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
fermentation of recombinant microorganisms 25
preparation of enzymatic biosensor 31
biocatalytic preparation of pharmaceutical precursor 36
biodegradation of environmental pollutant by recombinant bacterium 40
preparation and testing of microfluidic chip 45
preparation and transformation of liposomes 48
analysis of liposomes 51
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
PREPARATION AND TESTING OF MICROFLUIDIC CHIP
Lecturer: Mgr. Tomáš Buryška
Aims: fabrication of PDMS chip, preparation and microscopy of microdroplets,
microscopy of microdroplets with cells
FERMENTATION OF RECOMBINANT MICROORGANISMS
Lecturer: Mgr. Lukáš Chrást
Aims: fermentation of E. coli biomass and expression of recombinant protein,
technology EnBase
PREPARATION OF ENZYMATIC BIOSENSOR
Lecturer: Mgr. Šárka Bidmanová, Ph.D.
Aims: co-immobilization of recombinant enzyme and fluorescence probe to optical
transducer, detection of selected analyte
BIODEGRADATION OF ENVIRONMENTAL POLLUTANT BY RECOMBINANT
BACTERIUM
Lecturer: Mgr. Lukáš Chrást
Aims: engineered bacterium, synthetic metabolic pathway, biodegradation of
environmental pollutant, analysis of metabolites
BIOCATALYTIC PREPARATION OF PHARMACEUTICAL PRECURSOR
Lecturer: Mgr. Veronika Štěpánková, Ph.D.
Aims: biocatalytic synthesis of pharmaceutical precursor by using recombinant
enzyme
DESIGN OF RECOMBINANT SYSTEMS
Lecturer: Mgr. Andrea Schenkmayerová, Ph.D.
Aims: software tool Clone Manager, design of primers, restriction cleavage, cloning,
transformation
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
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
Doc. RNDr. Zbyněk Prokop, Ph.D.
EDUCATION
He obtained his Master´s degree in Environmental Science and
Ecotoxicology in 1998, Ph.D. degree in Environmental
Chemistry in 2001 and habilitation from Environmental
Chemistry in 2013 at the Masaryk University. Zbyněk Prokop
extended his expertise in enzyme kinetics and microfluidics
during his research stays at ETH Zurich in Switzerland,
University of Cambridge in United Kingdom, University of
Ghent in Belgium, University of Groningen in the Netherlands,
EMBL and University of Hannover in Germany.
PROFESSIONAL ASSIGNMENTS
Zbyněk Prokop works as an associate professor in the
Loschmidt Laboratories at Masaryk University were he leads a
team engaged in enzyme mechanism and kinetics. He received
the award for the Young Czech and Slovak Microbiologist of the
Year 2002 of the Czechoslovak Society for Microbiology, the
Excellence in Innovation Award of the Rector of Masaryk
University in 2005, the Alfred Bader Prize for Bioorganic
Chemistry of the Czech Chemical Society in 2005 and J. V.
Kostir Prize for Biochemistry of the Czech Society of
Biochemistry and Molecular Biology. He is co-author of 45
publications and three patents in the field of protein
engineering and industrial enzymology. He is founder of a
biotechnology spin-off company Enantis.
UČO 23696
zbynek@chemi.muni.cz
10Molecular Biotechnology – Practice | Bi7430c
Mgr. Šárka Bidmanová, 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 Bidmanová has been working as a researcher in
Loschmidt Laboratories at Masaryk University since 2009. 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 include immobilization and
characterization of enzymes and pH indicators. She is co-author
of eight publications and one book chapter. 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
bidmanova.s@seznam.cz
11Molecular Biotechnology – Practice | Bi7430c
Mgr. Veronika Štěpánková, Ph.D
EDUCATION
Veronika Štěpánková received her master’s degree in
Biochemistry in 2008 and a Ph.D. degree in the same field in
2013 from the Masaryk University in Brno. She has obtained an
international experience, theoretical knowledge and practical
skills during a four months research visit at the University of
Groningen in the Netherlands and several international
courses.
PROFESSIONAL ASSIGNMENTS
Veronika Štěpánková has been working as a researcher in
Loschmidt Laboratories at Masaryk University since 2008. Her
research interests are particularly related to the biocatalysis
using non-conventional media, such as organic solvents and
ionic liquids, and biocatalytical production of enantiopure
compounds. During her scientific carrier, she was author or coauthor
of eight publications, three book chapters and one
patent. She was awarded the Jean-Marie Lehn Prize for
talented young chemists (2nd place) in 2012. In 2013, she
received the Award of the Dean of the Faculty of Science of
Masaryk University and the Prize of the Czech Ministry of
Education.
UČO 106723
veronika@chemi.muni.cz
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
Mgr. Lukáš Chrást
EDUCATION
Lukáš Chrást received his master’s degree in Microbiology in
2012. He is currently studying for Ph.D. degree in Microbiology
at Masaryk University. He extended his experience with
fermentation and cultivation techniques during internship in
Contipro a.s., short stay at Institute of Microbiology of The
Czech Academy of Sciences and during his three month stay at
Institute for Biotechnology at Berlin Technical University in
2013. For the latter, he received Research Grant from the
Federation of European Microbiological Societies.
PROFESSIONAL ASSIGNMENTS
Lukáš Chrást started in Loschmidt Laboratories as Master
Student in 2010 and focused on characterization of haloalkane
dehalogenases from extremophilic microorganisms. His current
research interests are metabolic engineering of bacteria for
biodegradation of halogenated compounds and fermentation
technology. He is co-author of one research paper. He is
member of Czechoslovak Society for Microbiology and Czech
Biotechnology Society.
UČO 269981
lukchrast@gmail.com
14Molecular Biotechnology – Practice | Bi7430c
Mgr. Tomáš Buryška
EDUCATION
Tomáš Buryška received his master’s degree in Biochemistry in
2013. He is currently Ph.D. student of Microbiology at Masaryk
University in Brno. He gained substantial part of his knowledge
on a six month stay at the University of Cambridge in United
Kingdom and during shorter stays at the University of
Southampton and Korean Institute of Science and Technology
in Germany.
PROFESSIONAL ASSIGNMENTS
Tomáš Buryška has been working in the Loschmidt laboratories
at Masaryk University since late 2009. His specialization lies in
the high-throughput methods, microfluidics, protein
engineering and enzyme kinetics. He gained experience in
projects focused on identification of novel enzyme substrates
and inhibitors identified by in sillico methods. Since 2011 he
intensively focuses on application of microfluidics in life
sciences. Tomáš Buryška is a co-author of four research articles
and one review article.
UČO 323660
bary@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
PREPARATION AND TESTING OF MICROFLUIDIC CHIP
location: INBIT, Kamenice 34, ground floor, room 023
lecturer: Mgr. Tomáš Buryška (bary@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
17Molecular 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
18Molecular 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.
19Molecular Biotechnology – Practice | Bi7430c
FERMENTATION OF RECOMBINANT MICROORGANISMS
location: Loschmidt Laboratories, Kamenice 5/A13, 2nd floor, room 206
lecturer: Mgr. Lukáš Chrást (lukchrast@gmail.com)
I. WORKFLOW
– assembly of fermentation vessel
– calibration of probes
– setting up the fermentation
– sampling of cells from the fermentor
– downstream processes
II. MOTIVATION
Bioreactors (also known as fermentors or fermenters) are devices for cultivation of
microorganisms playing important role in research and especially production of
recombinant proteins or biomass in food and pharmaceutical industry. They can be
operated under batch, fed-batch or continuous conditions, while fed-batch cultures are the
most abundant in industrial applications. With high cell density cultures, great yields of
recombinant proteins can be achieved, which can lower the expenses and therefore also
the prices of final products.
III. THEORETICAL BACKGROUND
Bioreactor operation is very complex issue where several factors play 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. During the second
half of the practice, they will aseptically take culture samples from the fermentor, evaluate
cell density of the culture and perform the spread plate method for determination of the
number of living cells in the culture. At the end, downstream processes such as membrane
filtration and centrifugation will be applied to harvest cultured cells. The students will also
learn how to determine wet and dry cell weight as important indicators of the state of the
culture.
20Molecular Biotechnology – Practice | Bi7430c
IV. DESIGN OF EXPERIMENT
Solution and reagents:
– Escherichia coli BL21(DE3)
– Luria-Bertani broth
– Plate Count Agar
– Struktol SB2020
– 10 % citric acid
– 10 % NaOH
– lactose
– PBS buffer
– 10% ajatin solution
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)
– 10 mL syringes
– aluminum foil
– glass spreaders
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.
21Molecular Biotechnology – Practice | Bi7430c
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 alufoil.
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.
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. Ca. 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.
22Molecular Biotechnology – Practice | Bi7430c
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.
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. 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. Resuspend 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.
23Molecular Biotechnology – Practice | Bi7430c
V. HOMEWORK
1. Describe the difference between batch and fed-batch culture.
2. What does CFU/mL means? What method can be used to get these data?
3. Describe growth curve of bacterial culture. Label the phases and axes.
4. What are advantages and disadvantages of spectroscopic methods for determination
of growth parameters of bacterial cultures?
24Molecular Biotechnology – Practice | Bi7430c
VI. LITERATURE
1. 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.
2. Junker, B. H. (2004) Scale-up methodologies for Escherichia coli and yeast fermentation
processes. J. Biosci. Bioeng. 97: 347–364.
3. Kuprijanov, A., Schaepe, S., Aehle, M., Simutis, R., Lübbert, A. (2012) Improving
cultivation processes for recombinant protein production. Bioprocess Biosyst. Eng. 35:
333–340.
4. Hoskinsson, P. A., Hobbs, G. (2005) Continuous culture – making a comeback?
Microbiology. 151: 3153–3159.
25Molecular Biotechnology – Practice | Bi7430c
PREPARATION OF ENZYMATIC BIOSENSOR
location: Enantis, s.r.o., INBIT, Kamenice 34 (4th floor, room 4.24)
lecturer: Mgr. Šárka Bidmanová, Ph.D. (bidmanova.s@seznam.cz)
Ing. Markéta Kotlánová
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.
26Molecular 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
27Molecular 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.
28Molecular 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
29Molecular 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.
30Molecular Biotechnology – Practice | Bi7430c
BIODEGRADATION OF ENVIRONMENTAL POLLUTANT BY RECOMBINANT
BACTERIUM
location: Loschmidt Laboratories, Kamenice 5/A13, 2nd floor, rooms 208, 209, 227
lecturer: Mgr. Lukáš Chrást (lukchrast@gmail.com)
I. WORKFLOW
– whole-cell conversion of 1,2,3-trichloropropane to glycerol by recombinant
Pseudomonas putida KT2440 with synthetic biodegradation pathway
– detection and quantification of produced glycerol using enzymatic kit
– analysis of samples from the reaction by gas chromatography (GC)
II. MOTIVATION
Bacteria and other microorganisms have great potential for biodegradation of polluting
compounds (1). However, in case of some anthropogenic chemicals recently introduced into
the environment, natural catabolic pathways in bacteria are not yet evolved toward their
efficient degradation. Protein and metabolic engineering techniques can be applied in order
to assembly synthetic metabolic pathways that might catabolize problematic substrates.
III. THEORETICAL BACKGROUND
In this practice, students will get the opportunity to work with recombinant bacterium
Pseudomonas putida KT2440 carrying synthetic metabolic pathway for biodegradation of
important environmental pollutant of anthropogenic origin 1,2,3-trichloropropane (TCP;
2,3). The pathway consists of three enzymes: haloalkane dehalogenase DhaA from
Rhodococcus rhodochrous NCIMB 13064, and haloalcohol dehalogenase HheC and epoxide
hydrolase EchA from Agrobacterium radiobacter AD1 (Figure 1). The activity of DhaA with
TCP was improved 32-times by protein engineering resulting in mutant DhaA31 that is
currently used in the pathway instead of wild-type enzyme (4). The whole pathway was
assembled in form of synthetic operon and introduced on plasmid into the heterologous
host P. putida KT2440. In the first part of the Practice, students will use cells of P. putida
with expressed enzymes of the synthetic pathway to convert TCP to glycerol, a final product
that accumulates in the cells during the initial phase of reaction. In the second part of the
Practice, students will analyse samples from the reaction mixture and quantify TCP, glycerol
and two other metabolites from TCP pathway, that accumulate during reaction, using
enzymatic kit and GC.
31Molecular Biotechnology – Practice | Bi7430c
Figure 1. Scheme of the five-step synthetic metabolic pathway for biotransformation of
1,2,3-trichloropropane to glycerol. Used abbreviations of individual metabolites are shown.
DhaA, haloalkane dehalogenase from Rhodococcus rhodochrous NCIMB 13064; HheC,
haloalcohol dehalogenase; and EchA, epoxide hydrolase, both from Agrobacterium
radiobacter AD1. The final product glycerol can be utilized in glycolysis when the pathway is
assembled in vivo.
IV. DESIGN OF EXPERIMENT
Solutions and reagents:
– substrate 1,2,3-trichloropropane (Mr 147.43 g.mol-1, ρ=1.39 g.cm-3)
– sodium phosphate buffer (pH 7.0)
– quenching acetone with internal standard hexan-1-ol
– cell suspension of recombinant P. putida KT2440
– Free Glycerol Assay Kit (Biovision, USA) – buffer, enzyme mix, glycerol probe
Equipment:
– gas-tight glass vials with with a screw cap mininert valve
– Hamilton syringe (1 ml) and pipettes
– glass vials (1 ml) with silicon septa caps for GC and cramping pliers
– Eppendorf tubes (1.5 ml)
– 96-well microtiter plates
– shaking water bath
– thermoblock
– microcentrifuge
– vortex
– UV-VIS spectrophotometer
– GC with flame ionisation detector and FFAP column
32Molecular Biotechnology – Practice | Bi7430c
Protocol for whole-cell conversion of TCP (work in couples):
1. use 5 ml pipette and mix 5 ml of cell suspension with 5 ml of pre-incubated phosphate
buffer with 2 mM TCP in gas-tight glass vials
2. start counting the time of reaction and incubate vials with shaking (150 RPM) in
waterbath at 30°C
3. using Hamilton syringe, quickly withdraw 0.5 ml sample of reaction mixture and mix it
with 0.5 ml of acetone in prepared Eppendorf tube, vortex for 10 s
4. withdraw 0.1 ml sample of the reaction mixture and boil it in Eppendorf tube for 10
min at 95°C in thermoblock
5. centrifuge both samples at 13000 RPM for 90s
6. transfer supernatant from sample with aceton to GC vial using pipette, close the vial
with cap using cramping pliers
7. transfer the supernatant from the boiled sample to the new Eppendorf tube, keep the
tube and GC vial for further analysis
8. repeat the procedure of sample collection every 10 min for 30 min, sign samples 0, 10,
20, and 30 min
Protocol for detection and quantification of produced glycerol:
1. use boiled 0.1 ml samples collected at times 0, 10, 20 and 30 min
2. pipette 50 μl of sample from 0 min into one well of the microtiter plate
3. pipette 25 μl of each of samples from 10 – 30 min at three separate wells and mix with
25 μl of kit buffer
4. add 50 μl of glycerol detecting pre-mix (2 μl of glycerol probe, 2 μl of enzyme mix, 46 μl
of kit buffer) to each of four wells
5. incubate plate for 30 min in dark place at room temperature
6. analyse absorbance at 570 nm using UV-VIS spectrophotometer
7. use value from time 0 min as a blank and calculate concentration of glycerol in samples
from 10, 20 and 30 min of the reaction, use slope from Calibration curve 1 for
calculations (do not forget to include diluting factor)
33Molecular Biotechnology – Practice | Bi7430c
Quantification of TCP and other metabolites from synthetic pathway by GC:
1. samples quenched in acetone will be analysed by GC with help of instructor
2. one sample will be analysed as a demonstration and raw data (peak areas of individual
metabolites at certain time intervals) will be sent by instructor after the practice
3. calculate concentrations (mM) of TCP, 2,3-dichloropropan-1-ol (DCP) and glycidol (GDL)
in reaction mixture using the slopes of calibration curves you prepared in advance
(Homework 2) and raw data obtained from instructor, use internal standard to correct
your data, do not forget to include diluting factor
4. write final concentrations of TCP, DCP and GDL in Table 1, check whether the sum of
concentrations is always close to 2 mM
V. HOMEWORK
1. Calculate concentrations of metabolites from TCP pathway in reaction mixture.
Table 1. Concentrations of metabolites from TCP pathway in reaction mixture.
2. Calculate how much of TCP (μl) has to be added to 5 ml of phosphate buffer to achieve
final concentration of TCP in reaction mixture with recombinant cells equal to 2 mM.
Remember, that 5 ml of phosphate buffer with TCP will be mixed with 5 ml of cell
suspension to start the biodegradation reaction.
Result: μl
3. Use Microsoft Excel or alternative software. Prepare calibration curves (dependence of
peak area on concentration of compound) for TCP and two metabolites that accumulate in
the synthetic pathway during the biodegradation reaction. Use raw data from Table 2.
Calculate a slope for each of three calibration curves.
34Molecular Biotechnology – Practice | Bi7430c
time TCP DCP GDL glycerol SUM
0
10
20
30
Table 2. Peak areas of TCP, DCP and GDL determined for varying concentrations of
compounds by GC.
Glue the final graph with three calibration curves and corresponding slopes here:
VI. LITERATURE
1. Copley, S. D. (2009) Evolution of efficient pathways for degradation of anthropogenic
chemicals. Nat. Chem. Biol. 5, 559–66.
2. Bosma, T., Damborsky, J., Stucki, G., and Janssen, D. B. (2002) Biodegradation of 1,2,3trichloropropane
through directed evolution and heterologous expression of a
haloalkane dehalogenase. Appl. Environ. Microbiol. 68, 3582–7.
3. Kurumbang, N. P., Dvorak, P., Bendl, J., Brezovsky, J., Prokop, Z., and Damborsky, J.
(2014) Computer-assisted engineering of the synthetic pathway for biodegradation of a
toxic persistent pollutant. ACS Synth. Biol. 3, 172–81.
4. Pavlova, M., Klvana, M., Prokop, Z., Chaloupkova, R., Otyepka, M., Wade, R. C., Tsuda,
M., Nagata, Y., Damborsky, J. (2009) Redesigning dehalogenase access tunnels as a
strategy for degrading an anthropogenic substrate. Nat. Chem. Biol. 5, 727–33.
35Molecular Biotechnology – Practice | Bi7430c
conc. (mM) TCP DCP GDL
1.250 86.0 87.0 37.0
0.625 45.0 44.0 19.5
0.313 22.0 22.0 11.0
0.156 11.0 11.0 5.4
0.078 5.6 5.6 2.8
0.039 3.0 2.8 1.3
0.020 1.6 1.5 0.7
BIOCATALYTIC PREPARATION OF PHARMACEUTICAL PRECURSOR
(S)-2-BROMOPENTANE
location: Enantis, s.r.o., INBIT, Kamenice 34, 4th floor, room 4.24
lecturer: Mgr. Veronika Štěpánková, Ph.D. (veronika@chemi.muni.cz)
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
36Molecular 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.
37Molecular Biotechnology – Practice | Bi7430c
Protocol:
BIOCATALYTIC PREPARATION OF PHARMACEUTICAL PRECURSOR
(S)-2-BROMOPENTANE
Name:
Date:
MOTIVATION:
EXPERIMENTAL PART:
RESULTS AND CALCULATIONS:
DISCUSSION:
38Molecular 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.
39Molecular Biotechnology – Practice | Bi7430c
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).
40Molecular 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].
41Molecular 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.
42Molecular 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
43Molecular 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, …
44Molecular 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:
45Molecular 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?
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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
47Molecular 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.
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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
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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
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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.
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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
52Molecular Biotechnology – Practice | Bi7430c
Protocol template
Practice XY
Name:
Date:
Motivation:
Experimental part:
Results:
Conclusions:
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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.
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