Analyzing Lac Operon Response in E. coli Culture Mediums

This practical assignment investigates the Lac Operon in E. coli, focusing on the regulation of gene expression in response to different sugar sources. The experiment involves growing E. coli in three different culture mediums: one without sugar, one with lactose, and one with both lactose and glucose. The study uses the calorimetric assay method to measure the activity of β-galactosidase, an enzyme involved in lactose metabolism. The procedure includes treating the bacterial cultures with toluene to make the cells permeable, adding the substrate 2-nitrophenol-β-D-galactopyranoside (NPG), and measuring the absorbance at 410 nm using a spectrophotometer. The results show that the enzyme activity is highest in the presence of lactose, confirming the regulatory role of the Lac Operon. The discussion emphasizes the importance of the Lac Operon in energy conservation by E. coli, and the experimental results align with established understanding of the operon's function. The assignment also explores the use of enzyme inhibitors to determine if changes in enzyme activity are due to increased enzyme synthesis or activation of existing enzymes. The conclusion reiterates the role of the Lac Operon in lactose metabolism and its importance in controlling protein synthesis based on cellular needs.

Lac Operon 1
Response of lac operon to different cultural mediums
Name
Institution

Lac Operon 2
Introduction
Lac Operon in E coli is a combination of four genes to form a set that perform in unison
the in allowing the use of lactose from energy by the bacteria. (Esmaelli et al, 2015). These genes
helps in energy conservation by an organism in absence of lactose (Wheatley et al, 2015). This is
done by transcriptional control to reduce the amount of specific enzyme protein produced by the
cells. In lactose presence, the E coli activates the lac operon expression leading to formation of
mRNA coding for the three genes known as polycistronic mRNA (Goering, Dockrell,
Zuckerman & Chiodini, 2018). This helps in metabolism of lactose by the E coli. By calorimetric
essay method, it is easy to measure one of the enzymes produced known as β-galactosidase. This
practical entails measurement of the β- galactosidase expression by different cultures of E coli,
with lactose and or glucose and sugarless culture.
Materials
 2% Toluene in Ethanol
 0.08 M phosphate buffer pH 7.7
 4 mM 2-nitrophenol-β-D- galactopyranoside (NPG)
 Personal protective equipment (Safety glasses, gloves and lab coat)
Method
The reagents to be used in this practical are potentially harmful and therefore
personal protective equipment should be used. The experiment should also be done in
a fume chamber because the fumes produced by ethanol/toluene are highly
flammable. Any culture spill should be wiped using disinfectant and the contaminated
equipment placed in the bucket provided. 24hrs before the start of this practical,

Lac Operon 3
BL21, an E coli strain was grown in a little amount of media without glucose or
lactose. The culture was then divided into 3 equal parts and treated in the following
manner.
 1st bottle- Nutrients broth medium with no lactose or glucose.
 2nd bottle- Nutrients broth medium with 10ml lactose
 3rd bottle- Nutrients broth medium with 10ml lactose and 10 ml glucose
To allow the enzyme break out of the bacteria, they are should be treated with toluene
making the cells permeable hence making it possible to measure the amount of β-galactosidase
produced in each of the three media. β-galactosidase will hydrolyze 2-nitrophenol-β-D-
galactopyranoside (NPG) into yellow nitrophenol, a substance that is easily detected by use of
spectrophotometer. After obtaining the results, calculation of the enzyme activity is to be done
as follows.
Enzyme Activity of β- galactosidase= e c l
Where e= Molar extinction coefficient, c is the
c= Concentration (M)
l= Length of cuvette
e at 410 nm is 18.5 x 103 M-1cm-.
For the first bottle, the enzyme activity will be 18.5 x 103 x 1x4x0.00375= 277.5
nmoles/min/ml

Lac Operon 4
Procedure
i. 1.4 ml of each E coli culture was placed into different cuvettes of 4ml each and labeled
numbers 1,2 and 3
ii. 0.2ml of 2% toluene in ethanol was added into the cuvettes in fume chamber
iii. The cuvettes were incubated at room temperature for 10min
iv. 0.4ml phosphate buffer was added to each cuvette.
v. 1ml of 4 mM NPG was added to cuvette one to start the reaction and mixed by inversion
vi. The cuvette was immediately placed in a spectrophotometer and absorbance at 410 nm
measured.
vii. The absorbance was zeroed and recorded after every one minute for 5 minutes.
viii. The above procedure was repeated with cuvettes 2 and 3
ix. The content was then poured into the solvent waste bottle for recycling.
Results
Time(min) Without sugar Lactose Lactose+ glucose
0 0 0 0
1 0.001 0.053 0.005
2 0.002 0.102 0.007
3 0.003 0.155 0.008
4 0.003 0.202 0.008
5 0.004 0.247 0.011
Table 1

Lac Operon 5
Rate of change of absorbance at 410 nm, per minute
Bottle Rate (Absorbance/min)
1 0.00375
2 0.23375
3 0.007
Table 2
Values of the 3rd column of the table 2 below were calculated from Beer’s law which states that
the enzyme activity is a product of the extinction coefficient of the enzyme, its concentration
and the length of the cuvette (Oldham, and Parnis, 2017).
Bottle Rate (Absorbance/min) A (nmoles/min/ml)
1 0.00375 277.5
2 0.23375 17297.5
3 0.007 518
Table 3

Lac Operon 6
Control Lactose Lactose+glucose
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
Experimental treatmet
β-galactosidase activity
nmoles/min/ml
Figure 1
From the figure 1 above, the reaction rate of the enzyme in E coli is very high in culture
treated with lactose as compared to one without sugar and one treated with lactose and glucose.
Discussion of results
The results shows there is a higher rate of β-galactosidase activity in presence of lactose s
compared to presence of glucose absence of sugars. The results therefor confirms the lac operon
regulation established view. This is because of the lac operon activity of the E coli. The E. coli
lac operon has genes that are only involved in the metabolism of lactose (Ammar, Wang, and
Rao, 2018). These genes are only expressed in the presence of lactose and in absence of glucose
(Bidart, Rodríguez-Díaz, Pérez-Martínez and Yebra, 2018). This is made possible by the action
of lac repressor that acts as a sensor for lactose. It blocks the operon transcription in absence of

Lac Operon 7
the lactose and stops the repress action in presence of lactose (Fulcrand et al, 2016). The sensing
action occurs indirectly by use of allolactose isomer.
To determine, experimentally whether the changes in enzyme activity noted above are the
result of increased enzyme synthesis, or activation of existing enzyme inhibitors can be added in
the as one of the reagents in the experiment. Enzyme inhibitors are molecules that bind the active
enzyme sites and decrease its activity (Bateman et al, 2017). By so doing, it is possible to
determine whether the activity is as a result of production of new enzymes or activation of the
existing ones. This is because when all the enzymes present are bound by the inhibitors, there
will be no activity whatsoever if no new ones are synthesized. To ensure that no unused enzyme
inhibitor remains in the reagents that may affect the results, only the predetermined inhibitors
amount for a given amount of enzymes is added. If there is still some enzyme activities after
addition of enzyme inhibitors and after ensuring that all the enzymes are bound and no enzyme
inhibitor is remaining in Free State in the reagent, it means that new enzymes were synthesized.
If no activity is found, it means that no enzymes are synthesized and hence conclusion that the
enzymatic activity is only due to activation of existing enzymes.
Conclusion
Lac operon of the E. coli has a set of genes that are used in the metabolism of lactose and
only expressed in presence of lactose. This helps in control of protein synthesis according to the
cell requirements.

Lac Operon 8
References
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Bidart, G.N., Rodríguez-Díaz, J., Pérez-Martínez, G. and Yebra, M.J., 2018. The lactose operon
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Fulcrand, G., Dages, S., Zhi, X., Chapagain, P., Gerstman, B.S., Dunlap, D. and Leng, F., 2016.
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