Introduction

An operon is a functioning unit of genomic material containing a cluster of genes under the
control of a single regulatory signal or promoter. The lac operon is an operon required transport
and metabolism in Escherichia coli (E. coli), and some other enteric bacteria (Khanna, 2009).
It consists of three adjacent genes, a promoter, a terminator and an operator (Khanna, 2009), as
shown in Figure 1.

Figure 1: Structure of a lac operon. [Source: Khanna (2009)]

The three structural genes are lacY, lacZ and lacA. LacZ encodes for β-galactosidase, which is an
intracellular enzyme that cleaves the disaccharide lactose into glucose and galactose. LacY
encodes for β-galactoside permease, a membrane bound transport protein that pumps lactose into
the cell. Lastly, lacA is responsible for encoding β-galactoside transacetylase, an enzyme that
transfers an acetyl group from acetyl-CoA to β-galactoside (Khanna, 2009).
The DNA-binding protein that controls transcription of the lac operon is known as the lac
repressor protein, which regulates the lac operon (Kratz, 2009). The lac repressor has two
binding sites; a DNA-binding site that binds to the operator sequence of the lac operon and an
allosteric site that binds to an isomer of lactose (Kratz, 2009). The “blueprint” for the lac
repressor is contained in the I gene, which isn’t part of the lac operon. The I gene is
constitutively expressed so the lac repressor is always available in the cell. When the lac
repressor is active, the lac operon is shut down (Kratz, 2009). In the presence of glucose, the lac
operon is turned off and the cell uses exclusively the glucose. This means that the lac operon is

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subjected to glucose regulation. The inhibitory action of glucose on the operon is called
catabolite repression (Rao, 2006) and because the active repressor shuts down the operon,
regulation of the lac operon by the lac repressor is called negative control (Kratz, 2009).
Induction is the turning on of the lac operon. Induction allows E. coli to use lactose as a food
source. Therefore, lactose is the inducer of the lac operon (Kratz, 2009).
Beta-galactosidase or β-galactosidase is a hydrolase enzyme that splits lactose into glucose and
galactose, coded by the lacZ gene, in the lac operon of E. coli. This enzyme has a three-
dimensional structure. It is a tetramer with 222-point symmetry. It is a 1023-amino-acid chain
that folds into five sequential domains, with an extended segment at the amino terminus
(Jacobson et al, 1994). The active site of β-galactosidase catalyzes the hydrolysis of its
disaccharide substrate via “shallow” and “deep” binding (Mounsami et al, 2003). Monovalent
potassium ions as well as divalent magnesium ions are required for the enzymes optimal activity.
The beta-linkage of the substrate is cleaved by a terminal carboxyl group on the side chain of a
glutamic acid (Mounsami et al, 2003). In E. coli, Glu-461 was thought to be the nucleophile in
the substitution reaction, however it was later discovered that Glu-461 is an acid catalyst.
Instead, Glu-537 is the actual nucleophile that binds to a galactosyl intermediate. In humans the
nucleophile of the hydrolysis reaction is Glu-268 (Mounsami et al, 2003).

Global control systems respond to signals in the environment and regulate the expression of
many genes simultaneously (Madigan et al, 2005). Catabolite repression is a global control
system that helps cells efficiently use carbon sources. The lac operon is under the control of
catabolite repression as well as its own specific regulatory system (Madigan et al, 2005).
Catabolite repression is a positive control system that involves the control of transcription by an
activator protein. The binding of RNA polymerase to DNA occurs only if another protein called
catabolite activator protein (CAP), has bound first (Madigan et al, 2005). CAP only binds to
DNA of it has first bound to a small molecule called Cyclic AMP (cAMP). cAMP must be
synthesized from ATP by an enzyme called adenylate cyclase. When glucose is transported into
the cell, it inhibits the production of AMP and stimulates the cAMP transport out of the cell
(Madigan et al, 2005). When the levels of glucose rise in the cell, cAMP levels are decreased and
bind of RNA polymerase to the promoter doesn’t occur. Catabolite repression modulates several
unrelated regulatory systems (Madigan et al, 2005). Therefore it can be determined the for global

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control of the lac operon to occur, the levels of cAMP must be high enough so that CAP protein
binds to the CAP binding site; this is known as positive control and there must be an inducer,
such as lactose present so that the lactose repressor does not block transcription by binging to the
operator, which is known as negative control.
Natural inducers are those inducers that can be metabolized by β-galactosidase to produce
glucose and galactose. In this case the natural inducer in terms of the lac operon is allolactose, a
metabolic intermediate of lactose, because it can be metabolized by the β-galactosidase.
Artificial inducers are those that are used in assays in place of lactose but are cleaved in the same
way as the natural inducers. In this experiment the artificial inducer used was ortho-nitrophenyl-
β-galactosidease (ONPG) (van Hoek and Hogeweg, 2006).

β-galactosidase facilitates growth on carbon sources like lactose, by cleaving it into a molecule
of glucose, which the cell can catabolize and grow on. In the assay, the artificial chromogenic
substrate, ONPG, is used in place of lactose. When the β-galactosidase cleaves ONPG, ortho-
nitrophenol is released. ONPG is colourless, while the product compound has a yellow colour
(Jacobson et al, 1994). The rate of appearance of the yellow colour is used to measure the
enzyme activity.
lac+ is a term used to describe bacteria that are able to ferment and utilize sugar lactose whereas
lac- used to describe those that cannot use or ferment sugar lactose but instead use peptone as a
carbon source in Maconkey agar (McClane et al, 1999). The reason for this is that the lacZ gene
is not coded for and so β-galactosidase is not produced in the lac- (McClane et al, 1999).

Maconkey agar is used for the selective and identification of enterobacteriacaeae, and is a
selective and differential medium for the isolation of enteric gram negative bacteria (McClane et
al, 1999). Maconkey agar is also used to distinguish between lac-, that is, those bacteria that
cannot ferment sugar lactose and lac+ bacteria and coliforms (Hopwood, 1970). Organisms, such
as E. coli, are able to ferment lactose in the agar resulting in the individual colonies turning a
pink colour on the agar (McClane et al, 1999). This type of agar is usually composed of peptone,
lactose, bile salts (to inhibit the growth of most gram positive bacteria), agar, NaCl, crystal violet
(which also inhibits certain gram positive bacteria) and neutral red (which stains microbes
fermenting lactose). The agar is usually red in colour (Hopwood, 1970). Maconkey agar is both a
selective and differential medium. It is selective because it contains bile salts which are
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detergent-like molecules, similar to those found in the human gastro-intestinal tract. It is also
differential because it contains neutral red and high concentrations of lactose (McClane et al,
1999).

The aims of this experiment was to determine the activity of the β-galactoside enzyme in E. coli
cultures, to identify and differentiate between lac+ and lac- E. coli mutants and to determine how
different carbon sources affect β-galactosidase activity.

Methods and Materials

Experiment A: Assay for β-galactosidase activity.

Two samples of log-phase E. coli cultures were measured for their β-galactosidase activity.

The OD600 of each culture was measured to make sure that it was between 0.6 and 0.8. If it was
not, the sample was further diluted until it was within the range.

Figure 2: Flow Diagram of the Method for the β-galactosidase assay

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Experiment B: Identification of the lac- E. coli mutants.
A mixed culture of two E. coli strains, lac+ and lac-, were plated onto Maconkey Agar plates to
differentiate the lac+ and lac- strains.

Figure 3: Flow Diagram of the Method for lac- E. coli mutants.

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Experiment C: Effect of different carbon sources on β-galactosidase activity.
The K12 E. coli strain was grown in three different carbon sources, lactose (LMM), glucose
(GMM), and glycerol (GlyMM).

The OD600 of each culture was measured to make sure that it was between 0.6 and 0.8. If it was
not, the sample was further diluted until it was within the range.

Figure 3: Flow Diagram of the Method for lac- E. coli mutants.

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Results, Discussion and Conclusions.

The β-galactosidase activity was measure by using the following equation:

β-galactoside activity =
1000 x [OD 420 – (1. 75 x OD 550 )]
(Vol x ∆ Time x OD 600 )

Tables 1 and 4 show the OD600 readings for the cultures that were used in doing the experiments.

Table 1: OD600 Reading for Experiment A Cultures

Culture OD600
lac+ 0.638
lac- 0.225

Table 2: β-galactosidase Activity in E. coli cultures.

Tube Vol. OD420 OD550 Initial Initial ΔTime β-Gal

Cells time time (min)
(mL) (min) (min)

lac- 1a 0.5 0.019 0.018 0 8 8 -13.9

lac- 1b 0.5 0.121 0.110 0.08 8:05 7:57 -83.9
lac- 2a 0.25 0.054 0.079 0.16 8:10 7:54 -198.6
lac- 2b 0.25 0.383 0.210 0.25 8:15 7:50 -36.7

lac+ 1a 0.5 0.880 1.667 0 1:39 1:39 -4594.5

lac+ 1b 0.5 0.132 0.068 0.30 1:54 1:24 32.865
lac+ 2a 0.25 0.203 0.227 1:00 3:26 2:26 -538.8
lac+ 2b 0.25 0.078 0.104 1:30 3:25 1:55 -420.7

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Table 2 shows the β-galactosidase activities for lac+ and lac- strains of E. coli. The results show
negative values for the lac- strain. Any negative values were taken to be zero. This meant that
there was no β-galactosidase activity for this strain. This result was found to agree with what was
previously mentioned by McClane et al (1999), that lac- bacteria do not use lactose as a carbon
source where as there was some activity for the lac+ strain, showing that there was some activity.
There was a clear relationship between the absorbance and the activity. The higher the
absorbance, the lower the β-galactosidase activity. This experiment proved that lac- strains did
cannot use lactose as a carbon source.

Table 3: Colony Counts

Dilution 10-5 10-6 10-7 10-8

Number of Colonies 1336 728 96 3

Table 4: OD600 Reading for Experiment A Cultures

Culture OD600
lac+ GMM 0.628
lac+ GlyMM 0.588
lac+ LMM 0.640

Table 5: β-galactosidase Activity of Cultures Grown in Lactose, Glucose and Glycerol

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 Tube Vol. Cells OD420 OD550 Initial time Final time ΔTime β-Gal
(mL) (min) (min) (min)

A GMM 1a 0.25 0.396 0.323 0 10:46 10:46 -103.06

A GMM 1ba 0.25 1.079 1.575 0:30 10:34 10:04 -1064.1

B GlyMM 1a 0.25 0.433 0.283 1 10:17 9:17 -46.18

B GlyMM 1b 0.25 1.081 1.123 1:30 10:00 8:30 -724.7

C LMM 1a 0.25 0.741 0.232 2 3:54 1:54 1359.6

C LMM 1b 0.25 0.834 0.247 2:30 4:38 2:08 1207.2

The colonies seen on the agar plates were for the different dilution factors decreased as the
dilution factor increased as observed in Table 3. The colonies that were observed were the lac+
strain. They appeared purple on the Maconkey agar plates. These purple colonies fermented and
metabolized the lactose in the agar. There were supposed to be dull grey colonies also on the
agar but none were observed. This could have been due to them not growing or not having
sufficient time to grow and also not being as healthy as the lac+. If left to grow for a longer time
period they could have grown and been large enough to count. The dull grey colonies would
have been of the lac- strain. This strain does not ferment lactose but rather the peptone in the
agar. These properties are in agreement with what was previously mentioned in McClane et al,
(1999).
The β-galactosidase activity for the strains grown in different carbon sources, illustrated by Table
5, showed that the cells grown in minimal media containing lactose (LMM) had high activity for
both test tube 1a and 1b. This was because the lactose was converted to glucose and galactose.
The minimal media containing glycerol (GlyMM) and glucose (GMM) had no activity. The
activity for both these media was a negative number, which is taken as zero as mentioned before.
It is also observed that the time taken for the LMM reaction to be stopped was much shorter than
that of the GlyMM and GMM. The OD600 readings for the three media are shown in table 4. The
activity numbers obtained were of high. This is a good indication of the β-galactose activity.

Finally, taking all the abovementioned into conclusion, it can be concluded that the aims for this
experiment were achieved.

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References

Hopwood, D.A. (1970). Methods in Microbiology: Volume 3A, Chapter VI: The Isolation of
Mutants, Norris, pp 410-411, J.R. and Ribbons, D.W. (Editors), Academic Press Inc. Ltd.,
London.

Jacobson, R.H., Zhang, X.J., DuBose, R.F. and Matthews, B.W. (1994). Three-Dimensional
Structure of Beta-Galactosidase from E. coli, Institute of Molecular Biology, Eugene.

Khanna, P. (2009). Essentials of Genetics, Chapter12: Regulation of Gene Expression in

Prokaryotes, pp 286, I.K. International Publishing House Pvt. Ltd., New Delhi.

Kratz, R.F. (2009). Molecular and Cell Biology for Dummies, Chapter 19: Control of Gene
Expression, pp291, Wiley Publishing Inc., Indiana.

Madigan, M.T., Martinko, J.M., Parker, J. (2005). Brock Biology of Microorganisms, Chapter
10: Bacterial Genetics, 10th Edition, pp 217-218, Pearson Education, Inc., New Jersey.

McClane, B.A, Mietzner, T.A., Dowling, J.N. and Phillips, B.A. (1999). Microbial Pathogenesis:
A Principals-Orientated Approach, Chapter 12: Colonization I: Adherence, 1 st Edition, Fence
Creek Publishing, Madison.

Mounsami, D., Godmarhi, B.K.S. and Bisen, P.S. (2003). Molecular Diagnostics: Promises and
Possibilities, Chapter 5: Beta-Galactosidase: Reporter Gene System, pp 78, Springer, London.

Rao, M.N. (2006). Medical Biochemistry, Chapter 19: Regulation of Gene Expression, 2 nd
Edition, pp 461, New Age International Publishers Ltd., India.

van Hoek, M.J.A. and Hogeweg, P. (2006). In Silico Evolved lac Operons Exhibit Bistability for
Artificial Inducers but not for Lactose, Biophysical Journal, Vol.: 91 pp2833, Netherlands.

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