Bi7430c MOLECULAR BIOTECHNOLOGY - practiceBi7430c MOLECULAR BIOTECHNOLOGY
practice
table of contents
01 COURSE INFORMATION 3
syllabus 6
workplaces 7
02 LECTURERS 8
03 MANUALS 16
in-silico cloning of haloalkane dehalogenases 17
recombinant proteins: from small- to large-scale expression technologies 25
preparation and testing of microfluidic chip 40
biocatalytic preparation of pharmaceutical precursor 43
preparation and transformation of liposomes 47
analysis of liposomes 50
preparation of enzymatic biosensor 52
01
Bi7430c MOLECULAR BIOTECHNOLOGY - practice
course information
COURSE MOTIVATION
The practice provides an interesting combination of practical problems solved under the
guidance of experts from academia (Masaryk University and Veterinary Research Institute)
and business environment (a biotechnology company Enantis). At the end of the course,
students will be familiar with a variety of techniques of molecular biotechnology, such as
design of primers, DNA cleavage by restriction enzymes, transformation of bacterial cells,
methods of fermentation culturing E. coli, application recombinant microorganisms and
isolated enzymes in biosensing and remediation of hazardous substances or synthesis of
pharmaceutically pure chemicals. The course will enable students to deepen their
theoretical knowledge acquired in lectures Molecular Biotechnology. Students will become
familiar with the currently used methods and the principle and operation of modern
laboratory equipment. The course also aims to strengthen communication skills of students
in a discussion with experts of the field and exercise stylistic skills of students in a writing an
essays and protocols. The learning materials in English support the ability to work with
scientific texts, including strengthening the knowledge of the English terminology used in
this field.
4Molecular Biotechnology – Practice | Bi7430c
CAPACITY two groups of 10 students
LANGUAGE materials – EN
spoken language – CZ
protocols – student chooses between EN and CZ
ABSENCE official excuse in the Information System (IS) + written essay (2 pages A4)
INTERACTIVE SYNOPSIS available in Bi7430c in the Information System
PREREQUISITES
1. parallel attendance of Molecular Biotechnology lecture (Bi7430)
2. the course of Molecular Biology (Bi4020) and Basics of Molecular Biology (Bi4010)
completed
PRACTICE ORGANISATION
1. theoretical introduction (presentation given by lecturer)
2. introduction to practice and assignment control (homework)
3. experimental work in the laboratory
TEACHING AND ASSESSMENT METHODS
Students are expected to study the manual and prepare the homework before each
practice. In the laboratory, each student works independently or in pairs under the
guidance of the teacher. Each student works with own sample. The results of experiments
are evaluated by each student in separate essays and protocols based on the individual
practice. Students will obtain positive assessment of the practice for active participation in
exercises and preparation of protocols or essays from each exercise. Protocols should be
uploaded to IS within one week from each practice. Delay would result in extra written
essay (1 page A4).
5Molecular Biotechnology – Practice | Bi7430c
syllabus
IN SILICO CLONING OF A HALOALKANE DEHALOGENASE
Lecturer: Ing. Andrea Schenkmayerová, Ph.D.
Aims: software tool Clone Manager, design of primers, restriction cleavage, cloning,
transformation
RECOMBINANT PROTEINS: FROM SMALL- TO LARGE-SCALE EXPRESSION
TECHNOLOGIES
Lecturer: Ing. RNDr. Martin Marek, Ph.D.,
Ing. Andrea Schenkmayerová, Ph.D.
Aims: from DNA to recombinant protein production, small-scale expression screening,
large-scale (fermentor) overproduction, protein purification strategies, and quality
control of the purified proteins
PREPARATION AND TESTING OF MICROFLUIDIC CHIP
Lecturer: Mgr. David Kovář, Ph.D.
Aims: fabrication of PDMS chip, preparation and microscopy of microdroplets,
microscopy of microdroplets with cells
BIOCATALYTIC PREPARATION OF PHARMACEUTICAL PRECURSOR
Lecturer: Assoc. Prof. Radka Chaloupková, Ph.D.
Aims: biocatalytic synthesis of pharmaceutical precursor by using recombinant
enzyme
PREPARATION AND TRANSFORMATION OF LIPOSOMES
Lecturer: RNDr. Jaroslav Turánek, CSc.
Aims: preparation of liposomes, transformation, high-pressure extrusion, preparation
of SUV (small unilamellar vesicels), DLS (dynamic light scattering) analysis, ZetaSizer
ANALYSIS OF LIPOSOMES
Lecturer: RNDr. Jaroslav Turánek, CSc.
Aims: single particle tracking analysis by using Nanosight 500, transmission electron
microscopy
PREPARATION OF ENZYMATIC BIOSENSOR
Lecturer: Mgr. Šárka Nevolová, Ph.D.
Aims: co-immobilization of recombinant enzyme and fluorescence probe to optical
transducer, detection of selected analyte
6Molecular Biotechnology – Practice | Bi7430c
workplaces
LOSCHMIDT LABORATORIES
http://loschmidt.chemi.muni.cz/peg/
The Josef Loschmidt Chair was established at the Faculty of Science of Masaryk University
by a donation of Alfred and Isabel Bader to honour the name of great Czech scientist of the
19th century Jan Josef Loschmidt. Jiri Damborsky was appointed to the Chair in 2003 and
the Loschmidt Laboratories were founded two years later. The Laboratories hold prestigious
award from the European Molecular Biology Organization and the American foundation
Howard Hughes Medical Institute. The research is focused to the areas of protein and
metabolic engineering. Especially understanding the structure-function relationships of
haloalkane dehalogenase enzymes and improve their functionalities for bioremediation,
biocatalysis and biosensing.
ENANTIS, S.R.O.
http://www.enantis.com/
Enantis is a Czech R&D company founded in 2006 as the first biotechnology spin-off from
Masaryk University, Brno, Czech Republic. Since 2008, the company has its own fullyequipped
laboratories in the Biotechnology Incubator operated by the South Moravian
Innovation Centre, where research, service and production operations are being carried
out. Enantis provides consulting and development services in the field of enzyme
technologies and protein engineering for biomedical, environmental, agrochemical and
military-defence applications. Employing the latest technologies, the company offers its
own range of products based on proteins and microorganisms.
VETERINARY RESEARCH INSTITUTE
http://www.vri.cz/
The Veterinary Research Institute in Brno (VRI) was founded in 1955. Collection of Animal
Pathogenic Microorganisms which joined European Culture Collections Organisation in 1985
is a part of the Institute. The institution specializes in the veterinary medicine research. Its
activity was focused on performing exact experiments with the aim to solve health
problems in farm animals, protect people from zoonoses and to guarantee safety of
foodstuffs and raw materials of animal origin.
7Molecular Biotechnology – Practice | Bi7430c
Bi7430c MOLECULAR BIOTECHNOLOGY - practice
lecturers02
9Molecular Biotechnology – Practice | Bi7430c
Prof. RNDr. Zbyněk Prokop, Ph.D.
EDUCATION
Zbyněk Prokop is Professor in the Loschmidt Laboratories at
Masaryk University, where he leads research team engaged in
the study of fundamental principles of protein chemistry,
enzyme mechanism and kinetics. He received Ph.D. degree in
Environmental Chemistry from Masaryk University and
extended his expertise during his research stays at University
of Cambridge (UK), ETH Zurich (CH), TU Wien (A) and University
of Groningen (NL).
PROFESSIONAL ASSIGNMENTS
His current research interests are the development of
advanced biophysical methods for the structural and functional
study of proteins, enzyme technology development and
applications of microfluidics in life and biomedical sciences. He
has co-authored 80 papers in refereed journals (h-index 21),
five book chapters, five international patents and has given
more than 20 invited lectures at conferences and universities
worldwide. He received Alfred Bader Prize for Bioorganic
Chemistry in 2005 and Werner von Siemens Award for
Excellence in Innovation in 2015. He is a co-founder of Enantis
Ltd, a Masaryk University biotechnology spin-out company, and
a member of the Advisory Board of Centre for Technology
Transfer at Masaryk University. He teaches several courses,
such as Molecular Biotechnology, Methods in Biophysical
Chemistry and Summer School of Protein Engineering.
UČO 23696
zbynek@chemi.muni.cz
10Molecular Biotechnology – Practice | Bi7430c
Mgr. Šárka Nevolová, Ph.D.
EDUCATION
Šárka Bidmanová received her master’s degree in Microbiology
and Secondary School Teacher Training in Biology in 2007 and
Ph.D. degree in Environmental Chemistry in 2012 from the
Masaryk University in Brno. She extended her expertise during
her research stay at the Institute of Chemical Process
Fundamentals at the Academy of Sciences of Czech Republic
and at Institute of Analytical Chemistry/Chemo- and Biosensors
at the University of Regensburg in Germany.
PROFESSIONAL ASSIGNMENTS
Šárka Nevolová worked as a researcher in Loschmidt
Laboratories at Masaryk University from 2009 to 2016, she is
currently on maternity leave. She is focused on the
development, optimization and characterization of optical
enzyme-based bioanalytical devices for environmental
applications and military-defense. Her research interests also
included immobilization and characterization of enzymes and
pH indicators. She is co-author of twelve publications and two
book chapters. She obtained the Award of the Dean of the
Faculty of Science of Masaryk University in 2013 and the Award
of the Czechoslovak Microbiological Society in 2014.
UČO 77580
77580@mail.muni.cz
11Molecular Biotechnology – Practice | Bi7430c
Assoc. Prof. Radka Chaloupková, Ph.D.
EDUCATION
Radka Chaloupková received her master’s degree in
Theoretical and Physical Chemistry in 2001, a Ph.D. degree in
Biochemistry in 2008 and habilitation from Environmental
Chemistry in 2017 at the Masaryk University. She has gained an
international experience, theoretical knowledge and practical
skills during the three months’ research visit to the University
of Warwick (Coventry, UK) supported by Marie Curie
Fellowship, five months’ research visit at the Diamond Light
Source Ltd. (Didcot, UK) and several international and national
courses organized by EMBO, EPSRC, COST, Academy of
Sciences and Masaryk University.
PROFESSIONAL ASSIGNMENTS
Radka Chaloupková worked as a leader of a research team
focused on structure and thermodynamics of biomolecules
within the Loschmidt Laboratories from 2006 till 2018.
Currently she is working as Chief Science Officer in Enantis
company. She has more than 17 years’ experience with various
biophysical techniques exploited in protein research. She is an
expert in application of optical spectroscopic techniques,
microcalorimetry and X-ray crystallography for analysis of
structure-function relationships of enzymes. She has published
more than 60 scientific papers in international journals, 5 book
chapters and 2 international patents (h-index 19). She received
the Award of the Dean of the Faculty of Science, Masaryk
University in 2005 and the Award of the Rector of the Masaryk
University in 2007.
UČO 16143
radka@chemi.muni.cz
chaloupkova@enantis.com
12Molecular Biotechnology – Practice | Bi7430c
Ing. Andrea Schenkmayerová, Ph.D.
EDUCATION
Andrea studied Biotechnology at the Slovak University of
Technology in Bratislava, Slovakia and obtained her PhD in
Biotechnology while doing research at the Slovak Academy of
Sciences in Bratislava. Her studies were focused on
fermentation and biotransformation processes, whole-cell
immobilization techniques and construction of biosensors.
PROFESSIONAL ASSIGNMENTS
After finishing her PhD she was awarded a Claude Leon
Fellowship for her two year PostDoc in Biochemistry at
Stellenbosch University in South Africa. She was doing research
on new antistaphylococcal drug targets. She is currently doing
her second PostDoc in Protein Engineering at the Masaryk
University in Brno, Czech Republic, being employed by the
International Clinical Research Center of St. Anne's University
Hospital.
andrea.schenkmayerova
@fnusa.cz
13Molecular Biotechnology – Practice | Bi7430c
Ing. RNDr. Martin Marek, Ph.D.
EDUCATION
Martin Marek received his master’s degrees in Agricultural
Engineering (2002) from the Czech University of Life Sciences
(CULS) and in Molecular Biology (2007) from the Charles
University, and a PhD degree in Phytopathology and Plant
Protection from the CULS, all in Prague (CZ). He extended his
scientific expertise in recombinant multi-expression
technologies, biochemistry, biophysics and structural biology
(X-ray crystallography) during post-doctoral stays at the
Wageningen University (Wageningen, NL), Généthon (Paris, FR)
and IGBMC (Strasbourg, FR).
PROFESSIONAL ASSIGNMENTS
Martin Marek works as a senior researcher in the Loschmidt
Laboratories at the Masaryk University since 2017, where he
leads a team working in molecular and structural biology. In
2018, he was awarded by Marie Skłodowska-Curie Actions
individual fellowship. He is co-author >35 scientific
publications, three book chapters and one international patent
in the field of expression technologies, virology, molecular
structural biology and drug discovery.
UČO 241182
martin.marek@
recetox.muni.cz
14Molecular Biotechnology – Practice | Bi7430c
Mgr. David Kovář, Ph.D.
EDUCATION
David Kovář received his master’s degree in Analytical
Biochemistry in 2009 and Ph.D. degree in Biomolecular
Chemistry in 2015 at the Masaryk University. His Ph.D. studies
were focused on immunospecific sensors exploiting
nanoparticles and nanostructures with the main application of
magnetic nanoparticles. He then joined the group of Prof.
Maria Hepel at the State University of New York (SUNY;
Potsdam, NY) as a postdoctoral researcher in physical
electrochemistry.
PROFESSIONAL ASSIGNMENTS
In 2016, he joined Loschmidt Laboratories and has gained new
experience and knowledge in the field of protein engineering.
He extended his expertise in microfluidic during his research
stays at the ETH Zurich (Switzerland) in the group of prof.
deMello. Currently, his research activities relate to microfluidic
platforms for screening and selection methods in directed
enzyme evolution. He is also focused on development and
testing of new pH sensitive fluorescent assays.
UČO 150685
kovard@mail.muni.cz
15Molecular Biotechnology – Practice | Bi7430c
RNDr. Jaroslav Turánek, CSc.
EDUCATION
He graduated from the Faculty of Science at Masaryk
University, specialization biochemistry, and obtained the
degree RNDr. in 1982. In 1987, he terminated post-graduated
studies in the field of biochemistry and physical chemistry at
the Faculty of Science, MU and South Bohemian Biological
Centre, and obtained the degree CSc. Jaroslav Turánek
extended his expertise during a research stay at GTC, Imperial
College in London and by taking a number of specialist courses.
PROFESSIONAL ASSIGNMENTS
Jaroslav Turánek is Head of the Department of Pharmacology
and Immunotherapy, Veterinary Research Institute, Brno,
Czech Republic. His current research interests are development
of recombinant vaccines and molecular adjuvants,
immunotherapy of infectious diseases and cancer,
nanodelivery systems for drugs (e.g., liposomes, micelles and
dendrimers), and in vivo and in vitro pharmacological and
toxicological models. Dr. Turánek is a lecturer of the following
courses: Advanced Immunology (Masaryk University),
Immunochemistry (Technical University), and Cellular
Biotechnology (Technical University). He is a member of the
Committee for state examination (Masaryk University) and
supervisor of postgradual and pregradual students.
UČO 28301
turanek@vri.cz
Bi7430c MOLECULAR BIOTECHNOLOGY - practice
manuals03
IN SILICO CLONING OF A HALOALKANE DEHALOGENASE
location: Loschmidt Laboratories, Kamenice 5/A5 room 114 or A17/332
lecturer: Ing. Andrea Schenkmayerová, Ph.D. (andrea.schenkmayerova@fnusa.cz)
I. WORKFLOW
During this practical course/computer exercise we will perform all steps of the cloning of
the haloalkane dehalogenase gene (dhaA) from Rhodococcus rhodochrous. The gene will be
cloned in the pET21b vector for recombinant (over)expression in Escherichia coli.
– design of the primers for cloning the dhaA gene in pET21b
– in silico PCR reaction is performed using the gene sequence and the created primers to
create the RE site containing DNA fragment
– the restriction digest is performed on both the vector and insert using the chosen
restriction enzymes
– the two fragments are ligated to obtain the final plasmid
– finally the sequence obtained from sequencing is compared with the plasmid that was
made in CloneManager [1]
II. MOTIVATION
An important step in biochemical research and biocatalytic processes is the production of
the protein of interest. Therefore, the corresponding gene will be transferred into a vector
(e.g. plasmid) which can be transformed into expression hosts. This exploitation of nature
allows scientists to express their protein of interest at larger scale but also to alter it by
mutagenesis or attach specific tags to facilitate purification.
III. THEORETICAL BACKGROUND
What is Molecular Biotechnology? In its broadest sense, molecular biotechnology is the use
of laboratory techniques to study and modify nucleic acids and proteins for applications in
areas such as human and animal health, agriculture, and the environment. Thus, molecular
biotechnology is the branch of biology that deals with the formation, structure, and
function of macromolecules essential to life, such as nucleic acids and proteins, and
especially with their role in cell replication and the transmission of genetic information.
Molecular biotechnology results from the convergence of many areas of research, such as
molecular biology, microbiology, biochemistry, immunology, genetics, and cell biology
(Figure 1).
17Molecular Biotechnology – Practice | Bi7430c
Figure 1. Schematic relationships the different research areas in Molecular biotechnology
(biochemistry, genetics, and molecular biology).
More recently, much work has been done at the interface of molecular biology and
computer science, namely in bioinformatics and computational biology. Molecular
biotechnology is an exciting field fuelled by the ability to transfer genetic information
between organisms with the goal of understanding important biological processes or
creating a useful product. The central dogma (Figure 2) of molecular biology confirms the
importance of genetic information or DNA (and RNA) in nature and the biological sciences.
Figure 2. The Central dogma of molecular biology is an explanation of the flow of genetic
information within a biological system
The transfer, use or changing of genetic information (manipulation of DNA) is a very
important step – and in many cases the first experimental step – in biotechnological
research since this enables to introduce, delete or alter the DNA and hereby change and
study the characteristics of cells, proteins, enzymes, … Molecular biology is the branch of
biology that deals with the manipulation of DNA so that it can be sequenced or mutated or
used to study the biological effects of the mutation(s), gene product, …
In a standard molecular cloning experiment, the cloning of the desired DNA fragment can
be seen as a seven step process (Figure 3). Several types of DNA manipulations are used
here, including amplification, digestion and ligation by polymerase chain reaction (PCR),
restriction enzymes and a ligase, respectively. Standard protocols for molecular cloning and
DNA handling are well described in several (practical) handbooks [2].
18Molecular Biotechnology – Practice | Bi7430c
Figure 3. The different steps of a typical
molecular cloning process. Numbers
between brackets correspond to the
seven step mentioned in the text.
The seven steps of a standard molecular cloning experiment
1. Choice of a host organism and a cloning vector
Several host organisms and molecular cloning vectors are available, however, for the vast
majority of molecular cloning experiments a lab strain of the bacterium E. coli is used
together with an appropriate plasmid cloning vector. E. coli and its vectors are widely used
due to their relative simplicity, yet technically sophisticated nature, versatility, and wide
availability. In addition, they offer rapid growth of recombinant organisms with minimal
equipment.
2. Preparation of vector DNA
To prepare the cloning vector to accept the gene of interest it is treated with restriction
endonucleases in order to create (specific) sites at which the foreign DNA can be inserted.
3. Preparation of DNA to be cloned
The gene of interest can be picked up from genomic DNA extracted from the organism of
interest. Any tissues source can be used as long as the DNA is intact. In case of starting from
RNA, a reverse transcriptase step is needed to create complementary DNA (cDNA).
Nowadays, it is also possible to order synthetic DNA sequences, for example from hard to
amplify fragments, for variants containing multiple mutations, eukaryotic genes (cfr. introns
and RNA splicing) or unnatural sequences. Artificial gene synthesis has the advantage of to
build in or removal of specific restriction sites as well as codon optimization for your
specified host organism.
19Molecular Biotechnology – Practice | Bi7430c
PCR methods (Figure 4) are the techniques used for amplification of specific DNA or RNA
(RT-PCR) sequences prior to molecular cloning. While picking up the gene of interest one
can simultaneously ‘attach’ the desired restriction sites to the PCR product by using primers
containing these restriction sites.
Figure 4. Amplification of a DNA fragment is achieved by PCR
4. Creation of recombinant DNA
In this step, the vector and foreign DNA are mixed at appropriate concentrations and an
enzyme called DNA ligase will covalently link the ends together, creating a single DNA
molecule which can be introduced into the host organism. Nowadays, new techniques have
been developed like restriction/ligation free cloning, such as Gibson assembly [3] which
allows for the joining of multiple DNA fragments in a single, isothermal reaction.
5. Introduction of recombinant DNA into a host organism
The in vitro prepared DNA can now be inserted back into a living cell, namely the host
organism. Several options are available depending upon the experimental method and host
cells (e.g. transformation, transduction, transfection, electroporation).
6. Selection of organisms containing recombinant DNA
The introduction of recombinant DNA into host organisms is usually a low efficiency
process. Only a small number of cells will actually take up DNA. Therefore, efficient
selection of the good clones is needed. When using bacterial cells, this can easily be
achieved by a selectable marker on the vector. Most common are antibiotic resistance
markers which allow the good clones to survive in the presence of antibiotic while the other
cells die.
7. Screening for clones with desired DNA inserts and biological properties.
To make sure that the surviving colonies contain your gene of interest a very wide range of
20Molecular Biotechnology – Practice | Bi7430c
experimental methods is available, including the use of nucleic acid hybridizations, antibody
probes, polymerase chain reaction, restriction fragment analysis and/or DNA sequencing.
DNA sequencing is also used to confirm the absence of unwanted mutations and correct
integration of the DNA fragment.
PCR Primer Design Guidelines
(adapted from http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html )
1. Primer Length: The generally accepted optimal primer length for PCR primers is 18-22 bp.
This length is long enough to ensure adequate specificity and short enough for primers to
bind easily to the template
2. Primer Melting Temperature (Tm): Tm is the temperature at which one half of the DNA
duplex will dissociate to become single stranded and indicates the duplex stability. Tm in the
range of 52-58°C generally produce the best results. The formula Tm = 2°C x (A+T) + 4°C x
(G+C) is popular for its simplicity and roughly accurate prediction of oligonucleotide Tm.
3. Primer Annealing Temperature: Too high Ta will produce insufficient primer-template
hybridization resulting in low PCR product yield. Too low Ta may possibly lead to nonspecific
products caused by a high number of base pair mismatches.
4. GC Content: The GC content (the number of G's and C's in the primer as a percentage of
the total bases) of primer should be 40-60%.
5. GC Clamp: The presence of G or C bases within the last five bases from the 3' end of
primers (GC clamp) helps promote specific binding at the 3' end due to the stronger
bonding of G and C bases. More than 3 G's or C's should be avoided in the last 5 bases at
the 3' end of the primer.
6. Primer Secondary Structures: Presence of the primer secondary structures produced by
intermolecular or intramolecular interactions can lead to poor or no yield of the product.
Interactions can be intramolecular resulting to hairpin formation or self-dimerization or
intermolecular when different primers in the PCR mix form a dimer.
7. Repeats (repetitive presence of di-nucleotides) and Runs (long runs of a single base)
should be avoided because they can misprime.
8. 3' End Stability: It is the maximum ΔG value of the five bases from the 3' end. An unstable
3' end (less negative ΔG) will result in less false priming.
9. Avoid Template Secondary Structure, Avoid Cross Homology, …
21Molecular Biotechnology – Practice | Bi7430c
Luckily, these rules have been integrated in Molecular Biology software like CloneManager
and free online primer design tools.
In the case of this cloning experiment, specific sites have to be introduced where the
restriction enzymes (RE) can cleave the DNA for easy and correct fusing of the insert (gene
of interest) and plasmid vector. Therefore, it is important to choose the right restriction
enzymes for cloning and add the according RE sites to the primers. Some simple rules: the
chosen restriction enzymes cannot cleave anywhere else in the insert or vector fragment,
sticky ends are better than blunt ends, non-compatible ends are desired as they allow
unidirectional cloning.
IV. DESIGN OF EXPERIMENT
Equipment:
– PC with CloneManager software
– sequence of DhaA and pET21b plasmid
Workflow:
1. design of the primers for cloning the dhaA gene in pET21b
2. in sillico PCR reaction is performed using the gene sequence and the created primers to
create the RE site containing DNA fragment
3. the restriction digest is performed on both the vector and insert (created PCR
fragment) using the chosen restriction enzymes
4. the two fragments are ligated to obtain the final plasmid
5. finally the sequence obtained from sequencing is compared with the plasmid that was
made in CloneManager [1].
V. EXERCISES
1. Design primers for the cloning of the haloalkane dehalogenase gene (dhaA) from
Rhodococcus rhodochrous in the pET21b vector for recombinant expression in Escherichia
coli. Write down the primer and underline the restriction enzyme sites.
Fwd:
Rev:
22Molecular Biotechnology – Practice | Bi7430c
2. Calculate the melting temperatures of both primers using the A/T=2°C, G/C= 4°C rule
and compare it with the melting temperatures found using CloneManager.
Check the New England Biolabs website (www.NEB.com) for more information about the
restriction enzymes for question 3-5.
3. What is the advantage of using two different restriction enzymes for the cloning of a
gene into a vector, for example NdeI and EagI instead of NdeI at both ends? Which are the
two problems that are avoided?
4. What is the advantage of using for example NdeI and EagI for cloning rather than NaeI
and BsrBI?
23Molecular Biotechnology – Practice | Bi7430c
5. Below you find a protocol for mutagenesis. You created a mutant library and accidently
added DpnII to the PCR mix instead of DpnI. Do we have a problem now? Important to
know: E. coli has a system for DNA methylation, which is not present in a PCR reaction.
6. You are provided with two sets of sequencing data (Fwd + Rev). Which one confirms
the correct sequencing and which sample contains the error? What is the result of the error
at DNA level?
VI. LITERATURE
1. CloneManager (SciEd software, USA), http://www.scied.com/pr_cmpro.htm
2. Green MR & Sambrook J (2012) Molecular cloning: A laboratory manual, 4th ed. (Cold
Spring Harbor Laboratory Press, New York)
3. Gibson DG, Young L, Chuang R-Y, Venter JC, Hutchison CA, III, Smith HO (2009) Enzymatic
assembly of DNA molecules up to several hundred kilobases. Nature Methods 6:343-345
24Molecular Biotechnology – Practice | Bi7430c
RECOMBINANT PROTEINS: FROM SMALL- TO LARGE-SCALE
EXPRESSION TECHNOLOGIES
location: Loschmidt Laboratories, Kamenice 5/A13, 2nd floor, room 332
lecturer: Ing. RNDr. Martin Marek, Ph.D. (martin.marek@recetox.muni.cz)
Ing. Andrea Schenkmayerová, Ph.D. (andrea.schenkmayerova@fnusa.cz)
I. WORKFLOW
Selection of an expression host – purpose of protein production – combinatorial screening
in small-scale formats – bench-scale and large-scale (fermentor) overproductions –
downstream processing and purification strategies – quality/quantity check and final
formulation
II. MOTIVATION
Proteins are produced in heterologous systems because of the impossibility to obtain
satisfactory yields from natural sources. The production of soluble and functional
recombinant proteins is among the main goals in the biotechnological and pharmaceutical
industries. The selection of an optimal expression organism (host) and the most
appropriate growth conditions to minimize the formation of insoluble proteins have to be
done according to the protein characteristics and downstream requirements. Escherichia
coli is the most popular recombinant protein expression system despite the great
development achieved so far by eukaryotic expression systems. However, it is important to
mention that E. coli expression system possesses, in many cases, severe limitations for a
successful recombinant protein production. Therefore, eukaryotic systems, including
mammalian cells, insect cells, yeasts, filamentous fungus, and microalgae, are an important
alternative for the production of those difficult-to-express proteins. During this course we
will combine theoretical designs with practical demonstrations concerning all aspects of
recombinant protein (multi)-expressions, ranging from small- to large-scale (fermentor)
overproduction technologies. In the second part, we will focus on the downstream
processing and purification strategies. The students will get knowledge on:
How to select a heterologous system (expression host)
Pilot small-scale (transient) expression screening to determine optimal expression
conditions, and buffer screening strategies
Scaling-up of production process, bench-scale and large-scale (fermentor) productions
Harvesting process and timing
Downstream processing and purification strategies to yield highly pure recombinant
proteins in sufficient amounts
Control (quality/quantity) of the purified protein, final formulation and long-term
storage
25Molecular Biotechnology – Practice | Bi7430c
III. THEORETICAL BACKGROUND
Selection of expression organism (host): each heterologous expression system has
benefits and drawbacks with respect to their capacity for recombinant protein
production (Figure 1). The gram-negative bacterium Escherichia coli is frequently
the first expression host chosen for the production of a recombinant protein, owing
to the rapid, affordable and technically straightforward culturing associated with its
use. The E. coli system offers a mean for rapid, high yield, and economical
production of recombinant proteins (Figure 2). However, high-level production of
functional eukaryotic proteins in E. coli may not be a routine matter, sometimes it is
quite challenging. Techniques to optimize heterologous protein overproduction in E.
coli have been explored for host strain selection, plasmid copy numbers, promoter
selection, mRNA stability, and codon usage, significantly enhancing the yields of the
foreign eukaryotic proteins.
Yeasts is a single-celled eukaryotic organism capable of producing very large
quantities of recombinant protein. Pichia pastoris is the most used strain of first
choice for yeast expression. The baculovirus/insect cell expression vector system
(BEVS) is a popular choice for the production of recombinant proteins, particularly
those requiring complex post-translational modifications or integral membrane
proteins. Mammalian cell-based expression (e.g. HEK293 cell line) is the dominant
system for the production of therapeutic recombinant proteins. Their capacity to
handle complex post-translational modifications, folding and assembly of
recombinant proteins and protein complexes is superior to other systems. The
Leishmania tarentolae extract (LTE) in vitro translation cell-free expression system is
a rapid, convenient, flexible and cost effective tool to produce recombinant proteins
for biochemical, biophysical and structural analysis.
26Molecular Biotechnology – Practice | Bi7430c
Figure 1. Comparison of the most
industrially used expression
systems.
At the beginning of a protein expression project, small-scale expression screenings
in minimal volumes (2-6 mL) are recommended prior to running large-scale
overproductions. The pilot small-scale expression tests are designed to test different
expression parameters (host strain selection, timing of induction and harvesting,
expression temperature, pH, ionic strength etc.) to find an optimal condition and
buffer screening. Large-scale expression in bacteria, yeast, insect and mammalian
cell systems in culture volumes from 400 mL to 10 L are available in shake flask and
from 2 L to 20 L in a stirred-tank bioreactor or up to 25 L in a Wave bioreactor.
Large-scale production in bioreactor (fermentor) is very complex procedure where
several factors play a crucial role on the performance of the culture. Composition of
cultivation broth, sterilization efficiency, proper agitation and aeration, temperature
or feeding strategy can all together influence final yield of biomass and recombinant
protein from the fermentation. Since culture in fermentor is half opened system, it
is very susceptible to contamination. It is very important for the operator of the
fermentor to maintain sterility throughout the fermentation. In this practice, the
students will learn how to assemble the fermentor vessel and prepare it for
sterilization, how to calibrate pH and DO (dissolved oxygen) sensors and set up the
fermentation together with creation of recipes for fed-batch cultivations.
27Molecular Biotechnology – Practice | Bi7430c
Recombinant protein expression in E. coli: basic principles and advantages
versus limitations
ADVANTAGES
•Inexpensive setup and running costs
•High recombinant protein production levels
•Short timeline from cloning to protein recovery (1 week)
•Limited technical knowledge required for culturing
•Scalability from small (2 mL) to very large industrial culture (>10,000 L) volumes
DISADVANTAGES
•Inability to perform post-translation modifications (PTMs)
•Limited formation of disulphide bond
Figure 2. Schematic representation of the plasmid used for the protein expression in E. coli. (A) Map
of the typical expression plasmid. The gene of interest (DAC2) is inserted between NdeI and BamHI
restriction sites. Selection in E. coli is performed by the beta-lactamase ampicillin resistance gene
(ampR). Origin of replication sequence (ColE1) is available for maintenance in E. coli cells, and the lacI
gene is present for expression of the Lac repressor protein. (B) Details of the expression cassette. The
gene of interest (DAC2) is controlled by the T7 promoter and T7 terminator. The DAC2 gene is in
cloned in frame with a sequence coding for a C-terminal thrombin cleavage site followed by a polyhistidine
tag. (C) Overview of recombinant protein expression workflow in E. coli.
Recombinant protein purification: the aim of a purification procedure is to obtain a
highly pure and stable protein at an appropriate concentration in a buffer
compatible with the intended downstream application (Figure 3). Chromatographic
techniques are the most powerful and commonly used means of purifying
recombinant proteins. Each technique separates proteins based on different
properties, so it is often advantageous to combine several types to maximise
separation of the recombinant protein from host cell proteins. The use of fusion
tags (polyhistidines, Strep-tag, GST, MBP, SUMO, thioredoxin etc.) not only facilitates
affinity chromatography steps, but also can dramatically improve protein expression,
stability, resistance to proteolytic degradation and solubility. Variety of proteases
(thrombin, protease 3C, TEV etc.) for fusion tag removal during downstream
processing is available.
28Molecular Biotechnology – Practice | Bi7430c
Figure 3. Workflow of recombinant protein purification includes (i) the lysis of a cell pellet by
sonication or high-pressure homogenizer, (i) the clarification of cell lysate by high-speed
centrifugation, (iii) filtration, and (iv) one or more chromatographic purification steps.
Recombinant protein characterization: an aggregation problem. A key challenge in
recombinant protein production is to maintain and store the target protein in a
soluble and stable form. Protein aggregation can compromise protein function and
thus it is necessary to overcome this challenge to generate functionally active
protein (Figure 4). Protein aggregates can be detected by (i) analytical size-exclusion
chromatography (SEC), (ii) dynamic light scattering (DLS), and (iii) analytical
ultracentrifugation (AUC).
29Molecular Biotechnology – Practice | Bi7430c
Figure 4. Schematic representation of unwanted protein
aggregation problem that can compromise protein (enzyme)
function.
How to avoid (minimize) the protein aggregation problem:
• Culture conditions (e.g. reducing temperature)
• Buffer composition (ionic strenght, pH, reducing agents)
• Presence co-factors (Acetyl-CoA, metal ions)
• Fusion tags (Trx, MBP, SUMO)
• Minimising sample handling
• Avoiding time delays between purification steps
• Performing purification steps at 4°C
• Store purified proteins in -80°C
!!! Concluding remark: The recombinant protein production project is ultimately
determined by the end-use of the recombinant protein. The overall success of a
project lies in an effective project design.
IV. THEORETICAL DESINGS AND PRACTICAL DEMONSTRATIONS
Small-scale expression tests: finding optimal condition and buffer screening
1. Day 1, the E. coli BL21 (DE3) cells are transformed with the expression plasmid.
Transformed bacteria are seeded on a 6-well plates containing 2xLB agar medium with
ampicillin (100 µg/ml); the plates are then incubated at 37°C overnight.
2. Day 2, ampicillin-resistant colonies are inoculated in minicultures (2 ml 2xLB, 0.5%
glucose and 100 µg/ml ampicillin).
3. After incubation (275 rpm, 37°C) for 6h, induction is done at 22°C overnight by adding 2
ml of 2xLB medium with 0.6 % lactose, 0.5 mM IPTG, 20 mM HEPES pH 7.4 and
ampicillin 100 μg/ml.
4. Day 3, cells are centrifuged (3,500 rpm, 10 min), re-suspended in four different lysis
buffers (10 mM Tris-HCl pH 8.0 with 50 mM, 100 mM, 200 mM or 400 mM KCl) and
lysed by sonication (1 min, 40 % amplitude).
5. The lysates are clarified by centrifugation (4,000 rpm, 20 min), and then the
supernatants are loaded on Talon Superflow Metal Affinity Resin (Clontech) preequilibrated
in the lysis buffer.
6. The samples are incubated on roller shaker (4°C, 2 h), and the resins are then washed
with the corresponding lysis buffers twice by centrifugation (1,000 rpm, 2 min).
7. Finally, the resins are re-suspended in Laemmli Buffer (40 μl), and the proteins bound
on the resin are analysed by sodium dodecyl sulphate polyacrylamide gel
electrophoresis (SDS-PAGE) and visualised by Coomassie Brilliant Blue staining.
30Molecular Biotechnology – Practice | Bi7430c
Figure 5. The example of small-scale expression screening in 24-well plates that revealed induction
and harvesting times as the key parameters in successful production of soluble smHDAC8 protein.
Protocol for large-scale recombinant protein production in bioreactor (fermentor)
Protocol for assembly of the fermentation vessel:
1. Prepare and dissolve all components of the fermentation broth. The volume of the
medium should not be higher than working volume of the fermentor (usually 2/3 of
total volume).
2. Calibrate pH probes (See Protocol for calibration of pH probes).
3. Pour dissolved medium to the vessel, add antifoaming reagent Struktol SB2020 to the
final concentration 100 μL/L.
4. Insert baffles to the vessel. Make sure the O-ring is present on the top of the vessel.
5. Attach the lid to the glass part of the vessel by tightening large screws on the top of the
lid.
6. Attach small plastic tubes to reagent inlet ports and sampling port, and big tubes to the
sparger inlet and gas exhaust. Fix by tightening of screw clamps if necessary.
7. Attach the pH and DO probes to the vessel lid.
8. Place rubber septa to remaining ports and fix them with metal rings.
9. Close all plastic tubes with clamps, close sparger inlet tube with Hofman screw clamp.
10. Insert air filter to sparger inlet and gas exhaust tubing, cover ends with aluminium foil.
Cover all plastic tube ends with aluminium foil.
11. Make sure all ports and tubes are closed, except for exhaust tube.
12. Sterilize the fermentor in autoclave.
13. After sterilization, let the fermentor cool down to room temperature.
31Molecular Biotechnology – Practice | Bi7430c
Protocol for calibration of pH probe:
1. Connect the pH probe to the connector on fermentor control unit.
2. Select calibration mode on the control panel.
3. Put the probe in the container with calibration solution with pH 4.1.
4. Wait until the signal is stable, confirm the value.
5. Rinse the probe with distilled water.
6. Repeat measurement with calibration solution with pH 7 or 9.
7. Confirm new calibration slope.
Protocol for preparation of seed cultured:
1. Prepare night culture by picking one colony of freshly transformed E. coli
BL21(DE3) cells to 10 mL LB medium with respective antibiotics.
2. Cultivate the cells overnight at 37 °C and 200 rpm.
3. Approximately 8-10 hrs prior to fermentation, prepare seed culture by
transferring 2 mL of night culture to 200 mL of LB medium with respective
antibiotics.
4. Incubate at 37 °C and 200 rpm.
Preparation of the vessel for fermentation protocol:
1. Fill up the jacket with tap water if necessary.
2. Connect the hose connectors to the thermostat.
3. Connect all probes to appropriate connectors on the control unit.
4. Turn on heating by setting the fermentation temperature (30 °C in our case).
5. Attach the motor on the top of the lid.
6. Wait until the DO probe is polarized.
7. Calibrate DO probe (See Protocol for calibration of DO probe).
8. Connect tubes to appropriate pumps and solution bottles (citric acid and NaOH).
9. Connect the sparger inlet tube to mass flow control.
10. Release all clamps on tubing with exception of clamp on sampling tube.
11. Set the cultivation parameters on control panel or in computer.
12. Inoculate fermentor with seed culture. For this purpose, seed culture volume
corresponding to 1/100 of volume of medium in fermentor is used.
13. After 1 hr, add 2 g/L lactose to induce expression of heterologous protein. 14.
Let the fermentation run overnight, harvest the cells by centrifugation and store
the culture at -80 °C.
32Molecular Biotechnology – Practice | Bi7430c
Protocol for calibration of DO probe:
1. Wait until the temperature of the medium reaches demanded temperature. Be
sure the stirring is ON.
2. Connect bottle with pure nitrogen to sparger inlet tube via the sterile filter.
3. On control panel, select DO probe calibration module.
4. Wait until medium is fully saturated by nitrogen. Collect signal for 0 % O2.
5. Connect air tube to the sparger inlet.
6. Wait until the medium is saturated with air. Collect signal for 100 % O2.
7. Confirm new calibration slope.
8. This calibration has to be done prior to every fermentation! Oxygen solubility
differs with medium composition and viscosity and its temperature.
Settings for batch fermentation:
1. Temperature 30 °C
2. Stirring cascade parameters: 30 % saturation, setpoint 500, minimum 500,
maximum 1000 rpm
3. Aeration 0.5 vvm
4. 4. pH 7
Protocol for sampling from the fermentor:
1. Connect the sterile syringe to sampling tube. Release the clamp. Suck ca. 5 mL
to rinse the tube with fresh culture. Fix the clamp again. Unplug the syringe and
discard whole volume. Repeate this to take your sample. Transfer the culture
from syringe to sterile glass tube.
2. Transfer 1 mL of the culture to a plastic cuvette and measure optical density at
600 nm.
3. Prepare 6 tubes with 900 μL of PBS buffer. Transfer 100 μL of the culture to first
tube, mix properly. Transfer 100 μL of suspension from first to second tube,
repeat until the sixth tube. Take 100 μL of the final suspension and spread it
with glass spreader on two plates with Plate Count Agar. Incubate the plates
overnight at 37 °C.
4. Pre-weigh 15 mL plastic falcon, transfer 10 mL of the culture to the falcon and
centrifuge for 5 min at 5,000 g. Discard supernatant and weigh the cells.
5. Re-suspend the cells in 1 mL of water, transfer the suspension to pre-weighted
aluminium foil. Put the foil with suspension for 2 hrs to dryer (110 °C). Weigh the
foil again, calculate the dry cell weight.
Equipment: Biostat B Plus bench-top fermentor (Sartorius Stedim) – Labfors 3
bench-top fermentor (Infors HT) – table centrifuge – ultracentrifuge Avanti J30I
(Beckman-Coulter) – LabStak M10 membrane filtration system (AlfaLaval) –
spectrophotometer and plastic cuvettes – bench-top dryer STZ 5,4 (FALC)
33Molecular Biotechnology – Practice | Bi7430c
Purification of His-tagged recombinant protein
1. The following protocol is provided considering the use of cell pellets from 3 L of
cultures. First, if required, thaw the re-suspended cell pellets. Adjust the volume
of the cell re-suspension to 40 mL per liter of culture (i.e. final volume of 120 mL
for 3 L of culture) using the lysis buffer (identical to the resuspension buffer).
2. Lyse the cell suspension using a high-pressure homogenizer at high pressure
(18,000 psi) using a single round of lysis. After the lysis, centrifuge the disrupted
cell suspension at 210,000 × g for 1 h and collect the supernatant in an ice-cold
bottle.
3. Apply the supernatant to a column with 2 ml of Talon Metal affinity resin preequilibrated
in lysis buffer. Briefly, connect the column with pre-equilibrated
Talon resin to a peristaltic pump and pump the supernatant from Step 1 through
the column with Talon resin at a flow rate of 4.0-5.0 ml/min. Every 30 min,
disconnect the column from the peristaltic pump and mix the resin to release
the excess pressure. After the loading, wash the column extensively with
approximately 100 ml of the lysis buffer to remove non-specifically bound
proteins.
4. Release the protein from the Talon resin by thrombin treatment. Briefly, resuspend
the Talon resin with bound smHDAC8-His fusion protein with the lysis
buffer and transfer it to a new sterile 15-mL Falcon tube. The volume of the resin
suspension should be approximately 5 ml. Add 60 µl of thrombin (1U/ µl) and
place the tube on a rolling mixer overnight at 4°C.
5. Next morning, separate the released protein from the Talon resin particles by
applying the resin suspension onto an Econo-Pac column and collect the
unbound flow-through fraction into a fresh sterile 15-mL Falcon tube. Wash the
resin with additional 3 ml of the lysis buffer to harvest all thrombin-released
protein. Check the presence and concentration of protein in the flow-through
fraction by the Bio-Rad Protein Assay.
6. Load the flow-through containing smHDAC8 enzyme from the Step 4 onto a 1mL
HiTrap Q FF column pre-equilibrated with low-salt ion-exchange
chromatography buffer (10 mM Tris-HCl pH=8.0; 50 mM KCl). Elute the bound
protein with a gradient of KCl (50 mM to 1 M KCl): see Figure 5A for a typical
ion-exchange purification of smHDAC8. Identify fractions containing the protein
by SDS-PAGE.
7. Pool the peak fractions from the ion-exchange chromatography from Step 5 and
load this sample onto a gel filtration column (16/60 Superdex 200) equilibrated
with gel filtration buffer. Identify fractions containing the target protein by SDSPAGE.
See Figure 5B for a typical gel filtration purification of smHDAC8.
34Molecular Biotechnology – Practice | Bi7430c
8. Pool the peak fractions from gel filtration from Step 6, and concentrate the
smHDAC8 protein with an Amicon Ultra centrifugal filter unit to reach a final
concentration of 2.5 mg/ml. Check purity of the purified smHDAC8 enzyme by
SDS-PAGE and determine protein concentration by the Bio-Rad Protein Assay
reagent. Flash-freeze the final product with liquid nitrogen and store at -80°C.
Figure 5. (A) Chromatogram of ion-exchange purification of smHDAC8 protein. The gradient used for
this purification step is displayed (blue broken line). (B) Chromatogram of gel filtration purification of
smHDAC8 protein.
Quality/quantity control of the purified protein
1. Protein concentration is measured on DeNovixR DS-11 Spectrophotometer (DeNovix Inc.,
USA) using the absorbance A280 mode, where molar extinction coefficient (ε) and molar
mass (Mr) are provided.
2. Proper protein folding is detected by circular dichroism (CD). CD spectra are recorded at
protein concentration 0.2 mg.mL-1 and at 20 °C using a spectropolarimeter Chirascan
(Applied Photophysics). Data are typically collected from 185 to 260 nm, at 100 nm/min, 1 s
response time and 1 nm bandwidth using a 0.1 cm quartz cuvette. Each spectrum shown is
the average of five individual scans and is corrected for absorbance caused by the buffer.
Collected CD data are expressed in terms of the mean residue ellipticity (ΘMRE) using the
equation
where Θοbs is the observed ellipticity in degrees, Mw is the protein molecular weight, n is
number of residues, l is the cell path length, c is the protein concentration (in mg/ml) and
the factor 100 originates from the conversion of the molecular weight to mg/mol.
35Molecular Biotechnology – Practice | Bi7430c
3. Dynamic light scattering (DLS): The dynamic light scattering (DLS) experiments
are conducted typically with protein solutions (1-2mg/ml) in a corresponding
buffer containing using instrument DynaPro NanoStar (Wyatt). Protein solutions
are centrifuged (13,000 rpm/10 min) prior to DLS measurement in order to
remove impurities. Before measurement temperature is equilibrated to 20°C.
4. Differential scanning fluorimetry (nanoDSF) measurements: Thermal stability
of recombinant proteins is analysed by a label-free differential scanning
fluorimetry (DSF) approach using a Prometheus NT.48 instrument (NanoTemper
Technologies). Briefly, the shift of intrinsic tryptophan fluorescence of proteins
upon gradual temperature-triggered unfolding (temperature gradient 2095°C)
is monitored by detecting the emission fluorescence at 330 and 350 nm. The
measurements is carried out in nanoDSF-grade high sensitivity glass capillaries
(NanoTemper Technologies) at a heating rate of 1°C/min. Protein melting points
(Tm) are inferred from the first derivative of the ratio of tryptophan emission
intensities at 330 and 350 nm.
V. LITERATURE
Rosano G. L. and Ceccarelli E. A. (2014) Recombinant protein expression in
Escherichia coli: advances and challenges. Frontiers in Microbiology. 5: 172.
Young C. L., Britton Z. T., Robinson A. S. (2012) Recombinant protein expression and
purification: a comprehensive review of affinity tags and microbial applications.
Biotechnology Journal. 7: 620-34.
Sivashanmugam A., Murray V., Cui C., Zhang Y., Wang J., Li Q. (2009) Practical
protocols for production of very high yields of recombinant proteins using
Escherichia coli. Protein Science: 18(5): 936-48.
Diebold M-L, Fribourg S, Koch M, Metzger T, Romier C. (2011) Deciphering correct
strategies for multiprotein complex assembly by co-expression: Application to
complexes as large as the histone octamer. Journal of Structural Biology 175(2),
178-188.
Formenti, L. R., Nørregaard, A., Bolic, A., Hernandez, D. Q., Hagemann, T., Heins, A.L.,
Larsson, H., Mears, L., Mauricio-Iglesias, M., Krühne, U., Gernaey, K. V. (2014)
Challenges in industrial fermentation technology research. Biotechnol. J., 9:
727–738.
Kuprijanov, A., Schaepe, S., Aehle, M., Simutis, R., Lübbert, A. (2012) Improving
cultivation processes for recombinant protein production. Bioprocess Biosyst.
Eng. 35: 333–340.
36Molecular Biotechnology – Practice | Bi7430c
VI. HOMEWORK
Your goal is to recombinantly produce of 2 g of highly pure haloalkane dehalogenase
DhaA enzyme from Desulfobacterium autotrophicum. See amino acid and
corresponding nucleotide sequences below:
>ACN15444.1 DhaA [Desulfobacterium autotrophicum HRM2]
MVTRDPAEQSRNIKSPGIRRKINGTMVGTKDFYEIYPFVPHFMTLDRHKLHYLDLGKGSPVVMVH
GNPTWSFYFRRLARDLSVNHRVIVPDHMGCGLSDKPSTRDYDYTLASRVRDLDRLIQSLDLGKKITL
VVHDWGGMIGCAWALRHLDRIDRIIITNTSGFHLPGAKRFPLRLWLIKYLPWFAIPGIQGLNLFAR
AALYMAPKQSLSTTVRQGLTAPYNSWKNRIATLKFVQDIPLSPRDKSYELVNWVDTHLEGLKTVP
MMILWGRHDFVFDLSFLDEWNKRFPHAQTHIFEDAGHYLFEDKPDETSNLIKKFIEEY
>CP001087.1:2679075-2680040 Desulfobacterium autotrophicum HRM2, complete
genome
ATGGTAACCAGGGATCCAGCGGAGCAAAGCAGAAACATCAAAAGTCCGGGCATCAGAAGAA
AGATCAACGGCACCATGGTCGGCACCAAGGATTTTTATGAAATATATCCCTTTGTTCCCCATTT
CATGACCCTGGACCGGCACAAACTCCACTACCTTGACCTGGGTAAGGGAAGTCCAGTTGTCAT
GGTCCACGGTAATCCCACCTGGTCGTTTTATTTTCGCAGGCTTGCCCGGGATCTTTCGGTGAAC
CACCGGGTCATTGTTCCCGACCACATGGGGTGCGGCCTGTCTGACAAGCCGTCCACCAGGGAT
TACGACTATACCCTTGCATCAAGGGTCCGGGACCTGGACCGTCTGATCCAGAGCCTTGACCTTG
GAAAAAAGATCACCCTGGTCGTCCACGACTGGGGCGGTATGATCGGCTGCGCCTGGGCCCTTC
GTCACCTGGACAGGATAGACAGGATCATCATCACCAACACCTCGGGGTTTCATCTTCCCGGGG
CAAAACGATTTCCCCTGCGGCTTTGGCTGATCAAATACCTTCCCTGGTTTGCCATTCCAGGGAT
TCAGGGCCTGAATCTCTTTGCCAGGGCAGCCCTTTACATGGCTCCGAAACAATCACTTTCAACA
ACGGTCAGGCAGGGGCTCACGGCACCCTACAACTCGTGGAAAAACAGGATCGCCACCCTCAA
ATTTGTCCAGGACATTCCCCTTTCACCCAGGGACAAAAGCTACGAACTTGTCAACTGGGTGGA
CACCCACCTTGAAGGTCTTAAAACCGTTCCCATGATGATCCTATGGGGCAGACACGATTTTGTG
TTTGATCTGTCGTTCCTTGACGAGTGGAACAAACGGTTTCCCCATGCCCAAACACATATTTTCG
AGGATGCAGGCCATTATCTGTTTGAGGACAAACCCGATGAAACATCAAATCTTATCAAAAAAT
TCATAGAGGAGTACTAA
37Molecular Biotechnology – Practice | Bi7430c
1. Select a suitable expression host (heterologous system) for the DhaA enzyme
overproduction and explain why the selected host is the best choice:
2. Propose and design a strategy for the DNA template synthesis, including primer
design:
3. Propose a cloning strategy – ligation-dependent versus ligation-independent
cloning, selection of expression vector, affinity/solubility tags etc.? How will you
check the error-free clones?
38Molecular Biotechnology – Practice | Bi7430c
4. Briefly describe production process – how will you introduce a foreign gene into
the host, from pre-culture to large-scale overproduction, inducible versus stable
expression, cytotoxicity issue, timing, harvesting strategy etc.
5. How will you determine the quality and yield of the purified enzyme?
6. How will you determine oligomeric state of the DhaA enzyme?
39Molecular Biotechnology – Practice | Bi7430c
PREPARATION AND TESTING OF MICROFLUIDIC CHIP
location: INBIT, Kamenice 34, ground floor, room 023
lecturer: Mgr. David Kovář, Ph.D. (kovard@mail.muni.cz)
I. WORKFLOW
– preparation microfluidic chip
– basic chip operation
– droplet microfluidics and microscopy
II. MOTIVATION
Microfluidics can be defined as the science and technology manipulating and analysing fluid
flow in sub-millimeter dimensions. It is becoming important technology for many emerging
applications and disciplines, especially in the fields of chemistry, biology and medicine.
Concrete application examples are biosensor devices for molecular diagnostics, polymerase
chain reaction chips, high-throughput screening, controlled drug delivery systems, drug
discovery methods, forensic analysis instruments, and so on (1).
III. THEORETICAL BACKGROUND
Formation of water in oil droplets in microfluidic chip has several benefits when compared
to standard high-throughput technology. Amongst such benefits belong low volume of
reagents consumed, chip modularity, low cost and simple fabrication. When all pros
combined properly one may encounter drop costs of screening million fold (2, 3).
In this practice students will put hands on microfluidic chip fabrication on their own. During
the lesson they will go through cutting out and assembling a chip applying oxygen plasma
bonding. Prepared chips are to be used for water in oil droplet generation (4). An example
will follow with encapsulation of single E.coli BL21 DE3 cells to droplets. Finally, there will be
observation of single cells in emulsions generated.
IV. DESIGN OF EXPERIMENT
Solutions and reagents:
– Dow Corning Sylgard® 184
– HFE-7500 oil
– Trichloro(1H,1H,2H,2H-perfluorooctyl)silane
– 1.5M NaCl
– deionized distilled H2O
40Molecular Biotechnology – Practice | Bi7430c
– E.coli BL21 DE3 or other bacterial cells
– Percoll ®
– isopropanol
Equipment:
– Chemyx Fusion 200 syringe pumps
– gas-tight syringes (various volumes)
– PTFE tubing
– razor
– biopsy puncher
– glass slide
– USB-microscope
Protocol for PDMS chip assembling:
1. using razor carefully cut PDMS with chip out of plate
2. with the help of ruler cut out single chip and place them bottom up
3. punch holes for inlet and outlet holes with biopsy puncher
4. clean glass slide and PDMS chip with isopropanol
5. place chip on the tray and put inside the oxygen plasma
6. run oxygen plasma for 3 minutes at 50% generator power at oxygen flow around 5scsm
at 200 mTorr
7. immediately after oxygen plasma process ends, bond microfluidic chip and glass cover
and press with your fingers; NOTE: try to avoid having bubbles between PDMS and
glass
8. place bonded chip on hot plate at 60°C for another 5 minutes
9. let chip cool down to room temperature
10. flush channels with 1 % silane solution in HFE-7500, after 3 minutes replace by pure
HFE-7500 oil and air
Droplet formation protocol:
1. load syringes with HFE-7500 oil and 150 mM NaCl, 25 % (v/v) Percoll and properly
diluted cells
2. attach PTFE tubing to the syringe and remove any bubbles
3. put syringes into the syringe pumps, lock tight and set proper syringe diameter
4. connect syringe via tubing into the chip
5. set liquid flow – 300 µL.h-1 for oil phase and 30 µL.h-1 for the aqueous phase
6. observe droplet formation under microscope at various magnification
7. verify cell occupation in emulsions on inverted microsope
41Molecular Biotechnology – Practice | Bi7430c
V. HOMEWORK
1. Estimate volume in pico-/femto- litres for monodisperse droplet formed at channel
having dimensions 5, 10 and 20 µm, respectively (assume square cross-section forms
spherical droplets of the same diameter).
2. For calculated droplet volumes estimate approximate cell density in x.10y per mL, to
put single cell per droplet. There is approximately 1.108 cells in medium with OD600 0.5.
Cultivated cells have OD600 4.8. In case that grown culture has insufficient density, calculate
the factor for thickening cell media to sufficient level. Account for 10 per cent pipetting
error.
VI. LITERATURE
1. Dittrich, P. S., and Manz, A. (2006) Lab-on-a-chip: microfluidics in drug discovery., Nat.
Rev. Drug Discov. 5, 210–8.
2. Mazutis, L., Gilbert, J., Ung, W. L., Weitz, D. a, Griffiths, A. D., and Heyman, J. a. (2013)
Single-cell analysis and sorting using droplet-based microfluidics., Nat. Protoc. 8, 870–
91.
3. Yin, H., and Marshall, D. (2012) Microfluidics for single cell analysis., Curr. Opin.
Biotechnol. 23, 110–9.
4. Teh, S.-Y., Lin, R., Hung, L.-H., and Lee, A. P. (2008) Droplet microfluidics., Lab Chip, The
Royal Society of Chemistry 8, 198–220.
42Molecular Biotechnology – Practice | Bi7430c
BIOCATALYTIC PREPARATION OF PHARMACEUTICAL PRECURSOR
(S)-2-BROMOPENTANE
location: Loschmidt Laboratories, Kamenice 5/A13, 2nd floor, room 227
lecturer: Assoc. Prof. Radka Chaloupková, Ph.D. (chaloupkova@enantis.com)
I. WORKFLOW
– preparation of reaction mixtures
– enantioselectivity measurements
– gas chromatography analysis
– calculation of enantiomeric excess and yield
II. THEORETICAL BACKGROUND AND MOTIVATION
Enzymatic enantioselectivity may be defined as the ability of an enzyme to distinguish
between two enantiomeric substrates. The discriminating capacity of the enzyme is
quantitatively measured by the ratio E of the corresponding specificity constants Vm/Km (or
kcat/Km). The enantioselectivity of enzymes has been exploited in organic synthesis, for
example in kinetic resolution of racemic mixtures as well as in the synthesis of chiral
building blocks from achiral precursors. Enzymatic kinetic resolution of racemic mixtures is
an effective tool for the preparation of enantiomerically enriched compounds which is a
continuous social demand due to the clinical advantages that enantiopure drugs offer over
the racemic forms. Haloalkane dehalogenase DbjA from Bradyrhizobium japonicum
USDA110 showed high enantioselectivity with β-substituted bromoalkanes. This enzyme is
selectively acting on (R)-enantiomer, allowing the preparation of enantiopure (S)enantiomer
using kinetic resolution.
III. DESIGN OF EXPERIMENT
Solutions and reagents:
– glycine buffer (100 mM, pH 8.6)
– (rac)-2-bromopentane
– haloalkane dehalogenase DbjA (c = 1.5 mg/ml)
– diethyl ether
43Molecular Biotechnology – Practice | Bi7430c
Equipments:
– 25-ml Reacti Flask with Mininert Valves
– automatic pipette 5 ml
– Hamilton syringe 1 ml
– test tubes
– glass vials with crimp caps
– Pasteur pipettes
– water shaking bath
– vortex mixer
– gas chromatograph equipped with a chiral column
Procedure:
1. Inject 10 μl of (rac)-2-bromopentane into 15 ml of glycine buffer in 25-ml Reacti Flask
closed by Mininert Valves.
2. Prepare the required number of test tubes with aliquots of diethyl ether (1 ml).
3. Incubate the reaction mixture in a water shaking bath at the room temperature
(approx. 20 °C) for 15 min.
4. Withdraw 1 ml from the reaction mixture and mix it with 1 ml of diethyl ether in the
test tube to stop the reaction (sample at 0 min).
5. Vortex the sample for 30 s for the extraction.
6. Start the reaction by adding 0.5 ml of the enzyme solution (c = 1.5 mg/ml).
7. Withdraw 1 ml of the reaction mixture at 5, 10, 15 and 20 min and mixed samples with
1 ml of diethyl ether in test tubes. Do not forget to vortex each sample for 30 s for the
extraction!
8. Transfer the ether (upper) part of the sample into a clean vial.
9. Analyse samples by gas chromatograph equipped with a chiral column.
IV. HOMEWORK
1. Calculate:
a. concentration of enzyme (mM) in the reaction mixture
b. theoretical concentration of substrate (mM) at the beginning of measurement
c. enantiomeric excess (%) and yield (%) of (S)-2-bromopentane.
2. Prepare protocol.
3. Read article related to the topic of practice. The article can be found in study materials.
44Molecular Biotechnology – Practice | Bi7430c
Protocol:
BIOCATALYTIC PREPARATION OF PHARMACEUTICAL PRECURSOR
(S)-2-BROMOPENTANE
Name:
Date:
MOTIVATION:
EXPERIMENTAL PART:
RESULTS AND CALCULATIONS:
DISCUSSION:
45Molecular Biotechnology – Practice | Bi7430c
V. LITERATURE
1. Faber, K. (2000). Biotransformation in Organic Chemistry, Springer-Verlag, Berlin.
2. Straathof, J. J., Jongejan, J. A. (1997). The enantiomeric ratio: origin, determination and
prediction. Enzyme and Microbial Technology 21: 559-571.
46Molecular Biotechnology – Practice | Bi7430c
PREPARATION AND TRANSFORMATION OF LIPOSOMES
location: Veterinary Research Institute, Hudcova 297/70, 2nd floor, Department of
Pharmacology and Immunotherapy
lecturer: RNDr. Jaroslav Turánek, CSc. (turanek@vri.cz)
I. WORKFLOW
– preparation of liposomes
– transformation: high-pressure extrusion, preparation of SUV (small unilamellar vesicles)
– analysis: DLS (dynamic light scattering) analysis, ZetaSizer
II. MOTIVATION
Liposomes are very common delivery system in medicine because of their biocompatibility,
non-toxicity, encapsulation of hydrophilic and hydrophobic compounds (e.g. drugs) and
specific transport to the target region/area. Composition and surface of liposomes could be
modified for better targeting, long circulation in the body or better stability. These
modifications are a great tool for designing the liposomes according to current needs. After
preparation a verification of the result is needed.
III. THEORETICAL BACKGROUND
Liposomes (closed bilayer phospholipid system) are known for more than 50 years and as
delivery system they have many advantages. Their diameter is in the range of few
nanometers to few micrometers. They can be unilamelar or multilamelar or even
multivesicular and the bilayer is hydrophobic inside and hydrophilic outside. The process of
assembling is not spontaneous like in micelles, it must be controlled and certain steps must
be done. There are many possibilities of liposome preparation and many form to prepare,
also there are some following steps to attached ligands to them or just make them more
stabilized. In the method of Lipid film hydration the first step is to dissolve lipids in
chloroform and this mixture evaporates by vacuum rotary evaporator. The resulting lipid
film is hydrated by water PBS or other solution/buffer. Ultrasound or other techniques can
help during hydration. At this point the liposomes are polydisperse and other process is
needed to make them monodisperse.
Sonication and filtration through polycarbonate filters with defined pore size adjusts
liposome diameter and lamilarity (extrusion). Also a method of freezing and unfreezing
(FTMVL) can be used. Detergent removal method is used for preparation of unilamelar
liposomes. When the detergent reaches its critical micelle concentration, it solubilizes lipids.
47Molecular Biotechnology – Practice | Bi7430c
Dynamic light scattering is used for determining the size of small particles (proteins,
polymers, micelles, carbohydrates, and nanoparticles) in suspension. It is also possible to
measure the concentration and the size of surface. The principle is based on measuring the
fluctuation of particles, when the smaller particle the bigger fluctuations.
TEM (transmission electron microscope) uses a beam for electrons transmitted through an
ultra-thin specimen/sample. The electrons are interacting with the specimen and the
resulting image is detected by a sensor/camera. TEM is able to detect and imagine at higher
resolution than light microscope.
Preparation:
1. Lipid film hydratation
2. Detergent removal method
3. Sonication, extrusion, FTMVL
Analysis: DLS (size and polydispersity, zetapotential)
IV. DESIGN OF EXPERIMENT
Solutions and reagents:
– Lipids: 1-octadecanoyl-2-hexadecanoyl-sn-glycero-3-phosphocholine (SPPC), L-αphosphatidylcholine
(EPC)
– Sodium cholate
– NaCl
– Chloroform
– PBS
– Liquid N2
Equipments:
– vacuum rotary evaporator
– Liposofast
– Sonicator
– ZetaSizer
A) Lipid film hydration:
1. weight out: 100mg PPC-SPC
2. hydratation: 10ml 0,9% NaCl for infusio, final concentration of lipid: 10mg/ml
48Molecular Biotechnology – Practice | Bi7430c
B) Detergent removal method
1. sodium cholate – dilution with amount up to critical micelle concentration (CMC)
2. dialysis
C) Sonication, extrusion, FTMVL
1. extrusion: filters 200nm (app. 15x)
2. sonication : total time 1 min, pulse mode: 10s pulse/10s pause, amplitude: 60%
3. FTMLV: frozen-thawed 2 times
D) DLS (Malvern) – size, size distribution, PDI
MLV, after extrusion, after sonication
V. LITERATURE
1. Avanti Lipids
http://www.avantilipids.com/index.php?option=com_content&view=article&id=1384&
Itemid=372
49Molecular Biotechnology – Practice | Bi7430c
ANALYSIS OF LIPOSOMES
location: Veterinary Research Institute, Hudcova 297/70, 2nd floor, Department of
Pharmacology and Immunotherapy
lecturer: RNDr. Jaroslav Turánek, CSc. (turanek@vri.cz)
RNDr. Jana Plocková (plockova@vri.cz)
MVDr. Pavel Kulich (kulich@vri.cz)
I. WORKFLOW
– single particle tracking analysis by using Nanosight 500– size, size distribution
– transmission electron microscopy (TEM) – size, morphology
II. MOTIVATION
Single particle tracking is a method for measuring the movement of every particle in a
solution in time and for its quantifying. With this it is possible to study intracellular transport
of these particles and their kinetics in real time. NanoSight instruments accurately and
rapidly sort liposomes in water according to their size requiring only small volumes and very
little sample preparation. The system enables individual liposomes in suspension to be
visualized and their Brownian motion tracked. It anables specific and general nanoparticle
tracking (presence, size distribution, concentration and fluorescence of all types of
nanoparticles from 10nm to 2000nm depending on the instrument configuration and sample
type). The particle size will determine the distribution of the particles within the body and
the concentration and size of the particles will govern the amount of drug delivered.
III. THEORETICAL BACKGROUND
Single particle tracking is used to quantify specific behaviour of particles, e.g. liposomes, in
colloid solution. The trajectory of every particle is measured and then the volume,
hydrodynamic diameter and frequency of the particles are evaluated. This method is used to
determine different kinds of particles in the mixture of variable materials.
The rate of movement is related only to the viscosity of the liquid, the temperature and size
of the particle and is not influenced by particle density or refractive index. Absolute
numbers of particles can be measured and the relative number of monomer versus
aggregated particles is calculated in real time.
50Molecular Biotechnology – Practice | Bi7430c
IV. DESIGN OF EXPERIMENT
Solutions and reagents:
– liposomes prepared in the previous exercise
Equipments:
– NanoSight 500
– TEM
A) NanoSight 500
Using the solution of lipids from previous exercise, dilution as needed
B) TEM
1. Preparation of specimen for TEM
2. Imaging
V. LITERATURE
1. NANOSIGHT: http://www.nanosight.com/technology/nanoparticle-tracking-analysis-nta
51Molecular Biotechnology – Practice | Bi7430c
PREPARATION OF ENZYMATIC BIOSENSOR
location: Loschmidt Laboratories, Kamenice 5/A13, 2nd floor
lecturer: Mgr. Šárka Nevolová, Ph.D. (77580@mail.muni.cz)
Ing. Markéta Zámečníková
I. WORKFLOW
– preparation of biosensor discs
– preparation of reaction mixtures
– biosensor measurements
– evaluation of biosensor response
– calculation of detection limit
II. MOTIVATION
The need for simple, rapid, cost-effective, and portable screening methods has boosted the
development of practical biosensors with applications in medical diagnostics, food safety,
process control and environmental monitoring. Compared to traditional analytical methods,
enzymatic bioanalytical devices have several distinct advantages such as high sensitivity and
specificity, portability, cost-effectiveness, and the possibilities for miniaturization and mass
production. Additionally, they can be developed for real-time and high-frequency testing
without extensive sample preparation.
III. THEORETICAL BACKGROUND
Enzymatic biosensors employ the affinity and selectivity of catalytically active proteins,
towards their target molecules. The transducer converts the effect created by the
interaction of enzyme with the analyte, usually into an electrical signal. Depending on the
assay type, two fundamental classes of enzymatic sensors can be distinguished. First, the
enzyme detects the presence of a substrate, or co-substrate/co-factor. This is then, by way
of a transducer, used to monitor the increase of enzymatic activity. The second group is
based on the detection of inhibitors in the presence of a substrate. With this system the
decrease of signal (caused by enzyme inhibition) is monitored. In this practice, optical
biosensor based on enzyme haloalkane dehalogenase and fluorescent pH indicator will be
introduced. Haloalkane dehalogenase catalyses conversion of halogenated hydrocarbons to
a halide ion, an alcohol and a proton, the last being responsible for the signal change of
fluorescence pH indicator. Described biosensor is useful for assessment of contamination at
a particular environmental site or for monitoring the concentration of known halogenated
contaminant.
52Molecular Biotechnology – Practice | Bi7430c
IV. DESIGN OF EXPERIMENT
Solutions and reagents:
– enzyme haloalkane dehalogenase (EC 3.8.1.5)
– bovine serum albumin
– fluorescence pH indicator 5(6)-carboxynaphthofluorescein
– substrate 1,2-dibromoethane
– 1 mM HEPES buffer, pH 9.0: 1 mM HEPES, 20 mM Na2SO4, 1 mM EDTA
– 50 mM phosphate buffer, pH 9.0: 400 mM K2HPO4, 90 mM KH2PO4
– 25% (v/v) glycerol
– 70% (v/v) glutaraldehyde
– mixture of methanol with 1,2-dichloroethane as an internal standard
Safety precautions:
Glutaraldehyde, 1,2-dichloroethane and 1,2-dibromoethane are toxic chemicals. 1,2dichloroethane
and 1,2-dibromoethane are possibly and probably carcinogenic to humans,
respectively. Therefore, these chemicals must be handled carefully under appropriate
safety conditions. A fume hood with good ventilation is dedicated to preparation of the
samples and biosensor measurements. The material is handled using appropriate personal
safety equipment, including laboratory coats, glasses and gloves. All halogenated waste
needs to be disposed separately.
Equipments:
– EnviroPen device with glass stick
– laboratory stand with a burette holder
– analytical balances
– pH meter with calibration solutions
– stirrer with stirring bar
– 10 ml vials, GC vials, vial crimper
– set of automatic pipettes
– glass discs
– Eppendorf tubes
– minishaker
– 10 microlitre syringe
– beaker
– graduated cylinder
– tweezer
– exsiccator
– computer with software FluorPen and Origin
53Molecular Biotechnology – Practice | Bi7430c
Protocol for preparation of biosensor and measurement:
1. Weigh 4 mg of lyophilised enzyme and 8 mg of pH indicator in Eppendorf tube for
preparation of enzymatic layer.
2. Weigh 4 mg of bovine serum albumin and 8 mg of pH indicator in Eppendorf tube for
preparation of reference layer.
3. Dissolve each mixture in 65 µl of 25% glycerol by careful mixing.
4. Wash the glass discs with ethanol and distilled water.
5. Dry the glass discs with paper wipe.
6. Prepare enzymatic and reference discs - apply 5 µl of the corresponding mixture on
each glass disc.
7. Expose the glass discs to vapour of 70% glutaraldehyde for 30 min.
8. Store the prepared glass discs in a Petri dish with 50 mM phosphate buffer, pH 9.0.
9. Put the glass disc with enzyme layer on the glass stick and attached into EnviroPen.
10. Switch on the device by pressing the SET key for 1 s.
11. Press SET key to measure Ft.
12. Press MENU key to scroll down into the Main menu.
13. Find Setting and adjust the F-fulse to 0.90 using SET key.
14. Press SET key to confirm the selection and MENU key to return to measurement.
15. Switch on the computer and run the program FluorPen.
16. Check that the program FluorPen and the EnviroPen device are properly paired.
17. Adjust the pH of 50 mM phosphate buffer to 9.0 using 1 mM NaOH if necessary.
18. Pipette 5 ml of this buffer into vial and add stirring bar.
19. Fix the EnviroPen device into the laboratory stand and immerse the tip into the vial
with phosphate buffer.
20. Record the baseline while stirring for 10 min.
21. Dissolve chemicals for preparation of 1 mM HEPES buffer in 1 l of distilled water.
22. Adjust its pH to 9.0 using 1 M NaOH or 1 M HCl.
23. Pipette 5 ml of this buffer into vial and add stirring bar.
24. Inject 1 µl of substrate 1,2-dibromoethane into HEPES buffer and shake the reaction
mixture on vortex (20 s).
25. Transfer the EnviroPen tip from phosphate buffer to HEPES buffer with substrate.
26. Record the enzymatic reaction with substrate for 2 min.
27. Save the measurement record using menu option File and Export in .txt format.
28. Remove the used glass disc.
29. Put a new glass disc with a reference layer on the glass stick.
30. Repeat the whole measurement procedure as described for disc with enzymatic layer.
31. Prepare sample for GC analysis – pipette 0.5 ml of methanol with 1,2-dichloroethane
into GC vial.
54Molecular Biotechnology – Practice | Bi7430c
32. Pipette 0.5 ml of 1 mM HEPES buffer with substrate to the methanol.
33. Cap the GC vial using vial crimper and shake the mixture on vortex (20 s).
Protocol for data analysis:
1. Copy the raw data into Excel file.
2. Plot the signal from enzymatic and reference layer in dependence on time of
measurement in minutes.
3. Calculate a slope from the 1 min linear signal of enzymatic and reference layer.
4. Calculate biosensor response by substraction the reference slope from the enzymatic
slope.
5. Plot the biosensor responses in dependence on substrate concentrations using
software Origin and provided biosensor data.
6. Determine detection limit of biosensor for 1,2-dibromoethane.
V. HOMEWORK
1. Calculate amount of chemicals used for preparation of 1 l of 1 mM HEPES buffer: 1 mM
HEPES (Mr 238.30 g.mol-1), 20 mM Na2SO4 (Mr 142.04 g.mol-1), 1 mM EDTA (Mr 292.24
g.mol-1)
2. What is the concentration of 1,2-dibromoethane (Mr 187.86 g.mol-1; ρ=2.18 g.cm-3) if 1
µl of this chemical is dissolved in 5 ml of HEPES buffer?
3. Determination of halogenated pollutants using biosensor utilising haloalkane
dehalogenase is based on measurement of:
A: enzyme inhibition
B: increase of product concentration
55Molecular Biotechnology – Practice | Bi7430c
4. Which immobilization methods are commonly used in development of biosensors?
What are their advantages and disadvantages?
5. What is the difference between selectivity and sensitivity of the biosensor?
VI. LITERATURE
1. Wencel D., Abel T., McDonagh C. (2014): Optical chemical pH sensors. Anal. Chem. 86:
15-29.
2. Long F., Zhu A., Shi H. (2013): Recent advances in optical biosensors for environmental
monitoring and early warning. Sens. 13: 13928-13948.
3. 6Sassolas A., Blum L.J., Leca-Bouvier B.D. (2012): Immobilization strategies to develop
enzymatic biosensors. Biotechnol. Adv. 30: 489-511.
56Molecular Biotechnology – Practice | Bi7430c
Protocol template
Practice XY
Name:
Date:
Motivation:
Experimental part:
Results:
Conclusions:
57Molecular Biotechnology – Practice | Bi7430c
3Molecular Biotechnology – Practice | Bi7430c
VI. Homework
1. Estimate volume in pico-/femto- litres for monodisperse droplet formed at channel
having dimensions 5, 10 and 20 µm, respectively (assume square cross-section forms
droplets of same diameter).
2. For calculated droplet volumes estimate approximate cell density in x.10y per mL, to
put single cell per droplet. There is approximately 1.108 cells in medium with OD600
0.5. Cultivated cells have OD600 4.8. In case that grown culture has insufficient density,
calculate the factor for thickening cell media to sufficient level. Account for 10 per cent
pipetting error.
V. Literature
1. Dittrich, P. S., and Manz, A. (2006) Lab-on-a-chip: microfluidics in drug discovery., Nat.
Rev. Drug Discov. 5, 210–8.
2. Mazutis, L., Gilbert, J., Ung, W. L., Weitz, D. a, Griffiths, A. D., and Heyman, J. a. (2013)
Single-cell analysis and sorting using droplet-based microfluidics., Nat. Protoc. 8, 870–
91.
3. Yin, H., and Marshall, D. (2012) Microfluidics for single cell analysis., Curr. Opin.
Biotechnol. 23, 110–9.
4. Teh, S.-Y., Lin, R., Hung, L.-H., and Lee, A. P. (2008) Droplet microfluidics., Lab Chip, The
Royal Society of Chemistry 8, 198–220.
Copyright © 2018 Loschmidt Laboratories. All rights reserved. No part of this publication may be reproduced,
stored in a retrieval system, transmitted in any form or by any means, electronic, mechanical, photocopying,
recording or otherwise, without the prior written permission of the Publisher.