The Effect of Glucose, Lactose, and Galactose on the ...

1 journal of Experimental microbiology and Immunology (JEMI) Vol. 2:22-26 Copyright April 2002, M&I UBC The Effect of Glucose, Lactose, and Galactose on the Induction of -Galactosidase in E. coli ROB MONDIN, BRIAN JOSEPHSON, KELLY LIU, AND JACK (MIN-SOO) O Department of microbiology and Immunology, UBC The effects of lactose, Galactose , glucose, and glucose combined with Galactose on the induction of galactosidase by IPTG in Escherichia coli strain VC10, growing in a glycerol media, were compared against a control of glycerol plus IPTG. After inducing galactosidase production with the IPTG, the cultures were altered with the addition of one of the above carbohydrates or combinations.

2 This addition proved to alter the induction rate of the galactosidase, slowing it considerably in the case of glucose, lactose and the glucose combined with Galactose . These results show some consistencies with the model of catabolite repression. The addition of Galactose did not slow the galactosidase induction rate but instead seemed to provide an increase in this rate. Of the two known regulatory systems affecting the lac operon, the system involving the mechanism of catabolite repression in the presence of glucose will be the main focus of our investigation. It has been previously shown that the production of the lac operon enzymes can be induced to a high level using a lactose analog in place of lactose while growing in a media devoid of any sugars.

3 Upon addition of carbohydrates, the rate of induction was severely repressed when the carbohydrate added was glucose, then after a few minutes it began to increase again at a slower rate (2). This behaviour can be expected in the presence of glucose and can be explained by the mechanism of catabolite repression, which will slow the rate of induction for a while (1). When lactose is the carbohydrate added, it has been shown that the rate of enzyme induction is slowed even more and for a longer period of time than in the presence of glucose (2). The reason for this phenomenon has not been explained and it is this question that will be further explored in this experiment. Since lactose is required to induce the production of the lac operon enzymes, and the lac operon enzymes are needed for the breakdown of lactose, it is hard to see lactose itself as the cause of this phenomenon.

4 To test this, we will investigate the effects of glucose combined with Galactose since it is the combination of these two molecules that compose the disaccharide lactose. Lastly, we would also like to investigate as to whether Galactose itself is able to produce any of the effects of catabolite repression that are seen with glucose. MATERIALS AND METHODS Experiment was performed based on the procedure outlined on pages Exp A4-1-to-4 on microbiology 421 Laboratory Manual (2). Modifications in Growth and Sampling. After E. coli cells were incubated with 240 mL of M9 media with glycerol at 37 C for 35 minutes, 1 mL of IPTG was added (40 mL of E.)

5 Coli culture was set aside for a control before adding IPTG). E. coli culture was then divided into five flasks with 40 mL each. mL of Glucose, Galactose , and Lactose were added, one to each of three flasks as an additional source of nutrient. mL of Glucose plus mL of Galactose were added to the 4th flask and no additional nutrients were introduced into the last flask. All five flasks including the control were incubated in the water bath shaker for 12 minutes. At various times, turbidity readings were taken in the spectrophotometer at 460nm. Modifications in Beta-galactosidase assay of samples. At each time point 3 mL of sample was taken from each flask and mixed with 3 mL of toluene, immediately after the turbidity was read.

6 ML of permeabilized cells from below the toluene layer were transferred into a clean test tube and stored in the fridge overnight (for approximately 12 hours). Next day mL of Tris buffer was added to each test tube. mL of each mixture was transferred from each test tube to a new test tube, and then mL of ONPG was added to initiate the reaction. RESULTS The addition of any carbohydrate does not significantly affect the pattern of growth ( ). All cultures had a doubling time of approximately 50 minutes. On the other hand, the induction of B-galactosidase responded differently when different carbohydrates were added (Fig. 2). The control culture containing only the IPTG and glycerol showed an early rapid induction, which eventually levelled.

7 The culture in which Galactose was added, at first, only had a slight decrease in induction. However, at about 35 min, the induction level exceeded the control culture for the remainder of the experiment. The glucose-added culture had an initial inhibition, but was temporary. The first half of the glucose culture suppressed induction more than others, yet, halfway through the experiment, 22 journal of Experimental microbiology and Immunology (JEMI) Vol. 2:22-26 Copyright April 2002, M&I UBC synthesis resumed. The culture with lactose added had a relatively constant decrease in enzyme induction.

8 However, the rate approaches the control rate near the end of the experiment, because the control slowed. In contrast to the report on which this experiment is based, there was not as much suppression by lactose. In fact, the enzyme activity level was above the glucose culture. The culture containing both glucose and Galactose had a similar enzyme activity to the lactose culture. Effect of Carbohydrates on Culture Growth of E. (minutes)Turbidity (OD 460 nm)GlucoseLactoseGalactoseG & GControl The Effect of Different Carbohydrates on the Induction of B-galactosidase in E. (minutes)Enzyme Activity (milliunits per mLGlucoseLactoseGalactoseG & GControl 23 journal of Experimental microbiology and Immunology (JEMI) Vol.)

9 2:22-26 Copyright April 2002, M&I UBC The Effect of Different Carbohydrates on Specific Activity of B-galactosidase of E. (minutes)Specific Activity (milliunits per OD 460 - mL)GlucoseLactoseGalactoseG & GControl Figure 4. The Effect of Different Carbohydrates on the Activity of B-galactosidase of E. coli in Response to (OD 460 nm)Enzyme Activity (milliunits per mLIPTGG lucoseLactoseGalactoseG & G 24 journal of Experimental microbiology and Immunology (JEMI) Vol. 2:22-26 Copyright April 2002, M&I UBC The lactose and glucose combined with Galactose culture have fairly constant specific activity (Fig.))

10 3). The control culture has an early rapid increase and then reaches a plateau. The Galactose culture has a consistent increase, but then exceeds the control culture at approximately 35 minutes. The culture with glucose had an initial decrease and remained level for a while. A sudden inflection or increase is observed around 42 min, which eventually levels as well. The cultures with lactose, glucose & Galactose , and glucose have similar activity responses to changes in growth (Fig. 4). The control culture has a greater response than the previous three. The greatest response is observed in the culture with Galactose : at some points the activity is more than triple the first three cultures, and nearly double control.