Created: Sep 2021
For a short explanation of the theory concerning metabolism of lactose and glucose, jump to the bottom of this page.
In this experiment, E. coli will be cultivated in the presence of lactose, glucose and a mixture of lactose and glucose. Bacterial turbidity (OD600) and enzyme activity will be measured at the start and 60 minutes after the induction (cultivation in the presence of sugars). The enzyme used in this experiment is β-galactosidase, which is capable of breaking down lactose into glucose and galactose.
To determine the enzyme activity of β-galactosidase, the absorbance of the samples containing ortho-nitrophenol (the product of transformation of ONPG due to β-galactosidase) will be measured, and relative activity will be calculated.
The objective of this experiments is to demonstrate how β-galactosidase is induced and synthetized in presence of lactose, and suppressed by catabolite repression in the presence of glucose. Another aim is to determine the relative activity of β-galactosidase in different sugar solutions at different induction times.
Materials and Method
Equipment and Reagents
• Escherichia coli K-12 W3100 strain preculture
• Liquid LB culture medium, 4 tubes (plus 1 used as the black solution in the previous experiment Cultivation of Escherichia Coli )
• 1M lactose
• 0.5M glucose
• 3mM ortho-nitrophenyl-β-galactoside (ONPG) (100mM phosphoric acid buffer, 10mM KCl, 1mM MgSO4, 50mM mercaptoethanol, pH 7.5)
• 1M sodium carbonate (Na2CO3)
• Plastic cell
• Laboratory incubator shaker (37ºC)
Cultivate E. coli in LB culture medium at 37ºC for 12-16 hours. Place 1 ml of the LB black solution in a test tube (enzyme activity negative control sample) and 1 ml in a plastic cell (bacterial turbidity measurement). Prepare 9 microtubes with 0.5 ml of ONPG in each one.
b) Cultivation and sample collection
i. E. coli distribution
Add 1 ml of E. coli preculture to all of the 4 L-tubes containing 10 ml of LB culture medium. Place it in the laboratory incubator shaker at 37ºC for 30min.
ii. Addition of sugar solutions
Afterwards, add the following amounts of sugar solution:
Tube 1: 200 µL of 1M lactose (final concentration 20mM)
Tube 2: 400 µL of 0.5M glucose (final concentration 20mM)
Tube 3: 200 µL of 1M lactose and 400 µL of 0.5M glucose (final concentration 20mM of each)
Tube 4: don’t add anything
After adding the sugar solution to each tube, mix it immediately and place 1ml of each solution in a test tube and another 1ml in a plastic cell (induction time 0 min samples). Afterwards, return the L-tubes to the laboratory incubator shaker at 37ºC for another 60min. In the meantime perform the enzyme activity and bacterial turbidity measurements in c) and d).
iii. End of the incubation
After 60 min, collect 1ml of the samples from Tubes 1-4 into the test tubes and plastic cells again (same as in ii.) and measure the enzyme activity and bacterial turbidity following the methods described in c) and d).
c) Bacterial turbidity measurement
Cover the top of the plastic cell filled with the bacterial liquid with parafilm. Overturn the cell and measure the absorbance at 600 nm (use the liquid LB culture medium from the previous experiment as the blank solution). Overturn the cell and repeat the measurements 2 more times (3 times in total). Calculate the average and use it as the sample’s absorbance value OD600.
d) Enzyme activity measurement
i. Toluene treatment
After collecting the samples from the 4 L-tubes in b)ii. or iii. add one drop of toluene into each test tube, (including into the test tube with the blank solution from a)) immediately. Seal the test tubes with parafilm and put into a shaker at 37ºC for 15min, allowing the cells to dissolve to release β-galactosidase.
ii. β-galactosidase reaction
After the toluene treatment, place 200 µl of each sample from the 5 test tubes (including negative control sample) in a microtube containing 500 µl of ONPG at set time intervals e.g. every 1 minute. Mix immediately using the vortex mixer. Allow the reaction in each microtube to proceed for 8 min, then add 500 µl of 1M sodium carbonate and mix again.
iii. Absorbance measurement
Place all the microtubes (induction time 0 and 60 min) in a centrifuge at 12,000 rpm for 5 min. Collect 1 ml of the supernatant from each microtube and place it in plastic cells. Measure absorbance at 410nm, using the enzyme activity negative control sample as the blank solution.
e) Data analysis
Using the data collected in the experiment, calculate the relative enzyme activity for the samples from Tube 1-4 for induction time 0 and 60 min using the following method:
A: bacterial solution turbidity (= OD600)
B: volume of the collected toluene treatment sample that participated in the reaction (= 200 µl)
C: volume of the collected ONPG solution that participated in the reaction (= 500 µl)
D: volume of the collected 1M sodium carbonate solution that participated in the reaction (= 500 µl)
E: absorbance at 410nm caused by the presence of ortho-nitrophenol (= A410)
F: reaction time (= 8 min)
*volume of the toluene added to the samples can be ignored
1 U = [E × (B+C+D) / (B+C)] / [A × B / (B+C)] / 8
1U is the amount of ortho-nitrophenol produced (A410) in 1 minute while the reaction system’s bacterial turbidity equals 1. The units are [A410/ OD600 ⋅ min].
After performing the calculations, illustrate the relative activity values depending on the induction condition using one figure. In addition, discuss whether β-galactosidase is an inductive enzyme. Is it possible to determine the compound in the presence of which it displays induction considering the result of the sample without glucose?
Results and Discussion
E. coli has been cultivated in four tubes containing respectively lactose, glucose, a mixture of lactose and glucose and none of those sugars. Bacterial turbidity (OD600) and absorbance at 410 nm (ortho-nitrophenol concentration measurement) has been measured for all samples. The relative activity was calculated using the method outlined in the previous section. The results are shown in Table 1 and 2, and Figure 1.
As can be seen in Figure 1, relative activity of β-galactosidase is the greatest for Tube 1: lactose, for both cultivation times. Second highest relative activity is displayed in Tube 3: lactose + glucose. Third is Tube 4: no sugar. The lowest relative activity belongs to Tube 2: glucose sample. For Tubes 1,3 and 4, the relative activity of β-galactosidase decreased after 60 min of induction compared to the beginning of induction. The difference is particularly striking for Tube 3, where the relative activity decreased 2 times after 60 min of induction compared to 0 min.
Since the relative activity varies across the sugar solutions, it can be deduced that β-galactosidase is an inductive enzyme. Since the relative activity is greatest for Tube 1, it can be said that β-galactosidase is induced in the presence lactose.
β-galactosidase doesn’t tend to be synthetized in presence of glucose. When both lactose and glucose are present, less β-galactosidase is produced (compared to lactose only solution). This is because glucose is favoured over lactose as the energy source. It can be deduced that when glucose is present, β-galactosidase synthesis is suppressed by catabolite repression, regardless of whether lactose is present.
Theory Behind Lactose Metabolism- Lac operon transcriptional control system
Operon is a group of genes which are transcribed as a single mRNA. Lac operon contains 3 genes: lacZ, lacY and lacA that are transcribed as a single mRNA. Those genes encode proteins that allow E. coli bacteria to metabolise lactose.
However, since glucose is more easily metabolised, E. coli will use it as an energy source in preference to lactose. Therefore, if glucose is present, lac repressor will block the transcription of lac operon. When glucose is absent and lactose is present, the catabolite activator protein (CAP) will allow the transcription.
Both lac repressor and CAP bind to the DNA of the lac operon to control its transcription. Lac repressor detects lactose, and stops blocking the lac operon transcription in the presence of this sugar. CAP, on the other hand, is able to detect glucose, and turns on the lac operon transcription in low glucose levels. CAP detects glucose through the cAMP molecule, also called the “hunger signal” molecule.
The results of the experiment agree with the theory outlined above. β-galactosidase, which is encoded by lacZ gene in lac operon, displays highest relative activity in the presence of lactose. However, the relative activity decreases significantly in the presence of lactose and glucose solution. The lowest relative activity measured was that of the glucose sample. All three of those measurements agree with the theory. In the presence of lactose, lac operon is transcribed and β-galactosidase is synthesized in large quantity, so the relative activity is high. In case of lactose and glucose solution or glucose only solution, the transcription is being blocked to various degrees, so relative activity is lower.