Concept of lac-operon: Lactose induction of Beta galactosidase; Glucose Repression;

Diauxic growth curve of E. coli.

Introduction:
Lac operon is an operon or a group of genes with a single promoter that encode genes for the transport
and metabolism of lactose in E.coli and other bacteria.”
Gene regulation in prokaryotes can be explained with the help of the Lac Operon model. Here the alteration
in physiological and environmental conditions can be observed leading to an alteration in expression in
prokaryotes. It was observed by Jacob and Monod. The lac operon consists of:

 Regulatory gene i – It codes for the repressor protein.

 z gene – It codes for beta-galactosidase which catalyzes the hydrolysis of lactose into glucose and
galactose.
 y gene – It codes for permease which regulates the lactose permeability in the cell.
 a gene – It codes for transacetylase which assists the enzyme beta-galactosidase.
Hence, all these genes help in lactose metabolism. In lac operon, lactose acts as an inducer. If lactose is
provided in the medium for the bacteria, the regulatory gene is activated. The inducer will bind to the
repressor protein and render it inactive which allows transcription of the operon. Thus, the lac operon is
negatively regulated in this case.
Enzyme Induction is still considered a form of negative control because the effect of the regulatory molecule
(the active repressor) is to decrease or downregulate the rate of transcription.
 Catabolite repression is a type of positive control of transcription, since a regulatory protein affects
an increase (upregulation) in the rate of transcription of an operon. The process was discovered in E.
coli and was originally referred to as the glucose effect because it was found that glucose repressed the
synthesis of certain inducible enzymes, even though the inducer of the pathway was present in the
environment. The discovery was made during study of the regulation of lac operon in E. coli.

Glucose represses the induction of inducible operons by inhibiting the synthesis of cyclic AMP (cAMP), a
nucleotide that is required for the initiation of transcription of a large number of inducible enzyme systems
including the lac operon.

The role of cyclic a cAMP is complicated. cAMP is required to activate an allosteric protein called CAP
(catabolite activator protein) which binds to the promoter CAP site and stimulates the binding of RNAp
polymerase to the promoter for the initiation of transcription. Thus, to efficiently promote gene transcription
of the lac operon, not only must lactose be present to inactivate the lac repressor, but cAMP must be
available to bind to CAP which binds to DNA to facilitate transcription. In the presence of glucose,
adenylate cyclase (AC) activity is blocked. AC is required to synthesize cAMP from ATP. Therefore, if
cAMP levels are low, CAP is inactive and transcription does not occur. In the absence of glucose, cAMP
levels are high, CAP is activated by cAMP, and transcription occurs (in the presence of lactose).
Many positively controlled promoters, such as the lac promoter, are not fully functional in the presence of
RNAp alone and require activation by CAP. CAP is encoded by a separate Regulatory gene, and is present
in constitutive levels. CAP is active only in the presence of cAMP. The binding of cAMP to CAP causes a
conformational change in the protein allowing it to bind to the promoter near the RNAp binding site. CAP
can apparently interact with RNAp to increase the rate of operon transcription about 50-fold. Positive

control of the lac operon.

Bi-phasic or Diauxic growth is the phenomenon whereby a population of microbes, when presented with
two carbon sources, exhibits bi-phasic exponential growth intermitted by a lag-phase of minimal growth.
Originally, the phenomenon was described by Monod1 demonstrating diauxie with glucose and lactose in E.
coli . In his experiments Monod showed that the population first grows exponentially on glucose until all
glucose is exhausted, then stops growing for a considerable amount of time and subsequently resumes
exponential growth on lactose. The duration of the lag-phase can be substantial. There are two main
mechanisms responsible for two phase growth in bacteria: (i) Regulation of metabolic genes via global
transcription regulators, especially cAMP. (ii) Direct repression of uptake of the secondary nutrient in the
presence of glucose. Both of those effects are mediated by the phosphotransferase system (PTS)12, whereby
a phosphoryl group (P) is transferred from PEP to the carbohydrate in a five step phosphorelay. In
Enterobacteriaceae the PTS relay is catalysed by the generic “Enzyme I” (EI) and HPr as well as the
carbohydrate specific components EIIA, EIIB, EIIC. During glucose uptake the levels of dephospho
EIIAGlc increase. In its dephosphorylated form this enzyme directly interacts with the permeases of the
secondary sugars (i.e. lactose). In this way it prevents nutrient uptake and thus the induction of the relevant
uptake system (inducer exclusion). Diauxic growth has traditionally been interpreted as an adaptation that
allows the maximisation of growth in dual-nutrient environments. Intuitively, the reasoning is like so: When
there are two shared resources, one good, one less good, then sequential uptake of nutrient can be shown to
be a beneficial strategy under a wide range of conditions.
Aim: To study concept of lac-operon: Lactose induction of Beta galactosidase; Glucose Repression; Diauxic
growth curve of E. coli.

Requirements:
1. Sterile nutrient broth with 1.5 g glucose and 1g lactose in side arm flask
2. 18 hr old E.coli culture
3. Blank tube : sterile nutrient broth
4. Colorimeter with 620 nm filter

Protocol:
1. E.coli was freshly grown in nutrient broth and was kept for an 18 hr incubation period at 37 ̊ C the
day before.
2. 1ml of this culture was transferred into a side arm flask (500ml) with sterile nutrient broth (250ml)
containing glucose and lactose. This is mixed thoroughly and immediate reading was taken using
colorimeter set at 620 nm. This reading is termed as Tₒ.
3. The flask is placed at 37 ̊C in a shaking incubator.
4. After every half hour the reading was taken under aseptic conditions and an uninoculated broth was
Sr. Time Absorbance Sr. no. Time Absorbance used as control.
no. (620nm) (620nm) 5. This was done for 8 hrs till
1 8:00 11 1:00 second stationary phase isn’t
2 8:30 12 1:30 achieved.
3 9:00 13 2:00 6. Then a graph was plotted
4 9:30 14 2:30 showing absorbance vs. time.
5 10:00 15 3:00
Observation table:
6 10:30 16 3:30
7 11:00 17 4:00
8 11:30
9 12:00
10 12:30
Observation:

Graph:

Results and conclusion: