Cell Lysing


Cell Lysing

 Over the past few weeks, I've been learning how to lyse cells with my lab partner, Alex. We started with a paper (Kikuchi) on lysing Deinococcus radiodurans - then using the information from it's protocol to try and lyse cells that are Deinococcus aquaticus. 

Here's the protocol we pulled from the paper - 

MATERIALS AND METHODS

D. radiodurans MR., KR, (I1) and Sark were used in this experi-ment. One milliliter of overnight culture was inoculated into 200 ml of TGY medium (1). The bacterial cells were harvested after cultivation from 24 to 36 h at 30°C. The cells were washed twice with Multibuffer (50 mM sucrose, 10 mM Tris-HCI, 40 mM EDTA, 0.1% Triton X-100, pH 8.0) and were lysed by the incubation with lysozyme (1 mg/ml) for 5 min at 37°C. Plasmids were isolated by the alkaline lysis method (12).

For the analysis of plasmids, agarose gel electrophoresis was carried out using Tris-acetate buffer (12) at 100 V constant voltage. Two agarose concentrations, 0.9% for intact plasmids and 1.2% for digested plasmids, were used. When DNA was recovered after the electrophoresis, low-melting-point agarose was used (12). Charomid 9-20, 9-28, 9-36 and 9-42

(13) were used for the calculation of the molecular size of covalently closed circular DNA. Molecular sizes of DNA digested with restriction endonucleases were calculated using a molecular size marker (^ pHY) which was a mixture of pHY marker (TaKaRa Shuzo Co., Ltd., Kyoto, Japan) and HindIII-digested lambda phage DNA. ^ pHY contains DNA fragments of 23130, 9416, 6557, 4870, 4361, 2322, 2027, 2016, 1360, 1107, 926, 658, 564, 489, 267 and 80 bp.


First, we started with making an overnight culture and inoculating it. Then, while that incubated, we also made 400ml of TGY for the cells to grow in. The paper calls for 200ml, so we ended up having enough for two trials. In the first trial, we added 1ml of the overnight culture with an A600 reading of .22 to 200ml of TGY media. We planned to take it out around 38 hours later, but accidentally let it sit for about 52 hours. Later, we did a graham stain on it to still check how well we completed the procedure, and this is how it turned out - 


As you can see, there's nothing but Deinococcus aquaticus growing, so the environment and everything was quite sterile. Due to being left for so long, the A600 value read 2.66 after incubation. Before we did the graham stain, however, we started making the wash solution, and started the second trial of adding 1ml of overnight culture to 200 ml of TGY media. This time, the cells had a starting A600 of .70 (compared to .22 last time) and were left to grow for about 22 hours with a bit more shaking during incubation. 

    Now, I wasn't present, but my lab partner did the washing a lysing of the cells on Wednesday, and according to her, it was successful. Before I publish our results, however, I plan to further go over the results with her to make sure everything was done correctly.

    

    Overall, I've felt quite productive with this new project, and excited to see it through.  

Kieran

Citations

Kikuchi, M., Kitayama, S., Sjarief, S. H., & Watanabe, H. (1994). Plasmids in Several Strains Deinococcus radiodurans [Review of Plasmids in Several Strains Deinococcus radiodurans]. Radiation Research, 139(1), 123–125. JSTOR. https://www.jstore.org/stable/3578752

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