PHAR1422: Enzyme Induction in Bacterial Cells .

PHAR1422 Biochemistry/Biotechnology II

Laboratory 5: Enzyme Induction in Bacterial Cells

Introduction The enzyme β-galactosidase of E. coli strain K12 is an inducible enzyme - that is, its synthesis is stimulated by the presence of a metabolite (the inducer) in the growth medium. β-galactosidase hydrolyzes β-galactosides, e.g. the milk constituent sugar lactose. When E. coli is grown in the absence of lactose or other β-galactosides, it produces very small amount of β-galactosidase (only a few enzyme molecules per cell). On the other hand, when the same strain is grown in the presence of a β-galactoside (e.g., with lactose as the major carbon source), the rate of β-galactosidase synthesis is greatly increased. Therefore, the quantity of β-galactosidase in such induced cells is much higher than that in cells grown in the absence of the inducer. The inducer needs not to be a substrate for β-galactosidase. In the following experiment a gratuitous inducer, isopropylthio-β-galactoside (IPTG), which is not a substrate for β-galactosidase, is used to induce synthesis of the enzyme. The lactose operon is a gene cluster, which coordinates the regulation of the three enzymes needed for the metabolism of lactose. It consists of three structural genes encoding β-galactosidase, permease and transacetylase and a control region that contains a promoter and an operator site. RNA polymerase binds to the promoter site and, the binding is enhanced by a cyclic AMP receptor protein (CAP), which also binds in this region. Immediately adjacent to this area is the operator, which is the binding site for a repressor coded by lactose regulatory gene. In the absence of an inducer, the repressor binds to the operator site and blocks RNA polymerase so that the structural genes cannot be expressed. However, the inducer binds the repressor protein and converts it to an inactive form that can no longer recognize the operator site, so that the operon can be transcribed. The synthesis of β-galactosidase in E. coli K12 cells is regulated not only by the presence of inducers, but also by the availability of alternate carbon sources. Certain preferred carbon sources, such as glucose, have the capacity to repress the synthesis of β-galactosidase even in the presence of an inducer. This phenomenon is called catabolite repression. Other carbon sources, such as succinate, acetate, and glycerol, are poorer catabolite repressors. The compound o-nitrophenyl-β-galactoside (ONPG) is the substrate for assays of β-galactosidase activity in this experiment. The enzyme hydrolyses the colourless compound to o-nitrophenol (ONP), which instantly ionizes in alkali to the o-nitrophenolate anion, a yellow-coloured compound that absorbs at 420 nm.

PHAR1422: Enzyme Induction in Bacterial Cells .

Materials i) Growth of cells: Take an inoculum of E. coli K12S and grow it in a lactose-free

medium consisting of 1% tryptone, 0.5% NaCl and 1% glucose. Grow cells until it reaches an optical density of 0.3 - 0.6 at 660 nm (1 cm cuvette).

ii) Reagents: 0.08 M phosphate buffer, pH 7.7; 1.0 M Na2CO3; 0.02 M IPTG (freshly

prepared); 2.5 mM ONPG (freshly prepared); toluene. iii) Apparatuses: Mirco-centrifuge tubes, pipets for measuring volumes of 5, 0.5 and

0.05 ml, cuvettes, test tube racks, parafilm; wash bottle, ice bath, and 37°C water bath; spectrophotometer; droppers.

Experimental procedures 1. Label 10 tubes containing 0.5 ml of lactose-free medium as tube 1 to 10.

2. Add 0.05 ml of distilled water to tubes 1 to 3, and 0.05 ml of 0.02M IPTG to tubes

4 to10. Incubate all tubes in a 37°C shaking water bath for 5 min.

3. After incubation, quickly add 0.5 ml of E. coli K12S cells to each of the 10 tubes while keeping the tubes in the 37°C water bath. Immediately withdraw tube 4 (with IPTG) and tube 1 (no IPTG), and add a drop of toluene to the tubes. Shake gently for 10 sec and incubate the tubes on ice. (What is the function of toluene in this step?)

4. Repeat step 3 to the rest of the tubes according to the schedule listed in Table 1. While waiting for the other samples, proceed to step 5.

5. Prepare eleven test tubes each containing 1 ml of 0.08 M phosphate buffer (pH 7.7) and 0.2 ml 2.5 mM ONPG. Label the tubes as 1 to 10 and “Blank” (see Table 2).

6. Quickly add 0.2 ml of the toluene-treated cells obtained in steps 3 & 4 into the corresponding test tubes (complete this step within one minute). For “Blank”, add 0.2 ml of water instead of bacterial cells. Incubate all samples at 37°C for 15 min.

7. After incubation, terminate the reaction by adding 0.2 ml of 1 M Na2CO3 to all 11 tubes. Record the absorbance at 420 nm using the “Blank” for zero setting.

* Pay attention to biological safety regarding the handling and disposal of bacterial (E.coli) cells. *

PHAR1422: Enzyme Induction in Bacterial Cells .

Table 1: Induction of β-galactosidase in E. coli K12

Relevant procedures Samples

A. Label tube 1 2 3 4 5 6 7 8 9 10 B. Add lactose-free medium

(ml) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

C. Add distilled H2O (ml) 0.05 0.05 0.05 - - - - - - - D. Add 0.02 M IPTG

(ml) - - - 0.05 0.05 0.05 0.05 0.05 0.05 0.05

E. Shake at 37°C (5 min.) F. Quickly add (ml) E. coli

K12 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

0 min - - - - - - - -

5 min - - - - - - - - - Keep shaking at 37°C 10 min - - - - - - - - -

15 min - - - - - - - -

20 min - - - - - - - - -

25 min - - - - - - - - -

30 min - - - - - - - -

Withdraw the tubes indicated followed by adding one drop of toluene. Shake gently for 10 sec and

incubate the cell lysates on ice until the whole series of experiment is completed. ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

Table 2: β-galactosidase Activity Assay

Relevant procedures Samples

no IPTG with IPTG Sample no. 1 2 3 4 5 6 7 8 9 10

Blank Equivalent time interval (min) 0 15 30 0 5 10 15 20 25 30

A. Add 0.08 M PO4 Buffer (pH7.7) (ml) 1 1 1 1 1 1 1 1 1 1 1

B. Add 2.5 mM ONPG (ml) 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2

C. Add the cell lysates from the corresponding tubes 1 to 10 above (ml)

0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 dH2O

D. Incubate at 37°C for 15 min

↓ E. Add 0.2 ml 1 M Na2CO3 to terminate the reaction

↓ F. Read absorbance at 420 nm

PHAR1422: Enzyme Induction in Bacterial Cells .

Results and Calculations 1. For quantitative measurement of the β-galactosidase activity, one unit of enzyme

is arbitrary defined as the activity of the enzyme that hydrolyzes ONPG to generate an increase in optical density of 0.01 at the wavelength of 420 nm in 1 cm light path, in 30 min. Calculate the units of the enzyme per ml of the bacterial culture for the samples taken at different times following the addition of the inducer.

2. Plot a graph showing the time course of β-galactosidase induction. How does the

specific activity of the enzyme change during the incubation period? Questions for Discussion 1. In this experiment the growth of E. coli (or the increase in bacterial cell number or

cell concentration) was monitored by measuring the absorbance of the bacterial culture at 660 nm. Why is it possible to use absorbance to measure the bacterial growth? Can you suggest another method for estimating the cell concentration or the cell mass of a bacterial culture?

2. What is the experimental advantage of using IPTG instead of lactose as an inducer

of the lac operon? 3. What is the purpose of adding toluene to the cell suspension prior to the assay of

β-galactosidase activity? Why the mixing of the bacterial suspension with toluene should be done gently?

4. How would chloramphenicol and cycloheximide affect the induction of β-

galactosidase in E. coli.

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There is no Write-up Form for this practical. Please answer the following questions and the required calculations in the format of a Laboratory Report. The completed Laboratory Report (stapled with the Computer Grade Form) should be submitted to Ms Kaman Lee at BMSB Rm 410 by 17 March 2011, 5 pm.