9. (10 pts) Design an experiment that would test the effect of the presence of t
ID: 276377 • Letter: 9
Question
9. (10 pts) Design an experiment that would test the effect of the presence of the natural substrate, lactose, on the reaction. What would your data look like (you can compare it to data you collected). Include your hypothesis, controls and what different conditions/ratios you are going to test in your experiment (lactose:ONPG) What is your hypothesis? Do you think the absorbance will increase or decrease relative to control in the presence of lactose? Keep in mind why we are not using lactose in the current experiment. a. b. What different ratios of ONPG: lactose are you going to use? c. How your results compare from one ratio to the next? d. What are your controls?Explanation / Answer
Principle and hypothesis: ONPG (o-Nitrophenyl-?-D-galactopyranoside) is an alternate substrate (analog of lactose) to detect b-galactosidase activity. Upon clevage by b-galactosidase, o-nitrophenol is produced that gives yellow color to the reaction along with galactose. If one has to test the effect of lactose on a reaction, say in case of lactose fermenting bacteria. E.coli for example has lac operon that codes for both permease as well as galactosidase genes. Thus lactose gets utilised and broken down to glucose and galactose. If we lyse these cells to release b-galactosidase to look at its activity, we need a color reaction for it. ONPG gives yellow color and can be employed for this reaction.
When lactose is present, beta galactosidase activity is high but cannot be detected as there is no color reaction . However, if one incubates lactose with varying concentrations of ONPG, there will be change in color that can be colorimetrically detected. Proteus vulgaris, non-lactose fermenting bacteria can be taken as a negative control.
a. Absorbance will increase with an increase of ONPG concentration, yielding a yellow color. Lactose is broken down by b-galactosidase, but with an increase of ONPG, a competitor/analog of lactose will start yielding color reaction. Order in which one use lactose as a substrate or ONPG as a substrate matters. This reaction can be done in 2 ways. 1st: ONPG along with b-galactosidase followed by increasing the concentration of lactose. In that case yellow color will go down as compared to only ONPG and b-galactosidase. With a reduced color, it shows that there is presence of b-galactosidase as well as an active b-galactosidase is present.
b. For this experiment , one can use only ONPG, ONPG:lactose(1:0.5), ONPG:lactose(1:1), ONPG:lactose(1:2),ONPG:lactose(1:3),ONPG:lactose(1:4),ONPG:lactose(1:5), ONPG:lactose(1:10)
c. Without lactose, ONPG will be broken down to o-nitrophenol and galactose, former giving a yellow color. However, when lactose concentration is increased in the system, color intensity goes down that can be detected colorimetrically. Thus, color intensity is inversely proportional to lactose concentration. However, it shows that a functional b-galactosidase is present in the system.
d. Controls include buffer, buffer+b-galactosidase, only lactose without b-galactosidase, only ONPG without galactosidase
Positive control: ONPG+b-galactosidase
Note: The experiments to be followed can be either done with b-galactosidase or can be employed in bacteria where the principle is mentioned. One can correlate both biological as well as chemical experiment.
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