Week 5 DNA Extraction

After a reading up on the process of using the Omega DNA extraction kit I was shown a much faster yet not as efficient process to obtain the same results. Today I took lysozyme I have purified and run a DNA extraction side by side with the extraction process given to me. My process consists of using my unknown bacteria as the subject to which my DNA extraction will occur. The three DNA extraction tests I ran today were the use of a lysozyme I extracted from egg whites, lysome provided in a kit, and 40% ethanol in order to compare the results and effectiveness of each.

Week 4 Lysozyme Extraction

Egg Lysosome in DNA Extraction

The topic is significant because of Lysozyme’s ability to break chemical bonds on outer walls of bacteria. It is important to learn more about lysozymes to understand why and how they break down chemical bonds on outer walls of bacteria. It is important to conduct research in this area so we can find ways to apply lysozymes ability to break down chemical bonds in other scientific fields.

·        Experiment- I will run a procedure of partial purification of lysozyme.

1.     For the partial purification of lysozyme, egg whites, carefully separated from the egg yolks, were diluted 3- or 3.3-fold with 0.05M NaCl solution.

2.     To precipitate the egg white proteins other than lysozyme, the pH of this mixture was set to 4.0 by carefully adding several drops of 1 N acetic acid and it was diluted with an equal volume of 40%(v/v) ethanol. After 30-minute incubation at room temperature in the presence of ethanol, the mixture is centrifuged at 15,000xg for 15 minutes at 4 degrees C: then the precipitates were discarded.

3.     The supernatants were analyzed for their lysozyme activity and protein content to determine if lysozyme extraction was successful.

4.     A DNA extraction is performed using a kit the epicenter QuickExtract Bacterial DNA Extraction Kit (QEB0905T) and E.Z.N.A. Bacterial DNA Kit from Omega bio-tek (D3350-00) as controls

5.      A DNA extraction is performed using both kits but replacing the kit provided lysozyme with the student prepared egg white (EW) lysozyme.

Name	I/D/C	Units	Comment
Eggs	C	-	-
Lysozyme	D	-	-
Partial Purification of Lysozyme Protocol	I		Refer to Protocol

Is it possible to extract lysozyme from egg whites? My hypothesis is that the extraction of lysozymes from egg whites will be possible. The results I may discover are that the purity of the lysozyme from egg white may not be as pure as we need for a DNA extraction.

References

·        Alderton, G., Ward, W.H., & Fevold, H.L. (1945). Isolation of Lysozyme From Egg White [PDF]. Journal of Biological Chemistry, (157), 43-58. Retrieved from http://www.jbc.org/content/157/1/43.full.pdf

·        Fakharany, E. E., & Hassan, M. (2016). A universal method for extraction of genomic DNA from various microorganisms using lysozyme. New Biotechnology, 33(1), S210. http://dx.doi.org/10.1016/j.nbt.2016.06.1445

·        Ganong, B. (2007, June 11). Isolation of Lysozyme from Egg White. Retrieved October 20, 2016, from Barry Ganong's Homepage website: http://faculty.mansfield.edu/bganong/biochemistry/lysozyme.htm

·        QuickExtract™ bacterial DNA extraction kit [pdf]. (2012, December). Retrieved from http://www.epibio.com/docs/default-source/protocols/quickextract-bacterial-dna-extraction-kit.pdf?sfvrsn=8

·        Xue-li, C., Ting, L., & Ito, Y. (2007). Separation of Chicken Egg-White Lysozyme by High-Speed Countercurrent Chromatography using a Reverse Micellar System. Journal of Liquid Chromatography & Related Technologies, 30(17), 2593-2603. http://dx.doi.org/10.1080/10826070701540555

Week 3 Unknown Bacteria

Today I look forward to receiving my sample of unknown bacteria and attempting to identify it using the techniques previously discussed, including but not limited to Gram Staining and Ziel-Neelsen Staining. If the results prove to be effective i will proceed with the results to the next step, but if the tests are ineffective i will research a third way to which i can attempt to identify the bacteria.

After receiving my sample of unknown bacteria I have begun my identification process by using a three quadrant streak plate method.  I took a sample of the unknown and streaked 1/3 of the TSA petri-dish. I took a second sample of the same unknown bacteria and streaked a second time beginning in the prior quadrant for one drag of the loop with the bacteria and streaking another 1/3 of the petri-dish, finalized by a third sample of the same unknown. Dragging it once through the 2nd quadrant and continuing to streak the 3rd and final quadrant. I then placed the petri- dish in the incubator which will allow for the bacteria to colonize over the next 24 hours.

Week 2 Unknown Bacteria

To begin my first day here at the S-STEM internship I will begin research in regards to how to identify bacteria. From my knowledge of bacteria I know Gram Staining can be an effective form of identifying Gram Positive and Gram Negative bacteria. Also I understand that some bacteria have a "wax" like outer layer that would prove Gram Staining to be ineffective in the process of identifying the bacteria type. Therefore when Gram Staining proves ineffective another process of identification can be Ziehl-Neelsen Staining. This staining process proves effective only when identifying Mycobacteria in general. Ziehl-Neelsen Staining cannot provide specific identification of species within Mycobacteria, but can prove that the bacteria being tested are Mycobacteria. I understand the general concepts of the staining processes but have never attempted them nor do I know the specific process in which they are performed. My goal is to research the specific process for these staining methods and attempting them on unknown bacteria to better understand how the staining looks when performed. Once I have completed this, my following goal would be to research a way to differentiate between 2 different types of a bacteria genus. I also look forward to sharing my results and comparing with other interns our processes and findings in order to understand maybe other ways I can accomplish the same task or a more effective way or obtaining the same result. Also I would like to find a way to identify the sample without compromising it so I can conduct further testing with one sample.