with D, xinjiangensis and placed in the shaking incubator at 30 degrees Celsius for 24 hours. The flasks are Gram stained to check for contamination. 100 uL of media is taken from each flask and placed in their own separate Eppendorf tube. The OD values of these solutions are taken and normalized to 1. 450 uL of clean R2B is dispensed into 24 tubes. 24 sheets of parafilm are labeled with a letter (A-H) that corresponds to the amount of radiation given (see table below, numbers are the level of exposure measured in x100 uJ/cm^2) and a number indicating which flask. 

A

B

C

D

E

F

G

H

250

125

63

31

16

8

4

2

The bacteria were then exposed to the designated amount of radiation for two minutes. 20 uL of each droplet is then diluted in 450 uL of clean R2B before being inocluated on a plate of R2A. Plates are incubated at 30 degrees Celsius for 48 hours.

    Two R2A plates are inoculated with D. sonorensis from flasks of R2B. The cells are then lysed according to the instructions in the Zyppy Plasmid Miniprep Kit: 600 uL of culture is placed in a 1.5 mL centrifuge tube and pelleted. The supernatant is discareded and 100 uL of 7x Lysis Buffer is added to the tube, which is then mixed. The supernatant is placed in the Zymo-Spin IIN column, which is placed into a Collection Tube and centrifuged for 15 seconds. Following discarding the flow-through, the column is placed in the Collection Tube. The solution is then mixed with 200uL of Endo-Wash Buffer and centrifuged for 30 seconds. 400 uL of Zyppy Wash Buffer is added to the column, which is then centrifuged for one minute. The column is transfered into a tube and mixed with 30 uL of Zyppy Elution Buffer. The solution is centrifgued for 30 seconds. 2uL of the sample is mixed with 2uL of loading die and pippetted into a 10% loading gel. A 10k ladder is also pipetted into one of the wells. The gel is then examined after being run on 70 amps to determine if any plasmid was detected. 

Results

    D. xinjiangensis tested urease negative (figure 1), in line with results from our previous test. S. epidermis tested urease positive (figure 2), while B. subtilis tested urease negative (figure 3). 

Urease plates showing D. xinjiangensis. The absence of pink idicates a negative result, as when urea is hydrolized, it produces the weak base ammonia, which turns the phenolphthalein in the media pink.

A urea plate with S. epidermis. The pink indicated a positive result. S. epidermis was the positive control for the experiment.

A urea plate with B. subtilis. The absence of pink indicates a negative result. B. subtilis was the negative control for this experiment.


    D. xinjiangensis did not recover from any exposure. However, significant growth was observed from the control (non-irradiated) plate. This suggests that D. xinjiangensis is UV sensitive when grown in R2B. See figure 4 below.

The results of the UV test.


    No result was detected from the gel, showing that D. sonorensis lacks a plasmid under 25k bp in length. See figure 5 below.

The results of a gel electrophoresis of D. sonorensis.



Discussion

    The discrepency between the reported presence of urease in D. xinjiangensis and our experimental observation of its absence may be due to differences in the strains of D. xinjiangensis being tested. Different strains may have lost or re-evolved (possibly through lateral gene transfer) the ability to hydrolyze urea as adaptations to different local environments.

    R2B is less nutrient dense than TGY. The lower nutrient density may produce cells with weaker cell walls, leaving them sensitive to UV exposure. Additionally, R2B may lack necessary amino acids to build functioning DNA repair enzymes.

   D. sonorensis lacks plasmids under 25k bp in length.


Works Cited

Peng, F., et al. “Deinococcus Xinjiangensis sp. nov., isolated from desert soil.” INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, vol. 59, no. 4, 1 Apr. 2009, pp. 709–713, https://doi.org/10.1099/ijs.0.004564-0.

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