Name: Bhavi PatelPartners: Subhrangi Swain Title: Gene RegulationDate of Experiment: 11/17/11 and 12/1/11Due Date: 12/8/11Experiment #6Abstract: In this lab, we created an independent experiment to see how the lac operon is regulated. In our independent experiment, we decided to check the affect different pH has on the gene expression of the enzyme beta-galactosidase. We hypothesized that the level of absorption wouldbe highest when the pH level is 9. However, according to our data, our hypothesis was incorrect because my data did not conclusively prove this.Introduction: Over the years, scientists have seen that gene expression in prokaryotes is different from gene expression in eukaryotes, this is because “Prokaryotes frequently have a single regulatory region — called an operon — that controls several genes' expression” (Lab Protocol). The purpose of the lac operon is to “break down lactose into galactose and glucose, which is regulated by a repressor or a silencer mechanism” (Lab Protocol). However when lactose is not present, “the repressor binds to the silencer and blocks the expression of beta-galactosidase, the enzyme that breaks down lactose” (Lab Protocol). Therefore, beta-galactosidase would not be expressed.The pH level affects the production of the enzyme beta-galactose because “The concentration of hydrogen ions in solution affects the enzyme activity. Each enzyme has maximal efficiency under an optimum pH, since pH is one of the factors for the denaturation of proteins, if an enzyme is submitted to a pH level under which it is denatured there will be no enzymatic activity” (Enzyme Activity). Therefore we hypothesized that the level of absorption is dependent on the level of pH, so if the pH level is high then the absorption level will also be high.Methods: First my group labeled all the test tubes, we used 13, 23, and 33 for the pH level of 3, 15,25, and 35 for the pH level of 5, 17, 27, and 37 for the pH level of 7, and 19, 29, and 39 for the pH level of 9. We then added 1.5ml of growth media to each test tube with the correlating pH and changed the pipette every time we added a different pH level. After this we added 0.6ml of bacteria to all of the test tubes. Then we added 0.15ml of lactose to all the test tubes. After this we mixed all the test tubes by flicking the bottom. Next, we incubated the test tubes at 37 degrees Celsius for 85 minutes. Following this we added one drop of detergent to all of the test tubes. Then we added 0.8ml of 1% ONPG and incubated the test tubes at 37 degrees Celsius for 15 minutes. Next, we added 1ml of 1M Na2CO3 to each test tube to stop the reaction. After doing this, we used a spectrophotometer, Spectronic 20, to measure the level of absorption of each test tube and recorded our results.Results:After we took the test tubes from the incubator we put them into the spectrophotometer tocheck the absorption levels. We saw that our data did not follow a trend. In addition to this, we figured out the transmittance percentage by doing the following:(10^level of absorbance)/100 = % of transmittance And then we got the following results for our data:pH level and its effect on the absorption level of lac operonspH level Test Tube Absorption Transmittance3 13 .05 89.1%3 23 .04 91.2%3 33 .05 89.1%5 15 .1 79.4%5 25 .09 81.3%5 35 .1 79.4%7 17 .17 67.6%7 27 .165 68.4%7 37 .165 68.4%9 19 .08 83.2%9 29 .07 85.1%9 39 .1 79.4%Discussion:In our experiment we decided to test the effects of different pH levels on the beta-galactosidase. We had initially hypothesized that as the pH levels increased the absorption level would also increase, however our data disproved this. According to our data, the absorbance levels were not conclusive and we were not able to come to any conclusions using because our data because it does not support any trends. In addition to this, if the pH level is high then it would cause the enzyme to denature and would lower the production of beta-galactosidase and could even produce none at all. However, since our data does not support this, we do not see the absorbance levels increase when we used 3 then 5 then 7 and then 9 with the lactose. We are unsure about what had gone wrong, but we believe that we may have used either too much or toolittle of the different amounts of pH in the test tubes or we could have read the spectrophotometerincorrectly. In the end, our hypothesis was not supported with our data but we believe that as the pH level increases the production of beta-galactosidase would be lowered and the level of absorbance would increase.References:Barlow-Coleman, J. Zane. "Gene-regulation II Protocol | Intro Biology." Biology Computer Resource Center | Supporting Undergraduate Education in the Life Sciences. Web. 15 Nov. 2011. As found in<http://bcrc.bio.umass.edu/intro/content/gene-regulation-ii-lab-protocol>"Enzyme Activity." Biology Questions. Web. 07 Dec. 2011. <http://www.biology-questions-and-answers.com/enzyme-activity.html>.As found