Siberian Federal University
Mechanisms and applications of bioluminescence:
practical course
Course Guide
Krasnoyarsk, 2016
1
Table of Contents
1.
Course Description……………………………………………...… 3
2.
Lecturers and Tutors, Contact Information………………………..
3.
Prerequisites……………………………………………………….. 5
4.
Course Outline…………………………………………………….. 5
5.
Assessment………………………………………………………...
6.
Attendance Policy…………………………………………………. 12
7.
Required Course Participation………………………………….…. 13
8.
Annex 1 Example of Self-Study Assignment………………………………
9.
Annex 2 Example of Pre-Course Test Questions…………………………… 15
4
12
14
10. Annex 3 Outlines of selected Lab works …………………………………..
16
11. Annex 4 Examples of Final Test Questions………………………………...
19
2
Course Guide
1. Course Description
 Course overview
The practical course “Mechanisms and applications of bioluminescence“ is a
part of International Master Program “Biological Engineering” offered by Siberian
Federal University. It equips students with practical skills to perform different
types of bioluminescence reactions and acquaints them with the principles of
bioluminescent assays applied in environmental monitoring, medicine and other
practical fields.
The course consists of three parts (modules). In the first module the
biochemical basis of the most popular bioluminescent reactions are considered.
The second module covers various methods of expression, purification and
characterization of the bioluminescent and fluorescent proteins and their
application as BRET technique. The third module acquaints with the variety of
bioassays based on enzymatic systems.
 Special features of the course
The course is developed by one of the world’s leading schools of scientific
thought in the field of biological light-emitting systems and puts a strong emphasis
on practical laboratory work.
 Course aim
The practicum provides an opportunity to master main techniques for
measuring bioluminescence in vitro and learn to apply them in environmental
monitoring, medical diagnostics, pharmaceutical and food industries, etc. It aims to
enable students to master the basic techniques for performing of bioluminescence
reactions in vitro and to apply bioluminescence assays in various fields.
 Course objectives
The course objectives are:

to provide students with the opportunity to test the various chemical bases of
bioluminescent reactions and their kinetic properties;

to enable students to experience the whole set of methods starting from
expression and purification of target recombinant proteins to their
characterization and direct application;
3

to give students an understanding of principles of bioluminescent bioassays
design.
 Learning outcomes of the course
After completing the course students should be able to:

implement experimentally different types of bioluminescent reactions and
analyze their kinetic profiles;

extract, purify and characterize various types of bioluminescent and fluorescent
recombinant proteins;

understand the principles of bioluminescent assays as applied in environmental
monitoring, medical diagnostics, pharmaceutical and food industries, etc.
2. Lecturers and Tutors, Contact Information
Dr. Elena Nemtseva,
PhD (Biophysics),
Associate Professor of Biophysics Department,
Researcher in laboratory of Bioluminescent Biotechnologies,
SibFU,
e-mail: [email protected]
Dr. Elena Eremeeva,
PhD (Biophysics),
Researcher of Photobiology laboratory,
Institute of Biophysics,
Siberian Branch of Russian Academy of Science
e-mail: [email protected]
Dr. Elena Esimbekova,
PhD (Biophysics),
Associate Professor of Biophysics Department SibFU,
Researcher of Photobiology laboratory,
Institute of Biophysics,
Siberian Branch of Russian Academy of Science
e-mail: [email protected]
4
3. Prerequisites
Introductory courses in biochemistry, photobiology and molecular biology
techniques are essential prerequisites.
4. Course Outline
4.1. Course requirements
4.1.1. Required texts
Selected chapters of the books:

Lee J. Bioluminescence, the
(http://athenaeum.libs.uga.edu/)
Nature
of
the
Light.
–
2016.

Shimomura O. Bioluminescence: Chemical Principles and Methods, Revised
Edition. – 2012. (http://ebookcentral.proquest.com/lib/krasu-ebooks/)

Haddock S. H. D. Bioluminescence in the sea. / S. H. D. Haddock, M. A.
Moline, J.F. Case. // Annu. Rev. Mar. Sci. 2010. 2, PP. 443-493.
(http://www.annualreviews.org/)

Lakowicz J. R. Energy Transfer. In Principles of Fluorescence Spectroscopy.
3rd edition. 2006. Springer. ISBN: 978-0-387-46312-4 (Online)
(http://link.springer.com)

Nelson D. L. and Cox M. M. Lehninger Principles of Biochemistry. 5th
edition. (http://archive.org/)
Journal articles:

Esimbekova E., Kondik A., Kratasyuk V. Bioluminescent enzymatic rapid
assay of water integral toxicity. Environmental Monitoring & Assessment. Vol.
185 Issue 7, PP. 5909-5916. DOI: 10.1007/s10661-012-2994-1. (http://bik.sfukras.ru)

Esimbekova E. N., Lonshakova-Mukina V. I., Bezrukikh A. E., Kratasyuk V.
A. Design of multicomponent reagents for enzymatic assays. In: Doklady
Biochemistry and Biophysics. Vol. 461 Issue 1, PP.102-105. (http://bik.sfukras.ru)
Electronic articles:

Kratasyuk V. and Esimbekova E. Analytical Applications of Luminous
Bacteria Enzymes. In Photobiological sciences online / John Lee, Kendric C.
5
Smith, editors and webmasters. American Society for Photobiology. ISSN
2470-2749 (Electronic). (http://photobiology.info)

Branchini B. Chemistry of firefly bioluminescence. In Photobiological sciences
online / John Lee, Kendric C. Smith, editors and webmasters. American
Society
for Photobiology.
ISSN
2470-2749
(Electronic).
(http://photobiology.info)

Lin L. Y.-C. and Meighen E. A. Bacterial bioluminescence: biochemistry and
molecular biology. In Photobiological sciences online / John Lee, Kendric C.
Smith, editors and webmasters. American Society for Photobiology. ISSN
2470-2749 (Electronic). (http://photobiology.info)
4.1.2. Web page of the course
The Course Guide and all accompanying materials are collected on the
SibFU website for electronic courses: https://e.sfu-kras.ru/course/view.php?id=
8547.
4.1.3. Course materials
Updated lecture notes, home assignments, lab manuals and other materials
on corresponding topic will be available on the course web site at least one week
prior to each class session. The students can print and bring the notes to class,
because each laboratory work will be preceded by brief discussion of the key ideas
during the short lectures.
All the required reading texts are either open access or available from the
SibFU network.
4.1.4. Required feedbacks
The students must enroll the course on the website of the SibFU electronic
courses (https://e.sfu-kras.ru). This will allow the teachers to distribute class
announcements, readings, home assignments, etc. The contact with teachers
directly by e-mail is also possible.
All the tests will be carried out through the course website.
6
4.2. Time schedule and course outline
№
Topic
Week,
Learning Activities Hours
№
Home Assignments and Reading
Reading:
 Lee J. Bioluminescence, the Nature of the Light. –
2016. PP. 1-15, 89-122 (http://athenaeum.libs.uga.edu)
 Lee J. Basic Bioluminescence. In Photobiological
sciences online / J. Lee, K. C. Smith, editors and
webmasters. American Society for Photobiology
(http://photobiology.info)
 Scott D., Dikici E., Ensor M., Daunert S.
1.1 Responsive lecture
«Introduction into
bioluminescence
chemistry»
1
Bioluminescence and its impact on bioanalysis //
Annual review of analytical chemistry, 2011. 4, 297319. (http://www.annualreviews.org)
 Haddock S. H. D. Bioluminescence in the sea. / S. H.
D. Haddock, M. A. Moline, J.F. Case. // Annu. Rev.
Mar. Sci. 2010, 2, 443-493.
(http://www.annualreviews.org)
 Wilson T. and Hastings J. W. Bioluminescence //
1
Annu. Rev. Cell Devel. Biol., 1998, 14(1), 197-230.
(http://www.annualreviews.org)
1.
Useful websites:
 The Bioluminescence Web Page
«Mechanisms
of
bioluminescent
reactions»
(http://biolum.eemb.ucsb.edu)
 Video-lecture «Bioluminescence: Living Lights, Lights
for Living» J. Woodland Hastings, Paul C.
Mangelsdorf (https://www.youtube.com)
1.2 Pre-course test
1
 J. W. Hastings on iBiology.org
(https://www.ibiology.org/ibiomagazine/j-woodlandhastings-autoinduction-discovery-quorum-sensingbacteria.html)
Self-Study Assignment 1.1 (Annex 1)
Pre-course test questions set (Annex 2)
Lecture notes 1.1
Reading:
 Boyer R.F. Biochemistry laboratory: modern theory
and techniques / Rodney Boyer. — 2nd ed, PP.1-30
2
1.3 Lecture «Introduction
into laboratory
techniques»
1
Useful websites:
 http://web.expasy.org/protparam/
 http://www.biophysics.org/ProfessionalDevelopmen
t/SelectedTopicsInBiophysics/BiophysicalTechniques/ta
bid/2313/Default.aspx
 GraphPad Soft http://www.graphpad.com
/quickcalcs/
7
Reading:
 Shimomura O. Bioluminescence: Chemical Principles
1.4 Lab «Kinetics of the
bioluminescent reaction,
catalyzed by bacterial
luciferase»
and Methods, Revised Edition. – 2012. PP. 91-138
(http:// ebookcentral.proquest.com/lib/krasu-ebooks/)
3
 Lee J. Bioluminescence, the Nature of the Light. –
2016. PP. 22-43 (http://athenaeum.libs.uga.edu)
Нome Assignments № 1.1
Lab manual 1.1 (Annex 3)
Reading:
 Tu S.-C., Hastings J. W. Physical interaction and
activity coupling between two enzymes induced by
immobilization of one //Proceedings of the National
Academy of Sciences. 1980. V. 77. №. 1. PP. 249-252
(http://www.pnas.org/content/77/1/249.full.pdf)
3
1.5 Lab «Kinetics of the
coupled enzymes bacterial
bioluminescent reaction»
 Campbell Z. T., Baldwin T. O. Fre is the major flavin
3
reductase supporting bioluminescence from Vibrio
harveyi luciferase in Escherichia coli //Journal of
Biological Chemistry. 2009. V. 284. №. 13. PP. 83228328 (https://www.ncbi.nlm.nih.gov/pmc/articles/
PMC2659190/)
 Welham P. A., Stekel D. J. Mathematical model of the
Lux luminescence system in the terrestrial bacterium
Photorhabdus luminescens //Molecular Biosystems.
2009. V. 5. №. 1. PP. 68-76.
(https://core.ac.uk/download/pdf/1631074.pdf)
1.6 Reports discussion
1
Нome Assignments № 1.2
Lab manual 1.2
Reading:
 Shimomura O. Bioluminescence: Chemical Principles
and Methods, Revised Edition. – 2012. PP. 1-30 (http://
ebookcentral.proquest.com/lib/krasu-ebooks/)
4
1.7 Lab «Kinetics of the
firefly bioluminescent
reaction»
 Ford S.R., Leach F. R. Improvements in the
4
Application of Firefly Luciferase Assays (http://www.
springerprotocols.com/Abstract/doi/10.1385/0-89603520-4:3)
Нome Assignments № 1.3
Lab manual 1.3
Reading:
 Lee J. Bioluminescence, the Nature of the Light. –
2016. (http://athenaeum.libs.uga.edu) PP. 22-43
5
1.8 Lab «Kinetics of the
Ca-dependent
bioluminescent reactions»
 Shimomura O. Bioluminescence: Chemical Principles
3
and Methods, Revised Edition. – 2012. PP. 31-47 (http://
ebookcentral.proquest.com/lib/krasu-ebooks/)
Нome Assignments № 1.4
Lab manual 1.4
1.9 Reports discussion
1
8
Lecture notes 2.1
Reading:
 Shimomura O. Bioluminescence: Chemical Principles
2.1 Lecture «Apo-clytin
extraction from E.coli
RIPL cells»
1
and Methods, Revised Edition. – 2012. (http://
ebookcentral.proquest.com/lib/krasu-ebooks/)
 Haddock S. H. D. Bioluminescence in the sea. / S. H.
D. Haddock, M. A. Moline, J.F. Case. // Annu. Rev. Mar.
Sci. 2010. 2, 443-493. (http://www.annualreviews.org)
6
 Boyer R.F. Biochemistry laboratory: modern theory
and techniques / Rodney Boyer. — 2nd ed, PP. 308-322
2.2 Lab «Apo-clytin
extraction from E.coli
RIPL cells»
7
2.
«Bioluminesce
nt System of
Clytia
gregaria»
8
9
3
Lab manual 2.1
Home Assignment 2.1
Lecture notes 2.2
2.3 Lecture «Apo-clytin
purification using ionexchange chromatography
under denaturing
conditions»
1
2.4 Lab «Apo-clytin
purification using ionexchange chromatography
under denaturing
conditions»
3
Reading:
 Boyer R.F. Biochemistry laboratory: modern
theory and techniques / Rodney Boyer. — 2nd ed,
PP. 122-131
Lab manual 2.2
Home Assignment 2.2
Lecture notes 2.3
2.5 Lecture «GFP
extraction from E.coli
XL1-Blue cells and its
purification with affinity
chromatography»
1
2.6 Lab «GFP extraction
from E.coli XL1-Blue
cells and its purification
with affinity
chromatography»
3
Reading:
 Boyer, R.F. Biochemistry laboratory: modern
theory and techniques / Rodney Boyer. — 2nd ed,
PP.152-159
Lab manual 2.3
Home Assignment 2.3
Lecture notes 2.4
2.7 Lecture «Sodium
dodecyl sulfate
polyacrylamide gel
electrophoresis (SDSPAGE)»
1
2.8 Lab «Sodium dodecyl
sulfate polyacrylamide gel
electrophoresis (SDSPAGE)»
Reading:
Boyer, R.F. Biochemistry laboratory: modern
theory and techniques / Rodney Boyer. — 2nd ed,
PP.165-175
Lab manual 2.4
3
Home Assignment 2.4
Lecture notes 2.5
10
2.9 Lecture «Absorption
spectra of various
conformational states of
clytin and GFP»
9
1
Reading:
 Boyer, R.F. Biochemistry laboratory: modern
theory and techniques / Rodney Boyer. — 2nd ed,
PP. 201-219
2.10 Lab «Absorption
spectra of various
conformational states of
clytin and GFP»
Lab manual 2.5
3
Home Assignment 2.5
Lecture notes 2.6
2.11 Lecture «Methods to
determine protein
concentration»
Reading:
1
 Boyer, R.F. Biochemistry laboratory: modern
theory and techniques / Rodney Boyer. — 2nd ed,
PP. 67-70
11
Lab manual 2.6
Home Assignment 2.6
2.12 Lab «Methods to
determine protein
concentration»
3
Useful websites:
 ExPASy: SIB Bioinformatics Resource Portal
(http://www.expasy.org/)
12
Lecture notes 2.7
2.13 Lecture «Förster
energy transfer (FRET) in
the system clytincgreGFP: spectroscopic
studies»
1
2.14 Lab «Förster energy
transfer (FRET) in the
system clytin-cgreGFP:
spectroscopic studies»
3
Reading:
 Boyer, R.F. Biochemistry laboratory: modern
theory and techniques / Rodney Boyer. — 2nd ed,
PP. 220-225
Lab manual 2.7 (Annex 3)
Home Assignment 2.7
Lecture notes 2.8
13
2.15 Lecture «Förster
radius and energy transfer
efficiency»
1
2.16 Lab «Förster radius
and energy transfer
efficiency»
3
Reading:
 Lakowicz J. R. Energy Transfer. In Principles of
Fluorescence Spectroscopy. 3rd edition. 2006.
Springer. PP. 443-475 (http://link.springer.com)
Lab manual 2.8
Home Assignment 2.8
Reading:
 Nelson D. L. and Cox M. M. Lehninger Principles of
3.
«Bioluminescen
t Bioassays»
14
3.1 Lecture «Biological
methods for
environmental
monitoring»
1
Biochemistry, 5th edition. Chapter 6 Enzymes, PP. 183208 (http://archive.org/details/LehningersPrinciples
OfBiochemistry5e)
Lecture notes 3.1
Self-Study Assignment № 3.1
3.2 Lab «Effect of
pollutants on ADH and
LDH activity»
3
10
Lab manual 3.1
Reading:
3.3 Lecture «Principle of
bioluminescent enzymatic
toxicity assays»
1
15
 Kratasyuk V. and Esimbekova E. Analytical
Applications of Luminous Bacteria Enzymes. In
Photobiological sciences online / J. Lee, K. C. Smith,
editors and webmasters. American Society
for Photobiology (http://photobiology.info/)
 Esimbekova E., Kondik A., Kratasyuk V.
Bioluminescent enzymatic rapid assay of water integral
toxicity. Environmental Monitoring & Assessment, V.
185 Issue 7, PP. 5909-5916. (http://bik.sfu-kras.ru/)
Lecture notes 3.2
Self-Study Assignment № 3.2
3.4 Lab «Effect of
pollutants on activity of
coupled enzyme system
NAD(P)H:FMNoxidoreductase +
luciferase»
2
3.5 Reports discussion
1
Lab manual 3.2
Reading:
 Kratasyuk V. and Esimbekova E. Analytical
16
3.6 Lecture «How to
design new enzymatic
bioassays for toxicological
analysis»
Applications of Luminous Bacteria Enzymes. In
Photobiological sciences online / J. Lee, K. C. Smith,
editors and webmasters. American Society
for Photobiology (http://photobiology.info/)
1
 D. L. Nelson and M. M. Cox. Lehninger Principles of
Biochemistry, 5th edition. Chapter Cleaving the
Polypeptide Chain, (PP. 95-97), Chapter 18.1 Metabolic
Fates of Amino Groups, PP. 674-677. (http://archive. org
/details/LehningersPrinciplesOfBiochemistry5e)
Lecture notes 3.3
Self-Study Assignment № 3.3
3.7 Lab «Effect of food
preservatives on trypsin
activity»
3
Lab manual 3.3 (Annex 3)
Reading:
 Esimbekova E. N., Lonshakova-Mukina V. I.,
17
3.8 Lecture «Methods for
stabilisation of enzymes»
1
Bezrukikh A. E., Kratasyuk V. A. Design of
multicomponent reagents for enzymatic assays. In:
Doklady Biochemistry and Biophysics. V. 461 Issue 1,
PP.102-105 (http://bik.sfu-kras.ru/)
Useful websites:
 Enzyme Immobilization Methods and Applications
(Biotechnology Lecture Notes). Easy Biology Class.
(http://www.easybiolodgyclass.com/ enzyme-cellimmobilization-techniques/)
Lecture notes 3.4
Self-Study Assignment № 3.4
11
4.
Final
Assessment
18
3.9 Lab «Stability of
soluble and immobilized
enzymes to pH variation»
2
3.10 Reports discussion
1
4.1 Final Test
2
Lab manual 3.4
Final test questions set (Annex 4)
5. Assessment
The overall course percentage grade will be computed from the final test
results (20%) and the combined grades of 3 modules assignments (80%).
The each module scores are the sum of the following assignments:
• Module 1 (25% from 80%):
- pre-course test on the chemistry of bioluminescence (5%),
- reports on 4 lab works (each 5%, 20% total ).
• Module 2 (35% from 80%):
- lecture attendance (5%),
- written answers to the 8 question sets (each 2.5%, 20% total),
- instructor’s evaluation of student’s level of lab work participation and
success (10%).
• Module 3 (20% from 80%):
- lecture attendance (each 1%, 4% total),
- written answers to 4 question sets (each 1%, 4% total),
- reports on 4 lab works (each 3%, 12% total ).
The overall course percentage grade will be converted into a letter grade as
follows:
A = 91-100%
B = 81-90%
C = 71-80%
D = 61-70%
E = less than 61%.
6. Attendance Policy
Since almost every class contains practical work in laboratory, attendance of
all of them is obligatory. Missing one or more of laboratory work classes will
result in “No final grade”.
12
7. Required Course Participation
During interactive responsive lectures the topics concerning self-studied
questions are discussed by the students under the guidance of the teacher. Student
that demonstrates active participation in such discussion can be encouraged by
teacher with additional points.
After every lab session student should submit to teacher the lab report written
in accordance with the manual (see examples of report structure in Annex 3). The
form of submission (electronic or printed) and the deadlines will be defined by the
teacher of each module in the beginning of the course.
The written reports on Home Assignments 2.1-2.8 ought to be provided to
teacher during a week after corresponding lab sessions.
13
Annex 1
Example of Self-Study Assignment
Self-Study assignment № 1.1 (week 1, activity 1.1)
 Study the chapters and other materials listed below:

Lee J. Bioluminescence, the Nature of the Light. – 2016. (http://athenaeum.libs.uga.edu) PP. 1-15, 89122

Lee J. Basic Bioluminescence. In Photobiological sciences online / John Lee, Kendric C. Smith,
editors and webmasters. American Society for Photobiology. (http://photobiology.info/)

Lin L. Y.-C. and Meighen E. A. Bacterial bioluminescence: biochemistry and molecular biology. In
Photobiological sciences online / John Lee, Kendric C. Smith, editors and webmasters. American
Society for Photobiology. (http://photobiology.info/)

Wilson T. and Hastings J. W. Bioluminescence. Annual Review of Cell and Developmental Biology.
(http://www.annualreviews.org/)
Useful websites:


The Bioluminescence Web Page (http://biolum.eemb.ucsb.edu/)

J. W. Hastings on iBiology.org (https://www.ibiology.org/ibiomagazine/j-woodland-hastingsautoinduction-discovery-quorum-sensing-bacteria.html)
Video-lecture “Bioluminescence: Living Lights, Lights for Living” by J. W. Hastings, P. C.
Mangelsdorf (https://www.youtube.com/)
Answer the following questions:
1. What is definition for “bioluminescence”? How it is related with “chemiluminescence” and
“fluorescence”?
2. What is the origin of bright light emission from living organisms?
3. List the main functions of a bioluminescence in nature.
4. Give the definition for “luciferase”, “luciferin”, “photoprotein”.
5. Why the bioluminescent systems from different taxons can be called “colleagues”, but not
“relatives”?
6. Give the chemical equation of any two bioluminescent reactions. List the common features of
the chemically different bioluminescent reactions. Draw the generalized scheme of all
bioluminescent reaction.
7. What is the function of accessory or antenna proteins in bioluminescent systems?
8. Describe the mechanisms of “color change” in different types of bioluminescent reactions.
9. Evolutionary origins of the bioluminescence (main ideas).
 Self-Study Product:
Written answers to the questions. During responsive lecture 1.1 the questions concerning selfstudied material will be discussed by the students under the guidance of the teacher.
14
Annex 2
Example of Pre-Course Test Questions
 Pre-Course Test (week 1, activity 1.2)
Q.1-Q.3 carry one mark each
The bioluminescence emission maxima of most luminous marine organisms falls within
the range
(A) 370-450 nm
(B) 450-490 nm
(C) 450-620 nm
(D) 500-670 nm
Answer: B
Q.1
Q.2
Q.3
ATP molecule participates in bioluminescent reaction of
(A) bacteria
(B) fireflies
(C) fungi
Answer: B
(D) dinoflagellates
The structure of GFP molecule
(B)
(A)
(D)
(C)
Answer: C
Q.4-Q.6
Q.4
carry two or more marks each
The participants of the bioluminescent reaction of bacteria
(A) ATP
(B) FMNH2
(C) Ca2+
(D) R-CHO
(E) Mg2+
(D) ATP
(E) luciferin
Answer: B, D
Q.5
Photoproteins consist of
(A) Ca2+
(B) polypeptide
(C) O2
Answer: B, C, E
Q.6
The factors that determine the bioluminescence emission spectrum in vivo
(A) antenna proteins
(B) luciferase structure
(C) media pH
Answer: A, B
15
(D) type of inorganic ions
Annex 3.1
Outlines of selected Lab works
The laboratory work outline (module 1, week 2, activity 1.4)
Title: «Kinetics of the bioluminescent reaction, catalyzed by bacterial luciferase»
Aims: To register non-stationary bioluminescence kinetics of the reaction catalyzed by bacterial
luciferase, to compute the rates of different steps of reaction.
Materials: FMN, EDTA, bacterial luciferase, tetradecanal, phosphate buffer
Techniques, Instruments: Stopped-flow technique, analyzer SX-20 (Applied Photophysics)
Expected results: the dependence of rates of different reaction steps on reaction conditions
(temperature, pH, ionic strength).
Product: Lab report, containing:




the scheme of the reaction steps,
experimental decay curves (Fig. A.3.1),
computed rates of the reaction steps,
conclusion on obtained dependences.
6
1
С14
0,9
Intensity, arb.un.
5
0,8
C10
4
0,7
0,6
3
0,5
0,4
2
0,3
0,2
1
0,1
0
0
0
1
2
3
4
5
6
7
8
Time, s
Fig. A.3.1. Bioluminescence decay curves of reaction catalyzed by bacterial luciferase with different
concentrations of tetradecanal (C14: 14, 7 and 3.5 M) and decanal (C10: 6, 4 and 2 M).
16
Annex 3.2
Outlines of selected Lab works
The laboratory work outline (module 2, week 12, activity 2.14)
Title: « Förster energy transfer (FRET) in the system clytin-cgreGFP: spectroscopic studies»
Aims: To measure fluorescence and bioluminescence spectra of GFP and clytin, respectively,
and to calculate dissociation constant of clytin-GFP interaction using FRET technique
Materials: Clytin and GFP samples, Tris-HCl, EDTA, CaCl2, mirror cuvette
Techniques, instruments: Fluorescence spectroscopy, spectrometer Fluorolog 3-22 (Horiba Jobin
Ivon).
Expected results: plotted normalized fluorescence and bioluminescence spectra of GFP and
clytin, calculated dissociation constant of clytin-GFP interaction (Fig. A.3.2).
Fig. A.3.2. Experimental bioluminescence spectra of clytin with different GFP concentrations
Product: Lab report, containing:

Plotted normalized fluorescence and bioluminescence spectra of GFP and clytin

Plot of bioluminescence spectra of clytin with different GFP concentrations

Calculation of dissociation constant of clytin-GFP interaction.
17
Annex 3.3
Outlines of selected Lab works
The laboratory work outline (module 3, week 16, activity 3.7)
Title: «Effect of food preservatives on trypsin activity measured by bioluminescent assay»
Aims: To register trypsin activity in the presence of sodium benzoate to assess the safety of food
preservatives.
Materials: lyophilized preparation of highly purified enzymes, containing bacterial luciferase
(Photobacterium leiognathi) and NAD(P)H:FMN-oxidoreductase (Vibrio fischeri), lyophilized
preparation of trypsin, sodium benzoate, NADH, FMN, tetradecanal, phosphate buffer.
Techniques, instruments: Registration of trypsin activity by the bioluminescent method using the
decay constant of light intensity of coupled enzyme system NAD(P)H:FMN-oxidoreductase
+ luciferase; bioluminometer Lumat LB 9507 (Berthold Technologies).
Expected results: Inhibition of trypsin activity by sodium benzoate.
Product: Lab report, containing:



the figure showing how the trypsin activity depends on sodium benzoate concentration
(Fig A.3.3),
calculation of EC50 values for sodium benzoate,
conclusion on obtained dependence.
Fig. A.3.3. The relative activity of trypsin in the presence of sodium benzoate (example)
18
Annex 4.1
Example of Final Test Questions
 Final Test Questions
Q.1-Q.4 carry one mark each
Q.1
The structure bellow represents the luciferin of…
(A) bacteria
(B) fireflies
(C) fungi
(D) coelenterates
Answer: B
Q.2 The non-stationary kinetics of bacterial bioluminescent reaction in vitro is caused by…
(A) autoinducer
(B) luciferin
(C) luciferase
(D) product
absence
autooxidation
inactivation
inhibition
Answer: B
Q.3 The type of reactions catalyzed by oxidoreductases
(A) hydrolysis
(B) group transfer
(C) transfer of
reactions
reaction
electron
(D) transfer of groups
within molecules
Answer: C
Q.4
During bioluminescent reaction in bacteria the lucifern deprotonation occurs in the
following positions of the structure bellow:
(A) 1 and 2
(B) 1 and 2 and 3
(C) 4 and 6
Answer: C
19
(D) 4 and 5
(E) 4 and 5 and 6
Annex 4.2
Example of Final Test Questions
Q.5-Q.7
carry two or more marks each
The FRET rate depends on…
Q.5
(A) quantum
yield of the
donor
(B) refractive
index of the
medium
(C) lifetime of
the donor
(D) pH of the
medium
(E) lifetime of
the acceptor
Answer: A, B, C
Q.6
Colorimetry methods to determine protein concentration:
(A) Polyacrylamide
Gel Electrophoresis
Q.7
(B) Biuret test
(C) Gel-Exclusion
Chromatography
Answer: B, D, E
(D) Bradford
protein assay
(E) Lowry
protein assay
Methods for physical immobilization of enzymes:
(A) adsorption
(B) entrapment
(C) crosslinking
(D) encapsulation
Answer: B, D
Q.8-Q.10
Q.8
match the entries in Group I with the entries in Group II
Match the luciferin structure with the type of bioluminescent organisms
Group I
(A)
(B)
(C)
(D)
Group II
(1) bacteria
(2) fireflies
(3) coelenterates
(4) dinoflagellates
Answer: A-3, B-4, C-2, D-1
20
(5) fungi
Annex 4.3
Example of Final Test Questions
Q.9
Match the term and the definition
Group I
(A)
fluorescence
(B)
(C)
chemiluminescence
bioluminescence
Group II
(D)
photoluminescence
(1) luminescence as a result of electron transition between the energy levels of similar
multiplicity
(2) luminescence as a result of electron transition from excited state occupied after chemical
reaction
(3) luminescence as a result of electron transition from excited state occupied after chemical
reaction in active site of enzyme
(4) luminescence as a result of electron transition from excited state occupied after light
absorption
(5) luminescence as a result of electron transition from excited state occupied after heating
Answer: A-1, B-2, C-3, D-4
Q.10
Match the point of the dose-effect curve on the picture bellow and the name of toxicity
criterion
Group I
Group II
(A) 1
(B) 2
(C) 3
(1) EC50
(2) NOEC
(3) LOEC
(4) DNEC
Answer: A-2, B-3, C-1
21