BIOADSORBENTS: A GREEN WAY TO CLEAN

HEAVY METAL POLLUTION

A PROJECT REPORT IN BIOLOGY (044) SUBMITTED IN PARTIAL

FULFILLMENT OF THE REQUIREMENT FOR THE COMPLETION OF

AISSCE 2024-2025

BY

G.PHILIP
AISSCE Roll No: ………………..

Under the supervision of

Mr. S. NARESH
PGT Biology

JAIRAM PUBLIC SCHOOL

CBSE- SENIOR SECONDARY,
Chinnathirupathi (Po), Salem,
Tamilnadu.

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 JAIRAM PUBLIC SCHOOL,
CBSE- SENIOR SECONDARY,
Chinnathirupathi (Po), Salem -
636008

CERTIFICATE

This project entitled “BIOADSORBENTS: A GREEN WAY TO

CLEAN HEAVY METAL POLLUTION”, is the investigatory project work in

BIOLOGY (044), successfully completed by Master/Miss

THARANEESWAR, student of class-XII, Jairam Public School, Salem,

with AISSCE Roll No. , under the supervision of Mr.

S. Naresh (PGT Biology), for the partial fulfilment of requirements for the

course completion in pursuance of AISSCE 2022-23.

Teacher In-Charge Principal

Practical Examiner School

Stamp

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 ACKNOWLEGEMENT

I have taken efforts in this project. However, it would

not have been possible without the kind support and help of
many individuals.

I would like to thank my Principal Sir, Mr. Paul

Francis Xavier and school for providing me with facilities
required to do my project.

I am highly indebted to my Biology Teacher, Mr. S.

Naresh, for his invaluable guidance which has sustained my
efforts in all the stages of this project work.

I would also like to thank my parents for their

continuous support and encouragement.

My thanks and appreciations also go to my fellow

classmates and to the people who have willingly helped me out
with their abilities.

G.PHILIP

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 CONTENTS
S. NO. TOPICS PAGE NO.

1 INTRODUCTION

2 Structure

3 Genetic nomenclature

4 Regulation

5 Repressor structure

6 Lactose analogs

7 Development of the classic model

8 Regulation by cyclic AMP.

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Use in molecular biology

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 OBJECTIVE:

Lac operon contains genes involved in metabolism

1. INTRODUCTION

The lactose operon (lac operon) is an operon required for the transport and metabolism of lactose in
E. coli and many other

enteric bacteria. Although glucose is the preferred carbon source

for most enteric bacteria, the lac operon allows for the effective
digestion of lactose when glucose is not available through the
activity of beta-galactosidase. Gene regulation of the
lac operon was the first genetic regulatory mechanism to be
understood clearly, so it has become a foremost example of
prokaryotic gene regulation.

It is often discussed in introductory molecular and cellular biology

classes for this reason. This lactose metabolism system was used
by François Jacob and Jacques Monod to determine how a
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biological cell knows which enzyme to synthesize. Their work on
the lac operon won them the Nobel Prize in Physiology in 1965
Most bacterial cells including E. coli lack introns in their genome.
They also lack a nuclear membrane. Hence the gene regulation by
lac operon occurs at the transcriptional level, by preventing
conversion of DNA into mRNA.

Bacterial operons are polycistronic transcripts that are able to

produce multiple proteins from one mRNA transcript. In this case,
when lactose is required as a sugar source for the bacterium, the
three genes of the lac operon can be expressed and their
subsequent proteins translated: lacZ, lacY, and lacA. The gene

product of lacZ is β-galactosidase which cleaves lactose, a

disaccharide, into glucose and galactose. lacY encodes β-
galactoside permease, a membrane protein which becomes
embedded in the Plasma membrane to enable the cellular
transport of lactose into the cell. Finally, lacA encodes β-
galactoside transacetylase.

>It would be wasteful to produce enzymes when no

lactose is available or if a preferable energy source such as
glucose were available. The lac operon uses
a two-part control mechanism to ensure that the cell expends energy
producing the enzymes encoded by the

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 The lac operon. Top: Repressed, Bottom: Active.

1: RNA polymerase, 2: Repressor, 3:

Promoter, 4: Operator, 5: Lactose, 6: lacZ, 7:

lacY, 8: lacA lac operon only when necessary.

In the absence of lactose, the lac repressor, encoded by lacI, halts production of the
enzymes and transport proteins encoded by the lac operon. It does
so by blocking the DNA dependent RNA polymerase. This blocking/ halting is not
perfect, and a minimal amount of gene expression does take place all the time. The
repressor protein is always expressed, but the lac operon (enzymes and transport
proteins) are repressed. (But not completely stopped)

When lactose is available but not glucose, then some lactose enters the cell using pre-
existing transport protein encoded by lacY. This lactose then combines with the
repressor and inactivates it, hence allowing the lac operon
to be expressed. Then more β-galactoside permease is synthesized allowing even
more lactose to enter and the enzymes encoded by lacZ and lacA can digest it.

However, In the presence of glucose, regardless of the presence of lactose, the

operon will be repressed. This is because the catabolite activator protein (CAP),
required for production of the enzymes, remains inactive, and EIIAGlc shuts down
lactose
permease to prevent transport of lactose into the cell. This dual control mechanism
causes the sequential utilization of glucose and lactose in two distinct growth phases,
known diaux

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2. Structure

The lac operon consists of 3 structural genes, and a

promoter, a terminator, regulator, and an operator. The
three structural genes are: lacZ, lacY, and lacA.
 lacZ encodes β-galactosidase (LacZ), an intracellular enzyme
that cleaves the disaccharide lactose into glucose and
galactose.
 lacY encodes Beta-galactoside permease (LacY), a
transmembrane symporter that pumps β-galactosides
including lactose into the cell using a proton gradient in the
same direction. Permease increases the permeability of the
cell to β-galactosides.
 lacA encodes β-galactoside transacetylase (LacA), an enzyme
that transfers an acetyl group from acetyl-CoA to
thiogalactoside.

Structure of lactose and the products of its cleavag

3. Genetic nomenclature

Three-letter abbreviations are used to describe phenotypes in

bacteria including E. coli

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EXAMPLES INCLUDE:
Lac (the ability to use lactose),
His (the ability to synthesize
the amino acid histidine)
Mot (swimming motility)

 In the case of Lac, wild type cells are Lac + and are able to use
lactose as a carbon and energy source, while Lac − mutant
derivatives cannot use lactose. The same three letters are
typically used (lower-case, italicized) to label the genes
involved in a particular phenotype, where each different gene
is additionally distinguished by an extra letter. The lac genes
encoding enzymes are lacZ, lacY, and lacA. The fourth lac gene
is lacI, encoding the lactose repressor—"I" stands for
inducibility.

 One may distinguish between structural genes encoding

enzymes, and regulatory genes encoding proteins that affect
gene expression. Current usage expands the phenotypic
nomenclature to apply to proteins: thus, LacZ is the protein
product of the lacZ gene, β-galactosidase. Various short
sequences that are not genes also affect gene expression,
including the lac promoter,
 lac p, and the lac operator, lac o. Although cit is not strictly standard usage,
mutations affecting lac o are referred to as lac o ,
 for historical reasons.

4. Regulation

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 Specific control of the lac genes depends on the availability of
the substrate lactose to the bacterium. The proteins are not
produced by the bacterium when lactose is unavailable as a
carbon source. The lac genes are organized into an operon;
that is, they are oriented in the same direction immediately
adjacent on the chromosome and are co-transcribed into a
single polycistronic mRNA molecule. Transcription of all genes
starts with the binding of the enzyme RNA polymerase (RNAP),
a DNA-binding protein, which binds to a specific DNA binding
site, the promoter, immediately upstream of the genes.
Binding of RNA polymerase to the promoter is aided by the
cAMP-bound catabolite activator protein (CAP, also known as
the cAMP
 receptor protein)..4. However, the lacI gene (regulatory gene for lac
operon) produces a protein that blocks RNAP from
 binding to the operator of the operon. This protein can only
be removed when allolactose binds to it, and inactivates it.
The protein that is formed by the lacI gene is known as the lac
repressor. The type of regulation that the lac operon
undergoes is referred to as negative inducible, meaning that
the gene is turned off by the regulatory factor (lac repressor)
unless some molecule (lactose) is added. Once the repressor
is removed, RNAP then proceeds to transcribe all three genes
(lacZYA) into
 mRNA. Each of the three genes on the mRNA strand has its
own Shine-Dalgarno sequence, so the genes are
independently translated..5. The DNA sequence of the E. coli
lac operon, the lacZYA mRNA, and the lacI genes are
available from

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 The first control mechanism is the regulatory response to
lactose, which uses an intracellular regulatory protein called
the lactose repressor to hinder production of β-galactosidase
in the absence of lactose. The lacI gene coding for the
repressor lies nearby the lac operon and is always expressed
(constitutive). If lactose is missing from the growth medium,
the repressor binds very tightly to a short DNA sequence just
downstream of the promoter near the beginning of lacZ
called the lac
 operator. The repressor binding to the operator interferes
with binding of RNAP to the promoter, and therefore mRNA
encoding LacZ and LacY is only made at very low levels. When
cells are grown in the presence of lactose, however, a lactose
metabolite called allolactose, made from lactose by the
product of the lacZ gene, binds to the repressor, causing an
allosteric shift. Thus altered, the repressor is unable to bind to
the operator, allowing RNAP to transcribe the lac genes and
thereby leading to higher levels of the encoded proteins.

 The second control mechanism is a response to glucose, which

uses the catabolite activator protein (CAP) homodimer to
greatly increase production of β-galactosidase in the absence
of glucose. Cyclic adenosine monophosphate (cAMP) is a signal
molecule whose prevalence is inversely proportional to that of
glucose. It binds to the CAP, which in turn allows the CAP to
bind to the CAP binding site (a 16 bp DNA sequence upstream
of the promoter on the left in the diagram below, about 60 bp

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  upstream of the transcription start site),.6. which assists the RNAP in binding to the
DNA. In the absence of glucose, the
 cAMP concentration is high and binding of CAP-cAMP to the
DNA significantly increases the production of β-galactosidase,
enabling the cell to hydrolyse lactose and release galactose
and glucose.

 More recently inducer exclusion was shown to block

expression of the lac operon when glucose is present. Glucose
is transported into the cell by the PEP-dependent
phosphotransferase system. The phosphate group of
phosphoenolpyruvate is
 transferred via a phosphorylation cascade consisting of the
general PTS (phosphotransferase system) proteins HPr and
EIA and the glucose-specific PTS proteins EIIAGlc and EIIBGlc, the
cytoplasmic domain of the EII glucose transporter. Transport
of glucose is accompanied by its phosphorylation by EIIB Glc,
draining the phosphate group from the other PTS proteins,
including EIIAGlc. The unphosphorylated form of EIIAGlc binds to
the lac permease and prevents it from bringing lactose into
the cell. Therefore, if both glucose and lactose are present,
the transport of glucose blocks the transport of the inducer of
the lac operon

5. Repressor structure

 The lac repressor is a four-part protein, a tetramer,

with identical subunits. Each subunit contains a helix-
turn-helix (HTH) motif capable of binding to DNA. The
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 operator site where repressor binds is a DNA
sequence with inverted repeat symmetry. The two
DNA half-sites of the operator together bind to two of
the subunits of the repressor. Although the other two
subunits of repressor are not doing anything in this
model, this property was not understood for many
years.

 Eventually it was discovered that two additional operators are

involved in lac regulation..8. One (O3) lies about −90 bp upstream
of O1 in the end of the lacI gene, and the other (O2) is about
+410 bp downstream of O1
 in the early part of lacZ. These two sites were not found in the
early work because they have redundant functions and
individual mutations do not affect repression very much. Single

mutations to either O2 or O3 have only 2 to 3-fold effects.

However, their importance is demonstrated by the fact that a
double mutant defective in both O2 and O3 is dramatically de-
repressed (by about 70-fold).

Tetrameric LacI binds two operator sequences and induces DNA looping. Two dimeric LacI functional
subunits (red+blue and green+orange) each bind a DNA operator sequence (labeled).

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These two functional subunits are coupled at the tetramerization region (labeled); thus, tetrameric LacI binds

two operator sequences. This allows tetrameric LacI to induce DNA looping

In the current model, lac repressor is bound simultaneously to both the main operator O 1 and to

either O2 or O3. The intervening DNA loops out from the complex. The redundant nature of the two

minor operators suggests that it is not a specific looped complex that is important

One idea is that the system works through tethering; if bound repressor releases from O1 momentarily,

binding to a minor operator keeps it in the vicinity, so that it may rebind quickly. This would increase

the affinity of repressor for O1.

5.1 Mechanism of induction

The repressor is an allosteric protein, i.e. it can assume either one of

two slightly different shapes, which are in equilibrium with each
other. In one form the repressor will bind to the operator DNA with
high specificity, and in the other form it has lost its specificity.
According to the classical model of induction, binding of the inducer,
either allolactose or IPTG, to affects the distribution of repressor
between the two shapes. Thus, repressor with inducer bound is
stabilized in the non-DNA-binding conformation. However, this
simple model cannot be the whole story, because repressor is
bound quite stably to DNA, yet it is released rapidly by addition of
inducer.
Therefore, it seems clear that an inducer can also bind to the
repressor when the repressor is already bound to DNA. It is still not
entirely known what the exact mechanism
of binding is..9.

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5.2 Role of non-specific binding

 Non-specific binding of the repressor to DNA plays a

crucial role in the repression and induction of the Lac-
operon. The specific binding site for the Lac- repressor
protein is the operator. The non-specific interaction is
mediated mainly by charge-charge interactions while
binding to the operator is reinforced by hydrophobic
interactions. Additionally, there is an abundance of
non-specific DNA sequences to which the repressor
can bind. Essentially, any sequence that is not the
operator, is considered non-specific.
 Studies have shown, that without the presence of
non-specific binding, induction (or unrepression) of
the Lac- operon could not occur even with saturated
levels of inducer. It had been demonstrated that,
without non-specific binding, the basal level of
induction is ten thousand times smaller than
observed normally. This is because
 the non- specific DNA acts as sort of a "sink" for the
repressor proteins, distracting them from the
operator. The non-specific sequences decrease the
amount of available repressor in the cell. This in turn
reduces the amount of inducer required to unrepess the
system
Page | 15
6 Lactose analogs
 A number of lactose derivatives or analogs have been
described that are useful for work with the lac
operon. These compounds are mainly substituted
galactosides, where the glucose moiety of lactose is
replaced by another chemical group.
 Isopropyl-β-D-thiogalactopyranoside (IPTG) is
frequently used as an inducer of the lac operon for
physiological work..1. IPTG binds to repressor and
inactivates it, but is not a substrate for β-galactosidase.
One advantage of IPTG for in vivo studies is that since
it cannot be metabolized by E. coli. Its concentration
remains constant and the rate of expression of lac p/o-
controlled genes is not a variable in the experiment.
IPTG intake is dependent on the action of lactose
permease in P. fluorescens, but not in E. coli.

 Phenyl-β-D-galactose (phenyl-Gal) is a
substrate for β-galactosidase, but does not
inactivate repressor and so is not an inducer.
Since wild type cells produce very little β-
galactosidase, they cannot grow on phenyl-Gal
as a carbon and energy source. Mutants lacking
repressor are able to grow on phenyl-Gal. Thus,
minimal medium containing only phenyl-Gal as a
source of carbon and energy is selective
 for repressor mutants and operator mutants. If
108 cells of a wild type strain are plated on agar
plates containing phenyl-Gal, the rare colonies
Page | 16
 which grow are mainly spontaneous mutants
affecting the repressor. The relative distribution of
repressor and operator mutants is affected by the
target size. Since the lacI gene encoding repressor
is about 50 times larger than the operator, repressor
mutants predominate in the selection.
 Thiomethyl galactoside .TMG. is another
lactose analog. These inhibit the lacI repressor.
At low inducer concentrations, both TMG and
IPTG can enter the cell through the lactose
permease. However at high inducer
concentrations, both analogs can enter the cell
independently. TMG can reduce growth rates
at high extracellular concentrations
 Other compounds serve as colorful indicators of
β-galactosidase activity.

Page | 17
 ONPG is cleaved to produce the intensely yellow
compound, orthonitrophenol and galactose, and
is commonly used as a substrate for assay of β-
galactosidase in vitro.
Colonies that produce β-galactosidase are
turned blue by X-gal (5-bromo-4- chloro-3-
indolyl-β-D-galactoside) which is an artificial
substrate for B- galactosidase whose cleavage
results in galactose and 4-Cl,3-Br indigo thus
producing a deep blue color.

8. Regulation by cyclic AMP

Explanation of diauxie depended on the characterization

of additional mutations affecting the lac genes other
than those explained by the classical model. Two other
genes, cya and crp, subsequently were identified that
mapped far from lac, and that, when mutated, result in a
Page | 18
decreased level of expression in the presence of IPTG
and even in strains of the bacterium lacking the
repressor or operator. The discovery of cAMP in E. coli
led to the demonstration that mutants defective the cya
gene but not the crp gene could be restored to full
activity by the addition of cAMP to the medium.

The cya gene encodes adenylate cyclase, which produces

cAMP. In a cya mutant, the absence of cAMP makes the
expression of the lacZYA genes more than ten times
lower than normal. Addition of cAMP corrects the low
Lac expression characteristic of cya mutants. The second
gene, crp, encodes a protein called catabolite activator
protein (CAP) or cAMP receptor protein
(CRP)..20.

However the lactose metabolism enzymes are made in

small quantities in the presence of both glucose and
lactose (sometimes called leaky expression) due to the
fact that the RNAP can still sometimes bind and initiate
transcription even in the absence of CAP. Leaky
expression is necessary in order to allow for metabolism
of some lactose after the glucose source is expended, but
before lac expression is fully activated.

In summary:

Page | 19
 When lactose is absent then there is very little Lac
enzyme production (the operator has Lac
repressor bound to it).
When lactose is present but a preferred carbon
source (like glucose) is also present then a small
amount of enzyme is produced (Lac repressor is
not bound to the operator).
When glucose is absent, CAP-cAMP binds to a
specific DNA site upstream of the promoter
and makes a direct protein-protein interaction
with RNAP that facilitates the binding of RNAP
to the promoter.
The delay between growth phases reflects the time needed to
produce sufficient quantities of lactose-metabolizing enzymes. First,
the CAP regulatory protein has to assemble on the lac promoter,
resulting in an increase in the production of lac mRNA. More
available copies of the lac mRNA results in the production (see
translation) of significantly more copies of LacZ (β- galactosidase,
for lactose metabolism) and LacY (lactose permease to transport
lactose into the cell). After a delay needed to increase the level of
the lactose metabolizing enzymes, the bacteria enter into a new
rapid phase of cell growth

9. Use in molecular biology

Page | 20
 The lac gene and its derivatives are amenable to use
as a reporter gene in a number of bacterial-based
selection techniques such as two hybrid analysis, in
which the successful binding of a transcriptional
activator to a specific promoter sequence must be
determined..14. In LB plates containing X-gal, the
colour change from white colonies to a shade of
blue corresponds
 to about 20–100 β-galactosidase units, while
tetrazolium lactose and MacConkey lactose media
have a range of 100–1000 units, being most
sensitive in the high and low parts of this range
respectively... Since MacConkey lactose and
tetrazolium
 lactose media both rely on the products of lactose
breakdown, they require the presence of both lacZ
and lacY genes. The many lac fusion techniques
which include only the lacZ gene are thus suited to
X-gal plates.. or ONPG liquid broths..

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