Spring 2024: Week 10 - April 1, 2024 - April 5, 2024

 Introduction

Beta-galactosidase is an enzyme that breaks down the disaccharide lactose into the monosaccharides galactose and glucose. When used in a lab setting, if the enzyme successfully breaks down lactose, it will turn a yellow color. There have been questionable results when students perform the experiment. They have not been getting yellow tubes or the tubes turn more yellow at room temperature than they do at 37℃. Dr. Tuohy asked me to perform the experiment every day this week to see what results I get. They want to know if the enzymes are the problem or if the students are not performing the experiment correctly. I tested an enzyme that is 1 week old and one that is 2 weeks old.


Methods 

General Protocol:

I had 6 test tubes in total. To test the enzymes I used three test tubes per enzyme. Enzyme A was the 1-week-old enzyme (the newer one) and Enzyme B was the 2-week-old enzyme (the older one). Into each test tube, I added 2 mL of ONPG. ONPG stimulates lactose in this experiment. Into test tubes A1 and A2, I added 1 mL of mL of Beta-galactosidase (BG). Into the test tube labeled A3, I added 1 mL of buffer to serve as a control for Enzyme A. I replicated the same thing for the other three test tubes. Tubes B1 and B2 had 1 mL of 2-week-old BG while test tube B3 had 1 mL of buffer. In the end, each test tube had a total volume of 3 mL. After added all of the liquids into each tube, I covered them with parafilm and mixed them to homogenize the liquids. I then removed the parafilm from the tubes and incubated them at 37℃ using a water bath for 30 minutes. Once the tubes were done incubating, I took them out and checked them on the spectrophotometer at 420 nm by pouring some of the liquid into a cuvette. I used A3 to blank the spectrophotometer everyday


Tuesday:

On Tuesday, I was asked to add 3 mLs of ONPG into a test tube and test it along with the other tubes. If the tube had a similar reaction, we would be able to see if the ONPG is contributing to the problem. 


Results 

Monday BG Test Spectrophotometer Readings 

Tube 

Spec. Reading

A1

0.354

A2

0.919

A3

0.000

B1

0.057

B2

0.706

B3

0.017


Enzyme A - New Enzyme (1 week) after 30 minutes in the water bath.

Enzyme B - Old Enzyme (2 weeks) after being in the water bath for 30 minutes. 

3/26/2024 

Tuesday BG Test Spectrophotometer Readings 

Tube 

Spec. Reading 

A1

0.017

A2

1.16

A3

0.000

B1

2.50

B2

2.50

B3

0.00

C1

1.08

C2

0.071

ONPG

0.751


Enzyme A- New Enzyme (1 week) after being in the water bath for 30 minutes. 

Enzyme B - Old Enzyme (2 weeks) after being in the water bath for 30 minutes

ONPG after boiling the tube in a water bath for 30 minutes. 

Wednesday BG Test Spectrophotometer Readings 


Sample 

Spec. Reading 

A1

0.052

A2

1.16

A3

0.000

B1

1.04

B2

1.33

B3

0.013

ONPG

0.698


Discussion 

The results were inconsistent throughout the week and did not seem to have a pattern. I was asked to end the experiment after the third day since the results were inconclusive. I am not sure as to what could be causing the experiment to not work as expected. I made sure to be as clean and quick as possible in order to avoid user error. I worked in a pattern every day by first adding the ONPG and then adding either the BG or the buffer. 

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