Research Paper Rough Draft

Abstract:                         

In this project lysozyme will be extracted from egg whites and compared to commercially produced lysozyme.  Lysozyme is an enzyme that breaks down glycoproteins (amino acids) on the surfaces of bacteria and kills them. Large quantities of lysozymes are found in egg whites and can be used in the process of DNA extraction from bacteria.  The lysozyme extracted from egg whites will be used in DNA extraction to determine the effectiveness of the lysozyme.  The extracted lysozyme will be utilized in DNA extractions over several intervals of time after the initial extraction to determine the shelf life of the enzyme and compared it to commercially produced lysozyme. Egg white lysozyme will be extracted according to Ganong, (2007).

Background Information:

Researching the shelf life of egg white lysozyme is important because egg white lysozyme can substitute expensive commercially produced lysozyme.  The proficiency of DNA extraction is crucial in order to perform consistent duplicable results. (Ettenauer , 2012).  Research in DNA extraction can lead to diagnosis of illnesses and development of new drug treatments.

 Introduction: 

Research Question: 
What is the shelf life of lysozyme extracted from egg whites or will the effectiveness of egg white extracted lysozyme decrease with time in its ability to aid DNA extraction from Providencia stuartii?

Hypothesis: 
It is anticipated that the shelf life of egg white lysozyme will produce comparable DNA extraction results from Providencia stuartii as the commercially produced lysozyme.

Methods:

Variable Table
Name	I/D/C	Units	Description
Egg White Lysozyme	I	ng/ul	Lysozyme extracted from eggs
Kit Lysozyme	I	ng/ul	Lysozyme from kit Ready-Lyse-Lysozyme
Water No Lysozyme	C	ng/ul	No lysozyme added only bacterium and  di water
Time	C	15 minutes	Amount of time to control
Temperature	C	Room Temp	Experiment was conducted at room temperature

Apparatus   

Nano Drop Machine (Used to measure DNA yields from egg whites, kit & water samples)

Centrifuge Machine (Used to pellet microbial DNA)

Pipettes (Used to measure exact amounts of fluids into centrifuge tubes)

Procedures:

Get a fresh egg from refrigerator, crack egg carefully separate egg yolk from egg white, without breaking the yolk, transfer 10mL of egg white into sterile container with lid/label egg white.  Add 3.3 fold (30mL) of 0.05mNaCl/di H₂O solution to egg white volume/stir.  Add 2mL of Acetic Acid (vinegar) to egg white solution/stir, add 2mL of 40% ethanol/stir and measure pH level.  Add remaining 2mL of Acetic Acid and 2mL 40% ethanol to egg white solution/stir, measure pH level, it should read pH 4.  If not keep adding equal amounts of Acetic Acid and 40% ethanol to egg white solution until pH 4 is reached.  Incubate egg white solution for 30 minutes at room temperature, centrifuge 1mL of egg white solution at 12,500rpm (max speed) for 15 minutes at 4°C (In refrigerator).  Pipette 1mL of Providencia stuartii bacteria into 3 separate labeled (Egg White, Kit, H2O) centrifuge tubes , centrifuge the bacteria tubes at 5,000rpm for 3 minutes, to pellet the cells.  Carefully remove and discard supernatant from centrifuged tubes, do not touch cell pellet.  Wash the Providencia stuartii bacteria cell pellet by pipetting 0.5mL of di H2O into each centrifuge tube, centrifuge the tubes at 5,000rpm for 3 minutes, carefully remove supernatant, do not touch cell pellet.  Pipette 100µl of Quick Extract Bacterial DNA Extraction Solution into each centrifuge tube (Egg White, Kit, H2O).  Pipette 1µL of Egg White Solution (centrifuged at 4°C) to centrifuge tube labeled Egg Whites, pipette 1µL of Ready-Lyse-Lysosome Solution to centrifuge tube labeled Kit, pipette 1µL of di H2O to centrifuge tube labeled H2O.  Incubate centrifuge tubes at room temperature for 15 minutes, use Nano drop machine to measure DNA yields from the centrifuge (Egg White, Kit, Water) tubes.

 

Results:

Analysis

Data is inconsistent, but does show that the shelf life of egg white lysozyme does produce comparable DNA extraction results from Providencia stuartii as the commercially produced lysozyme.  Adjustments (increase) will be made to the concentration of Ready-Lyse-Lysosome Solution used for the commercial Kit to obtain higher DNA yields. Also contamination of one egg white lysozyme centrifuge tubes was discovered that might have skewed the results for that particular sample. The length of incubation time for the TSB tube of Providencia stuartii needs to be increased to at least two days, to allow DNA pellets to form when centrifuged.  Consistency in fluid amounts when washing DNA pellet with di water needs to be adjusted.   With the above adjustments taken into considerations data will continue to be gathered and analyzed.  PCR will have to be done to gather data and provide consistency.

Conclusion:

Conclusion to come, data is still being gathered and analyzed.  

References:

Ettenauer, J. D., Piñar, G., Lopandic, K., Spangl, B., Ellersdorfer, G., Voitl, C., & Sterflinger, K. (2012). Microbes on building materials — Evaluation of DNA extraction protocols as common basis for molecular analysis. Science of The Total Environment, 439, 44-53. doi:10.1016/j.scitotenv.2012.09.005

Ganong, B. (2007, June11). Isolation of Lysozyme from Egg White. Retrieved March 09, 2017, from Barry Ganong’s Homepage website: http://faculty.mansfield.edu/bganong/biochemistry/lyszyme.htm

QuickExtract^TM bacterial DNA extraction kit [pdf]. (2012, December). Retrieved March 09, 2017 from: http://www.epibio.com/docs/default-source/protocls/quickextract-bacteria-dna-extraction-kit.pdf?sfvrsn=8