Bacterial Culture Techniques & Lac Operon (pdf)

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