Gene induction: ß-galactosidase in E. coli
Class practical
Escherichia coli (E. coli) can produce the enzyme β-galactosidase which breaks lactose into galactose and glucose. However, the gene for β-galactosidase is normally switched off, except in the presence of lactose.
In this procedure, a sample of E. coli is treated with lactose, and then the β-galactosidase activity of this sample and an untreated sample are compared. ONPG (ortho-nitrophenyl-?-D-galactoside) is used as a substrate for the enzyme action which produces galactose and a compound which is yellow in alkaline conditions. The intensity (or optical density) of the yellow colour produced is a qualitative indicator or quantitative measure (with a colorimeter) of the β-galactosidase activity.
The procedure indicates that the gene which produces β-galactosidase in E. coli is induced or ‘switched on’ by the presence of lactose.
This practical is adapted from protocols developed for Salters-Nuffield Advanced Biology (SNAB) by Science and Plants for Schools (SAPS) and the NCBE. Links to all three original protocols are given at the bottom of this page.
Lesson organisation
The practical doesn’t require strict aseptic technique, but does require good microbiology practice when handling the E. coli. In the final stage you follow the colour change with a colorimeter. If you have only one spectrophotometer/ colorimeter (or suitable datalogging device), you could ask all students to follow the changing colour by eye, and one group to produce quantitative data to share with the group.
Apparatus and Chemicals
For the class - set up by the technician/ teacher
Access to a fume cupboard (Note 1)
Colorimeter or spectrophotometer to measure optical density at 420 nm (or 440 nm if 420 nm is not possible)
Cuvettes for colorimeter
Water bath, set to 37 °C
Pasteur pipettes in the cultures, sterile, attached to 1 cm3 syringes
Discard pot for Pasteur pipettes (Note 2)
Disinfectant for cleaning the work area and for the discard pot (Note 2)
E. coli culture, in nutrient broth or LB broth
(Note 3), 0.1 cm3 per working group
E. coli culture, in nutrient broth or LB broth with added lactose (Note 3), 0.1 cm3 per working group
Nutrient broth with added lactose, no E. coli introduced, 0.1 cm3 per working group
Z buffer, about 10 cm3 per working group
(Note 4)
ONPG solution, concentration of 4 mg per 10 cm3 in Z buffer (Note 5)
β-galactosidase solution, 0.1 cm3 per group
Methylbenzene (toluene), 1 drop per test tube (Note 1)
For each group of students
Access to hairdryer in fume cupboard
5 cm3 syringe with Pasteur pipette tip (to dispense ONPG solution)
Test tubes, 5
Stoppers for test tubes or parafilm
Marker pen/ labels
Health & Safety and Technical notes
E. coli (K12) strain is safe for work in schools but should still be handled using good microbiological practice. See Standard technique Aseptic techniques.
Methylbenzene is highly flammable and harmful. Avoid skin contact and inhalation.
1 Evaporating methylbenzene: this is essential if your colorimeter cuvettes are made of plastic. Methylbenzene will permanently damage the plastic, by clouding the surface, or by damaging other plastic components inside your colorimeter. If your cuvettes are glass and have stoppers, you may be able to remove this stage from the procedure. Methylbenzene is described on the CLEAPSS Hazcard as HIGHLY FLAMMABLE and HARMFUL – IRRITATING and TERATOGENIC. It is absorbed through the skin, and vapours may cause drowsiness and dizziness.
2 Suitable disinfectants include sodium chlorate(I) (hypochlorite) at concentrations providing 1000 ppm available chlorine for general surface cleaning or 2500 ppm chlorine for discard pots, or Virkon (follow manufacturer’s instructions).
3 E. coli: use a strain with the lacZ (β-galactosidase) gene, for example, the K12 strain. Make up the culture 24-48 hours before use in LB broth, or nutrient broth. For LB broth, add 10 g tryptone, 5 g yeast extract, and 10 g sodium chloride to 800 cm3 H2O. Adjust pH to 7.5 with 1.0 M sodium hydroxide. Adjust the volume to 1 litre with more water. Sterilize by autoclaving. Add lactose at a concentration of 0.01 g per cm3 of broth (or 10g per litre) to one sample of the broth.Label the two kinds of broth clearly ('with lactose' or 'without lactose').
4 Z buffer is a phosphate buffer at pH7. To make 100 cm3, take 1.60 g Na2HPO4.7H2O, 0.55 g NaH2PO4.H2O, 0.075g KCl and 0.012g MgSO4. Bring to approximately 80 cm3 with distilled water. Dissolve all the salts. Adjust the pH to 7.0 with 1 M or 2 M NaOH. Add more water to bring to 100 cm3. Store in a refrigerator at 4 °C. It will keep for several months at this temperature. The buffer once made up is LOW HAZARD – see CLEAPSS Hazcard. All the salts are low hazard. Sodium hydroxide is described on the CLEAPSS Hazcard as CORROSIVE at this concentration.
5 ONPG solution is best prepared no more than 24 hours ahead of time, and should be stored in a fridge at 4 °C in a bottle wrapped in foil. Add 0.55 g of ONPG to 100 cm3 of Z buffer. ONPG may be harmful if inhaled and may cause respiratory tract irritation, so make up solutions in a fume cupboard.
6 β-galactosidase: Make up a 1% solution of the enzyme. The CLEAPSS Hazcard advises treating enzymes as irritants – both in powder form and 1% solution.
Procedure
SAFETY:
Handle the E. coli using good microbiological practices. See Standard techniques Aseptic techniques and Maintaining and preparing cultures of bacteria and yeasts.
Avoid skin contact with methylbenzene, and work with good ventilation to reduce inhalation of vapour.
Avoid skin contact with the enzyme solution.
Preparation
a Make up enough Z buffer (phosphate buffer at pH7) – Note 4.
b 24 to 48 hours in advance, make up two cultures of a the K12 strain of E. coli (or another that can produce lactose), and one without lactose (Note 3).
c No more than 24 hours in advance, make up the ONPG solution (Note 5).
d Make up 1% β-galactosidase solution (Note 6).
e Make up Virkon (or other disinfectant) for discard pots and cleaning the work area. (Note 2).
Investigation
f Disinfect the working area with Virkon or other suitable disinfectant (Note 2).
g Set out and label 5 test tubes, 1 to 5.
h Add 0.1 cm3 of E. coli in nutrient broth (that has not been induced with lactose) to tube 1.
i Add 0.1 cm3 of E. coli in nutrient broth (that has been induced with lactose) to tube 2.
j Add 0.1 cm3 of β-galactosidase enzyme solution to tube 3.
k Add 0.1 cm3 of nutrient broth with lactose to tube 4.
l Add 0.1 cm3 of distilled water to tube 5 to equalise the volume for comparison with other tubes.
m Add a drop of methylbenzene to each tube. This kills the cells and disrupts the cell membranes which will allow the ONPG and enzymes to meet. Cover with parafilm or a stopper, and shake to mix the contents thoroughly.
n In a fume cupboard, use a hairdryer to evaporate the methylbenzene. It is less dense than water and appears as a greasy film on the surface. When it has all evaporated, you can proceed to the next step (Note 4).
o Add 2 cm3 of ONPG in Z-buffer to all five tubes.
p Transfer to a water bath at 37 °C, and record the time.
q Measure the depth of colour in the samples (either transmission or absorbance) at 420 nm (or 440 nm) at 10 minute intervals until there is no further change.
r Plot a graph of depth of colour against time. (The value will decrease with time if you are measuring transmission, or increase if you are measuring absorbance.)
The contents of the tubes are as follows:
| Tube | E. coli in nutrient broth (not exposed to lactose) | E. coli in nutrient broth with lactose | β-galactosidase | Nutrient broth with lactose – no E. coli |
Methyl-benzene |
Z buffer with ONPG |
| 1 | 0.1 cm3 | 1 drop | 2 cm3 | |||
| 2 | 0.1 cm3 | 1 drop | 2 cm3 | |||
| 3 | 0.1 cm3 | 1 drop | 2 cm3 | |||
| 4 | 0.1 cm3 | 1 drop | 2 cm3 | |||
| 5 | 1 drop | 2 cm3 |
Teaching notes
Salter’s-Nuffield Advanced Biology suggests running this procedure as a qualitative investigation, in which case you don’t need to use a colorimeter and don’t need to evaporate the methylbenzene. The NCBE protocol describes an alternative qualitative procedure using E. coli grown on agar.
The qualitative difference will be noted by comparing tubes 1 and 2 with tube 5 (the control) and noting that the bacteria previously exposed to lactose have been able to change the ONPG, whereas the bacteria not previously exposed have not been able to.
Using a colorimeter, measuring over a 10 minute time period, you will see whether the previously exposed bacteria have simply got a 'head start' in the breakdown of ONPG.
Every nucleus in the cells of an organism contains the full complement of genes of that organism. Differentiation of tissues involves switching genes on or off, and expressing selected genes to produce the proteins that distinguish each tissue. In some tissues, some genes can be switched on or off in reaction to the surrounding conditions.
Only expressing useful genes at any time means that energy is not wasted, since protein synthesis requires energy. It also means that cells (or microorganisms) can react to changing conditions by producing useful proteins, including enzymes only, when the substrates are available for them.
Health & Safety checked, May 2009
Downloads
Download the student sheet 
Gene induction: ß-galactosidase in E. coli (57 KB) with questions and answers.
Web links
www.ncbe.reading.ac.uk/NCBE/PROTOCOLS/DNA/bgalactosidase.html
This is the NCBE version of this protocol, including a qualitative method that shows the induction of enzyme activity without the use of a colorimeter to quantify the level of activity.
www.saps.org.uk/secondary/teaching-resources/95-investigating-the-effect-of-competitive-and-non-competitive-inhibitors-on-the-enzyme-ss-galactosidase
This is the SAPS version of the protocol, including some background to the Jacob-Monod hypothesis of gene induction. The main difference is that the SAPS protocol uses simple sodium phosphate rather than the complex Z buffer described above, and larger volumes of solution.
www.bbc.co.uk/scotland/learning/bitesize/higher/biology/control_regulation/genetic_control_rev1.shtml
BBC Bitesize revision of the Jacob-Monod operon model for the control of gene expression.
(Websites accessed October 2011)