UNIVERSITI

TUNKU
ABDUL
RAHMAN
FACULTY OF ENGINEERING & SCIENCE
(FES)

UESE 2204 MOLECULAR BIOLOGY

Bachelor of Science (Hons) Biochemistry

Year 2 Semester 2

Name: Sudhashini John

ID No: 07UEB06355
Group: 2
Group Members: Ooi Khai Zheng
She Wei Wei
Lee Hui Yee
Chua Hui Ying
Yap Pun Jing
Cheah Wei
Exp: 5
Title: Enzyme Synthesis Regulation in Escherichia coli
(The Lactose Operon)
Date: 26th of March 2009
Lecturer: Dr.Teh Yok Lan

Objective: To study the regulation of enzyme synthesis in Escherichia coli.

Results:
Inducer Absorbance
Control 0.000
ddH2O 0.016
GLU 0.119
LAC 0.459
IPTG 1.554

Discussion:

In this experiment based on the results obtained, IPTG shows the highest value

compared to the lactose and glucose. The order is as follows:

IPTG>lactose>glucose.

IPTG has a higher absorbance value compared to lactose and glucose because the

concentration of the IPTG in the cell does not change as it is not consumed or degraded

due to the chemical bond of its sulphur atom which is not hydrolyzed. Thus, the induction

is able to persist and sustains, providing a higher reading for the presence of the enzyme

β -galactosidase whereas the lactose shows lower absorbance value compare to IPTG

because the lactose will gradually decrease in amount as it is a substrate to the β -

galactosidase enzyme where the β -galactosidase enzyme will degrade the lactose into

the glucose and galactose. Moreover, the conversion of lactose to allolactose is catalyzed

by the β -galactosidase enzyme. The glucose shows the lowest reading because the

enzyme for glucose degradation was not synthesized by the lac genes.This is because the

operon is activated in the presence of lactose and not glucose and distilled water was

used as a blank in this experiment.

 This experiment was carried out to investigate the effects of different inducers on

the lactose operon of Escherichia Coli that synthesizes the enzyme β -galactosidase. The

lac operon consists of three structural genes, which code for permeases (carries lactose

into cell), β -galactosidase (converts lactose into glucose and galactose) and

transacetylase (transfers acetyl group). The regulator gene codes for a repressor molecule.

The repressor molecule binds to the operator gene, overlapping and blocking attachment

of RNA polymerase to the promoter region beside it. Hence, transcription of the operon

does not occur. Inducers bind and dislodge the repressor molecule from the operator

gene, making the promoter region available for binding and transcription by RNA

polymerase.

In this experiment, beside glucose and lactose, an artificial inducer isopropyl beta-

thiogalactoside (IPTG) was also used to investigate their effects on the operon. The

presence of the β -galactosidase enzyme was detected by its degrading action on the

ortho-nitrophenyl-β -galactoside (ONPG) to galactose and ONP. The ONP produced are

yellow in colour at high pH and are tested for absorbance at 420nm. The standard

conditions are created using the sodium phosphate buffer and the reaction was stopped at

high pH using sodium carbonate. The higher the amount of enzyme present, the more

ONPG is converted to ONP (yellow compound), the more intense the compound and the

higher is the absorbance. Although ONPG is used to determine whether β -galactosidase

has been synthesized in the cell or not, the compound will not quickly pass through the

living cell membrane. Therefore, sodium deoxycholate and toulene an organic solvent is

used to destroy the selective permeability of the membrane so that the ONPG can enter

the cell and react with the enzyme.

 The glucose shows a lower reading indicating that the enzyme was not

synthesized by the lac genes. This is because the operon is activated in the presence of

lactose and not glucose. Glucose is the preferred energy source for E. Coli, as the

enzymes for glucose metabolism are made constantly in the cell. On the contrary, the

lactose metabolism enzymes are only made in the presence of lactose. The regulatory

proteins are useful to switch a group of genes on and off for the bacteria to adapt to the

environment. This saves the cellular resource and energy.

In the presence of both glucose and lactose, E. Coli uses the glucose first. This is

due to the readily available enzymes and to avoid energy wastage in synthesizing

enzymes for the lactose metabolism. The concentration of cyclic AMP in E. coli is

inversely proportional to the concentration of glucose: as the concentration of glucose

decreases, the concentration of cyclic AMP increases.The presence of glucose causes a

drop in cellular levels of cyclic AMP (adenosine monophosphate) which inactivates the

catabolite activator protein (CAP), which is needed for rapid transcription of the operon.

When the glucose is all used up, cAMP levels increase and this stimulates the CAP

attachment to the chromosome. Once attached, there is rapid transcription and rapid

synthesis of the enzymes that transport and catabolize lactose.

Conclusion:

The absorbance reading for IPTG was the highest followed by lactose and

glucose. The absorbance reading for the IPTG is 1.554, followed by lactose 0.459 and

glucose 0.119. The ONPG was used to determine the presence of the ß-galactosidase.
References:

• Watson, Baker, Bell, Gann, Levine, Losick. (2004). Techniques of Molecular

Biology. Molecular Biology of the Gene (5th Ed). pp 488-496.

• www.phschool.com/science/biology_place/biocoach/lacoperon/intro.html

• oregonstate.edu/instruction/bb492/lectures/Regulation.html

• users.rcn.com/jkimball.ma.ultranet/BiologyPages/L/LacOperon.html