S-STEM/TRAIN Scholar Jasmine Patricio

    Lab Proposal: Lysozyme purification from egg-white. 1. An explanation of why you selected your research topic.  Consider issues like: Q: Why is the topic significant? A: Every bacteria carries peptidoglycan which is a layer outside the plasma membrane of most bacterias. Peptidoglycan is the specific site that lysozyme targets, lysozyme is an enzyme found in tears, saliva, sweat and other body fluids. It is capable of breaking the chemical bonds in the outer cell wall of the bacteria. Q: Why is it important to learn more about this topic? A: Prevent other bacterias from spreading or get rid of a international bacteria. Q: Why is it important to conduct research in this area? A: It is important to conduct a research in this area because the advancement of lysozyme can perhaps in the future become more advanced and have a cure to other bacterias. 2 . A description of how you will be researching your topic.  For example, you might be planning to: Observe:

Q: What is Lysozyme?   A: A n enzyme found in saliva and sweat and tears that destroys the cell walls of certain bacteria . N ow that I have created what could possibly be lysozyme , my next step would be purifying i t. Meaning I have to now find a protoc ol that would help me purify l ysozyme that was extracted really is pure.    This is one possible protocol:   Materials and Methods: A solution of egg white diluted to ¼ with 0.1 M phosphate buffer pH 7 and filtered though glass wool is used as the source for lysozyme (Laboratory Manual. 2007). The solution is put through size exclusion chromatography with G-50 Sephadex column (fractionation range of 1,500-30,000 da) to produce 24 test tubes of equal egg white fractionations of 0.75 mL (Laboratory Manual. 2007). Numerous assays are conducted with varying pH and micrococcus (substrate) concentration to determine the optimal conditions for the highest enzymatic activity of lysozyme. After the collected column fractions and prep

The Protocol that Josh had handed me was much more understandable than the first one that I was working on during the summer, the only problem with this was that the measurements were not listed on the protocol, so I had to solve for those myself, (with the help of Brian). 1. Measure 120 mL of DI water in 100 mL graduated cylinder. 2. Measure .3502 g of NaCl 3. Pour the .3502 g of NaCl into the 120 Ml of DI water 4. Pour the 120 ML of DI water + NaCl into 400 mL with 40 mL of the egg white 5. In a 50 mL measure 12 mL 15 mL of DI water place in Falcon Tube 6. Measure 10 mL of 100% Ethanol 7. Put it in the Falcon Tube 8. Grab a pipit and add add drops of Acetic Acid into teh 120 mL of DI water + .3502g NaCl+ 40 mL eggwhite, all into the 300 mL beaker. 9. Poured Acetic Acid until its PH was 4.06 What I have written down was mostly the measurements of everything since the protocol I was given didn't really have ant measurements of how much of everything I was suppose to use.

So this week I was continuing the protocol to making Lyzosome. Josh, Mat and Cory and I had decided that we needed a new needed to look at a different Protocol because the one that we had done during the summer had a lot of difficult stuff to it. And because of my poor note taking during the summer we were not able to duplicate what i had done over the summer. Also because during the summer the Protocol that we had, we mostly took educated guesses on what we were adding and contributing to the experiment.  Josh was able to find a new Protocol for me that he had found : http://www.epibio.com/ applications/nucleic-acid- purification-extraction-kits/ buccal-swabs-and-dna- extraction/quickextract- bacterial-dna-extraction-kit http://omegabiotek.com/store/ product/bacterial-dna-kit/ And that made it much simpler for me to follow. The only problem with that Protocol was that it didn't have that much any measurements for the chemicals that we were adding on. So we had to com