Gene technologies1
GENE TECHNOLOGIES
Model organisms:
Model organisms used in biotechnology - bacteria (E. coli), yeasts (Pichia, Saccharomyces) and fungi (Penicillium),
Caenorhabditis elegans (nematode), Drosophila melanogaster, Danio rerio (Zebra fish), house mouse, animal cell cultures,
Arabidopsis thaliana, viruses (bacteriophages, retroviruses).
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Model Organisms
̶ DNA is found in all living organisms and viruses
̶ Only a fraction of so-called model organisms are studied in detail
̶ In model organisms, we now know the complete genome
̶ We use model organisms:
- as a model for studying similar organisms
- in a wide range of biotechnological processes
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Bacteria
̶ Master of model organisms
̶ Makes up approx. 50% of all living organisms (5 x 1030)
̶ Ability to survive in extreme conditions -temperature (Thermus
aquaticus), pH (Acidothiobacillus)
̶ Escherichia coli is the most commonly used:
- Gram-negative rod
- has about 10 flagella and thousands of pili on its surface
- most strains are harmless
- E. coli O157:H7 - two toxins responsible for bloody diarrhea
Clark and Pazdernik, 2016
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Bacteria
Clark and Pazdernik, 2016
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E. coli
̶ Rapid growth of culture
̶ Can only grow in a medium containing mineral salts
and sugar
̶ Liquid culture will last for weeks in the refrigerator
̶ Can be frozen at -70°C for up to 20 years
̶ Can grow under both aerobic and anaerobic conditions
̶ Has one circular chromosome containing about 4000
genes
Clark and Pazdernik, 2016
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Plasmids
̶ Survival strategy requires cooperation with other
organisms
̶ A number of bacteria secrete toxins called bacteriocins
̶ E. coli produces so-called colicins (E1, M) - perforation
of the plasma membrane, DNA/RNA degradation
̶ The bacteria's immune proteins neutralise the effect of
the toxins
̶ The ability to produce colicins is due to the presence of
plasmids (ori site)
̶ These plasmids have been modified for biotechnological
purposes
Clark and Pazdernik, 2016
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Plasmid pET28
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̶ Bacillus subtillis - production of proteases
and amylases
̶ Pseudomonas putida – the ability to
degrade a range of aromatic compounds
̶ Streptomyces coelicolor - degrades
cellulose and chitin, production of a range
of antibiotics (Clorobiocin,
Undecylprodigiosin, Actinorhodin)
̶ Corynebacterium glutamicum - production
of L-glutamate and L-lysine
̶ Streptococcus zooepidemicus - production
of hyaluronic acid
Bacteria in Biotechnology
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Eukaryots
̶ The entire line of eukaryotes is diploid (two copies of each
chromosome)
̶ In contrast, a whole range of plants are polyploid (wheat =
hexaploid, tomato = tetraploid)
̶ In animals, there is a difference in germ and somatic cells
- diploid germ lines give rise to haploid gametes (eggs and
sperm)
- somatic cells are diploid
- somatic mutations are transmitted within the organism
- somatic mutations are not transmitted to offspring
̶ In most plants, cells are totipotent
̶ In animals, only stem cells carry this property
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iPSC (induced Pluripotent Stem Cell)
Abbar et al., 2020
̶ Method first described in Takahashi and Yamanaka (2006) for induction of iPSCs from fibroblasts
̶ Requires the expression of 4 transcription factors - octamer-binding transcription factor 3/4 (Oct3/4), SRY
(sex determining region Y)-box 2 (Sox2), Krüppel-like factor 4 (Klf4) and cellular-Myelocytomatosis (cMyc)
(OSKM).
Gene technologies
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Somatic mutations
Clark and Pazdernik, 2016
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Yeasts and Fungi
̶ Fungi are traditionally used in biotechnology - Penicillium roqueforti
(Roquefort), P. candidum, caseicolum and camembertri (Camembert),
Aspergillus oryzae (soy sauce), Penicillium notatum (Penicillin),
Aspergillus niger (citric acid)
̶ Usually cultivated in bioreactors
̶ Yeasts have the advantages of both bacteria and eukaryotes
̶ The most commonly used yeast is Saccharomyces cerevisiae
̶ The yeast genome is separated by a nuclear membrane
̶ S. cerevisiae has 16 chromosomes containing telomeres and
centromeres
̶ Some yeasts have extrachromosomal elements, the so-called
2.micron circle.
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Yeasts
̶ Yeasts multiply by budding
̶ Budding produces identical cells - division
by mitosis
̶ Yeasts have diploid and haploid phases
within the life cycle
̶ Under critical conditions, yeast undergo
meiosis - formation of haploid spores,
called ascospores in the ascus)
̶ Under favorable conditions, spores
germinate and conjugate to form diploid
cells
̶ In yeast, conjugation can only occur
between two different mating types (a, a) Clark and Pazdernik, 2016
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Pichia pastoris
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Caenorhabditis elegans
̶ Small nematode (nematodes) living in soil with mainly root vegetables
̶ It has two sexes - 99.9% hermaphrodite (self-fertilizing) and 0.1% male
̶ Body consists of a simple tube covered with a cuticle
̶ Inside the body - 959 somatic cells including about 300 neurons
̶ The head has a variety of sensory organs (taste, smell, temperature,
touch)
̶ Body is translucent = easy to use fluorescence techniques, generation
cycle 3 days
̶ RNA interference performed for the first time - ideal tool for reverse
genetics
̶ First known complete genome of a multicellular organism (100 Mbp)
https://www.hsph.harvard.edu/mair-lab/c-elegans/
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Drosophila melanogaster (fruit fly)
̶ A widely consumed organism in genetic studies
̶ Easy to grow, 2-week life cycle
̶ Egg hatches into a larva (24h), several larval stages
after adult
̶ Many mutants available - identification of genes
involved in development (homology with humans)
̶ Genome is 165 Mb - 3 pairs of autosomal and X/Y
chromosomes
̶ Polytene chromosomes during rapid larval
development
Clark and Pazdernik, 2016
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Danio rerio (Zebra fish)
̶ A simple model vertebrate used in molecular biology
̶ Easy to grow and propagate in aquaria, availability of a wide
range of mutants
̶ Embryonic development outside the mother's body, development
from a single cell to an individual takes 24 hours
̶ Embryo is translucent - easy to monitor the effect of mutations on
development
̶ Genome contains 25 pairs of chromosomes (1700 Mb), 70% of
protein-coding genes in humans have orthologs in Danio
̶ Model for studying a range of human diseases
̶ Embryos are often used for screening new drugs
https://theconversation.com/animals-in-research-zebrafish-13804
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Arabidopsis thaliana
̶ The most widely used model organism in plant genetics
and molecular biology
̶ Similar response to stress factors and diseases as
economic crops
̶ Many of the genes responsible for development and
reproduction are identical to those of economic crops
̶ Easy to grow, space-saving, generation time 6-10
weeks, many seeds
̶ Can be maintained in a haploid state
̶ Small genome - five chromosomes (125 Mb), 25 000
genes
- Rice (430 Mb), 40-50 thousand genes
- wheat (17 Gb), tomato (950 Mb), tobacco (4.5 Gb)
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Viruses
̶ Entities at the edge of the definition of life, pathogens attacking host cells
̶ Consists of a protein envelope called a capsid that encases the DNA/RNA
genome
̶ Found in all living organisms (bacteria, plants, animals)
̶ Bacterial viruses = bacteriophages (phages)
- attach to the host
- entry of the viral genome
- replication of the viral genome
- production of new viral proteins
- assembly of a new viral particle
- release of virions from the host
̶ Many viruses go through a latent phase - lysogeny in bacteria
̶ Integration of the virion into the host genome often occurs - provirus
(prophage) formation
Clark and Pazdernik, 2016
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Viruses
̶ We can divide based on the shape of the
capsid (spherical, complex, fibrous)
̶ Complex = bacteriophages (T4, P1, Mu)
̶ ssRNA viruses have a positive (+) or
negative (-) genome
̶ Retroviruses contain reverse transcriptase
(transcription of RNA to DNA), integrate
into the genome using long terminal
repeats (LTRs)
Clark and Pazdernik, 2016
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The Life Cycle of RNA Viruses
Mechanism of SARS-CoV-2 (+RNA) replication Mechanisms of retroviruses replication
Dubois et al. 2018
V’kovski et al. 2021
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GENE TECHNOLOGIES
Manipulation of DNA, RNA, and proteins
Cell fractionation, isolation of proteins and nucleic acids.
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Isolation of DNA and RNA
̶ Different types of samples = different strategies
• blood, brain tissue, heart tissue,
liver tissue
• stool, urine, swabs from the
urethra, throat, vagina, rectum,
conjunctiva, cerebrospinal fluid
• seeds, leaves, roots,
wood
• gram positive and negative
bacteria, yeasts, fungi
• food (cheese, meat, egg, milk)
• soil, water, manure
Plants tissues Animal and human tissues Bacteria and environmental samples
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Desintegration of sample
̶ Soft animal tissues - lysis at 50-60°C by Proteinase K
̶ Proteinase K
- digests preferentially after hydrophobic amino acids
- active in a wide range of temperatures (20 and 60°C ), pH and buffers
- activity is stimulated when up to 2% SDS or up to 4 M urea are included in the reaction
̶ Solid animal tissues and plant tissues - must be crushed mechanically
̶ Microorganisms - grinding with sea sand or garnet beads, lysozyme (G+)
̶ Mechanical grinding
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Mechanical grinding
̶ Liquid nitrogen and mortar and pestle
̶ Retsch mill, Precellys, Cryomill
̶ Garnet beads
https://www.youtube.com/watch?v=Z8UvIQXRJFY
https://www.youtube.com/watch?v=k6mPWPuR8PY
https://youtu.be/OwoUAO7vaJA?list=TLGGIXBeSy4AvBcyODA5MjAyMg
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Lysis buffer
̶ The goal of lysis buffer is to suppress the activity of nucleic acid-degrading
enzymes and to separate proteins from nucleic acids
̶ EDTA – chelating of Mg2+ ions = inhibition of nucleases
̶ RNAsin – inhibitor of RNAses
̶ Detergents - sodium and lithium salts of lauryl sulfate or Triton X-100 and
Tween20 - nuclease inhibitors and at the same time release the nucleic acid
from its binding to the proteins/histones
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Deproteinization
̶ Phenol - one of the most effective denaturing agents, but phenol can
degrade nucleic acids with repeated use.
̶ Chloroform mixed with isoamyl alcohol – effectively denatures proteins
(chloroform denatures proteins and isoamyl alcohol reduces foaming)
̶ Guanidine hydrochloride – breaks the structure of proteins and biologically
inactivates them. It can be used to isolate both DNA and RNA.
̶ Sodium perchlorate – removes detergents from extraction solutions by
forming their complexes with proteins
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Removing of saccharides
̶ Cetrimonium bromide (CTAB) - can be used to precipitate DNA and RNA,
while the saccharide remains in the liquid.
̶ Tetraethylammonium bromide (TEAB) –isolation of RNA from the
saccharide of a 50% ethanol solution of TEAB. The saccharides precipitate
and the RNA remains in the liquid. The saccharides are removed by
centrifugation.
̶ 2.5 M LiCl - LiCl precipitation is useful following RNA isolation or in vitro
transcription, because RNA is efficiently precipitated, while protein,
carbohydrates, and DNA are very inefficiently precipitated or are not
precipitated at all
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Phenol-chloroform isolation of NA
̶ The phenol-chloroform extraction method is most often used to isolate NA from plant tissues
or enviromental samples and large amounts of DNA from blood.
̶ A mixture of phenol, chloroform and isoamyl alcohol is added to the sample.
̶ TriReagent, TRIZOL - A mixture of phenol, chloroform and GuHCl
̶ Chloroform does not mix with the aqueous solution of the cell lysate, so the mixture is divided
into two phases - upper aqueous and lower chloroform. By shaking, the phases are mixed,
during which the phenol precipitates the proteins present in the aqueous lysate.
̶ Using of acidic phenol (pH≈4) – isolation of RNA to upper aqueous phase/DNA in interphase
̶ Using of basic phenol (pH≈8) – isolation of DNA to upper aqueous phase
̶ DNA/RNA is precipitated from aqueous phase by isopropanol
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Phenol-chloroform isolation of NA
Acidic phenol
Basic phenol
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NA precipitation
̶ Precipitation of RNA and DNA can be facilitated by addition of co-precipitant
– Glycogen, GlycoBlue
GlycoBlue - dye covalently linked to glycogen, a branched
chain carbohydrate, which is useful as a nucleic acid
coprecipitant.
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Isolation of NA using commercial kits
̶ Types of isolation techniques used by commercial kits:
- resins - bind DNA specifically
- membranes (filters)
- silica columns – specific binding of nucleic acids
- paramagnetic particles with a differently modified surface
silica columns membranesparamagnetic particles
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Silica columns
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Paramagnetic particles (MPs)
̶ One of the methods of isolation of nucleic acids, which has become more
widespread
̶ MPs are particles with a size of 5 nm–100 μm formed from a metal core, which is
most often gamma-Fe2O3 (maghemite) or Fe3O4 (magnetite).
̶ The core is covered by a layer that has a prepared specific surface. This can be
adjusted according to which molecules we want to isolate from the given material.
̶ The size of MPs itself can be adjusted according to what we are isolating: 5-50 nm
proteins; 20 –450 nm nucleic acids, viruses; 10–100 μm cells.
̶ The principle of isolation is based on the physico-chemical properties of MPs.
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Paramagnetic particles
Binding of DNA fragments depends
on the concentration of ethanol
https://www.beckman.com/resources/technologies/spri-beads?wvideo=kh244puadj
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Purification of DNA from RNA
̶ For some applications it is necessary to have RNA without DNA
contamination
̶ Precipitation of DNA with 1/10 volume of isopropyl alcohol - DNA precipitates
and RNA remains in solution; however, the method is not 100%
̶ Treatment of sample with DNAse I (RapidOut DNA removal kit)
- DNase I binds to Inhibition reagent (beads)
- special DNase I with lower Km
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Quantification and Purity
̶ Measure of concentration and purity by spectrophotometer (NanoDrop)
- RNA: A260/280 = 2.0, A260/230 >1.5, e = 40 (μg/mL)-1cm-1
- DNA: A260/280 = 1.8, A260/230 >1.5, e = 50 (μg/mL)-1cm-1
̶ Measure of concentration by Qubit (fluorometry)
̶ Measure of RNA integrity by Fragment Analyzer or TapeStation (Electrophoresis)
- RIN (RNA integrity number) > 7
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Fragment Analyzer (RIN)
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Protein isolation
̶ RIPA buffer (from tissue cultures) - 30mM HEPES, pH 7.4,150 mM NaCl, 1%
Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 5mM EDTA, 1mM
NaV04, 50mM NaF, 1mM PMSF, 10% pepstatin A, 10 μg/ml leupeptin, and 10 μg/ml aprotinin
̶ Homogenization in SDT buffer - 4% SDS, 0.1M DTT, 0.1M Tris-HCl pH=7.6
̶ Homogenization in Urea buffer - 9M Urea, 20mM HEPES pH 8.0
The hydrogen bond interaction between urea and the peptide groups
opens the entrance for water and contributes to the unfolding
denaturation of protein.
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Proteins quantification
̶ Bradford assay (A595) - interferuje SDS
̶ Bicinchoninic assay (BCA) (A562)
- strong interference –SH group and EDTA
- no interference with SDS (up to 5%)
̶ Folin assay (A750)
̶ Measurement of Trp fluorescence (280/350 nm)
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Manipulation of DNA, RNA and proteins
PCR techniques. DNA sequencing, high-throughput sequencing methods
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Chemical synthesis of DNA
̶ H. Gobind Korana synthesized the first active tRNA molecule of 72
nucleotides (1970)
̶ Artificial DNA synthesis is in the 3' → 5' direction
- attaching the first base to CPG (controlled pore glass)
- the 5' end is blocked with DMT (dimethyloxytrityl)
- the DMT group is removed using a weak acid (TCA)
- another nucleotide is added in the form of so-called phosphoramidite
activated by tetrazole
- 5'- OH ends of unreacted nucleotides are acetylated using acetic
anhydride
- repeating the process
(from the left) Har Gobind Khorana, Robert W Holley,
Luis W Alvarez, Marshall W Nirenberg, Lars Onsager
and Yasunari Kawabata at the awarding of the Nobel Prize
in 1968.
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Chemical synthesis of DNA DMT-Dimethoxytrityl
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Chemical synthesis of DNA
https://www.youtube.com/watch?v=1S0x3aRCviM
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Polymerase Chain Reaction
K Mullis, F Faloona, S Scharf, R Saiki, G Horn, H Erlich.
Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction.
Cold Spring Harb Symp Quant Biol;1986;51
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Modifications of PCR
̶ Inversion PCR
̶ Reverse Transcription PCR (RT-PCR)
- 5´RACE, 3´RACE
̶ PCR mutagenesis
̶ Emulsion PCR
̶ Droplet Digital PCR
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RACE PCR
Rapid Amplification of cDNA Ends
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Emulsion PCR
Used in NGS technology (454, ion torrent)
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Droplet digital PCR
https://www.youtube.com/watch?v=lAVVoyZxlTU
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Sequencing of human genome
̶ at the time of the beginning (the year 1990) a monumental task
̶ started in 1990 with the participation of the DOE and NIH
̶ sequencing done using contig maps and BACs
̶ the initial plan envisaged duration of 15 years
̶ finally, sequencing using the Sanger method was almost completed already in 2000
̶ resulting sequence map published on April 14, 2003, with 99.99% accuracy (National Human Genome
Research Institute)
̶ total cost of the project 3 billion dollars
̶ in 2000, President Bill Clinton asserted the unpatentability of DNA
https://https://www.genome.gov/25019885/online-education-kit-how-to-sequence-a-human-genome//
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Celera Genomics Project
̶ founded by scientist Craig Venter and started a sequencing project in 1998
̶ the total cost of 300 million dollars was fully covered by private sources
̶ the "whole genome shotgun sequencing" method was used for the first time
̶ used an approach developed by Gene Myers to analyze the sequencing data
̶ this approach required extreme computational demands
̶ final calculation performed on 7000 processors to obtain 1000 times the speed
of Pentium computers
̶ this innovative approach allowed sequencing to be completed in just 9 months
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The strong role of diplomacy
It is hard to imagine today’s politicians reminding
scientists that cooperation has as much value as
competition.
In 26 June 2000, US President Bill Clinton and UK Prime
Minister Tony Blair presided over a carefully choreographed
piece of scientific theatre. Through a video link connecting
Washington DC and London, they announced to the world that
scientists had completed a rough first draft of the human
genome sequence. Craig Venter (left), Francis Collins, Bill Clinton (right)
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Sanger sequencing
• Synthesis of DNA in-vitro using "terminators" - dideoxynucleotides that prevent further elongation after
being incorporated into DNA.
Deoxyribosa Dideoxyribosa
• It requires the use of an initial primer, DNA polymerase and a mixture of
dNTPs with labeled ddNTPs
• The synthesized strands are separated using polyacrylamide gel
electrophoresis or capillary electrophoresis
• Possibility of fully automated separation using fluorescently labeled
ddNTPs
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Sanger sequencing
Throughput/Performance by Run Module
XLRseq: 768 samples per day (690 Kbases)
LongSeq: 1152 samples/day (980 Kbases)
StdSeq: 2304 samples/day (1550 Kbases)
FastSeq: 2304 samples/day (1600 Kbases)
RapidSeq: 3840 samples per day (2100 Kbases)
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• it enables rapid sequencing of short stretches of DNA - sequencing of 30 to 50 bases takes
approximately 30 to 45 minutes.
• it is bio-luminometric DNA sequencing based on the detection of inorganic pyrophosphate (PPi)
released during nucleotide incorporation.
Pyrosequencing (1990)
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454 a GS Junior system
Průchodnost 1 miliarda bazí za den
Doba analýza 10.0 hodin
Délka čtení 400
Počet čtení/analýzu 1 000.000
Správnost >99.0% správnost jednoho čtení na 400
bazích
Potřebné množství DNA Méně než 100 ng DNA
Multiplexování Až 192 vzorků/běh
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Qiagen – PyroMark instruments
̶ https://www.labtube.tv/video/MTAxNzE1
̶ https://www.qiagen.com/us/knowledge-and-support/knowledge-hub/explainer-
videos-and-demos/pyrosequencing-cascade-reaction
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Preparation of Sequencing Library
DNA sample – fragmentation (Covaris, fragmentase)
End-repair (DNA polymerase)
Adaptor ligation (ligase)
Selection of fragments (SPRI beads)
Amplification of fragments
Sequencing (Illumina, IonTorrent, Nanoballs)
Fragmentase
• A mixture of endonucleases (NEases) cleaving
one strand and then the opposite one
• A mixture of two enzymes (DNase I and SD
(strand-displacement) polymerase)
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Nextera technology
https://doi.org/10.1038/nmeth.f.272
• uses in vitro transposition
• transposases catalyze the random
insertion of excised transposons
• transposase makes random, staggered
double-stranded breaks in the target DNA
and covalently attaches the 3′ end of the
transferred transposon strand to the 5′ end
of the target DNA.
• for integration only free transposon ends
are sufficient
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Targeted Enrichment
PCR enrichment DNA capture Inversion probes
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Amplicon sequencing
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Quantification of NGS library
Electrophoretic methods
• Fragment Analyzer (Adv. Anal.)
• TapeStation (Agilent)
• BioAnalyzer (Agilent)
Fluorometric methods
• Qubit (Thermo Scientific)
• Quantus (Promega)
Real-Time PCR
• KapaBiosystem
• NEB
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Illumina sequencing system
MiniSeq, MiSeq, NextSeq, HiSeq, NovaSeq
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Sequencing in clusters
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1st step – hybridization on flow-cell
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2nd step – bridge PCR
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2nd step – bridge PCR
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2nd step – bridge PCR
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2nd step – bridge PCR
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2nd step – bridge PCR
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3rd step - linearisation
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4th step – separation of reverse strand
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5th step – blocking of 5´end
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6th step – hybridization of seq. primer
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Reverse terminators
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Sequencing by synthesis (SBS)
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7th step – pair-end sequencing
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7th step – pair-end sequencing
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7th step – pair-end sequencing
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7th step – pair-end sequencing
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Index read
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Single index read
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Dual index read
iSeq, MiniSeq, NextSeq,HiSeq
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PGM analyser (ion torrent)
Application
Targeted Exome Transcriptome Genome
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DNBSeq (MGI) https://en.mgi-tech.com/products/
https://youtu.be/xUVdJN0m38c
• it uses phi29 polymerase for amplification of
one-strand template
• this process creates nanoballs
• sequencing cell contains regions with
positive charge for binding of nannoballs
• different technology of sequencing
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Current techniques of 2nd generation
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3rd generation of sequencers (PACBIO)
Sequel System PacBio RS II
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PACBIO
• Sequencing based on Single Molecule, Real-Time (SMRT®) technology
• It uses so-called Zero-Mode Waveguides (ZMWs) enabling the illumination of only the lower
part of the well, in which the DNA polymerase is immobilized at the bottom
• The main advantage is the possibility of long reads (up to 20 kb)
• Another advantage is the possibility of direct detection of methylated bases (epigenome)
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Library preparation
https://www.youtube.com/watch?v=v8p4ph2MAvI
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3rd generation – Oxford Nanopores
• The technology is based on nanopores
• At the beginning of sequencing, NA is bound to a nanopore formed by a protein
• It is then denatured and passes through the nanopore, generating a change in current
• Based on the observed change, individual bases are read in real-time
• Enables sequencing of very long chains (tens to hundreds of kilobases)
• The disadvantage is a higher error rate, correctness >95%
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GENE TECHNOLOGIES
Methods of studying gene expression and function
Mapping techniques, DNA libraries, gene expression, metagenomics
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Mapping techniques
̶ Genome maps provide a series of markers for assembling sequence data:
̶ Creation of a genome map:
- genetic maps (crossbreeding, pedigree analysis, gene transfer) - linkage maps
- physical maps (radiation hybrid panel, FISH)
̶ Genetic maps based on linkage = the probability that two mapped markers will separate from each
other in a cross
̶ To determine the relative distance of markers, the percentage of times they are found together is crucial
̶ A variety of markers are used today
Type of mapping Markers Methods of localization
Genetic Gene, biochemical properties, DNA markers (RFLP,
VNTRs, microsatellite, SNPs)
Linkage analysis using crossing or mating
Kinship analysis
Physical STSs, EST, VNTRs, microsatelites Restriction analysis, Radiation hybrid panel, FISH,
Cytogenetic mapping
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Genetic markers
̶ RFLP analysis of related individuals, easy identification
̶ Variable Number Tandem Repeat (VNTR, minisatellites) – tandem
repeats with a length of 9-80bp (forensic testing, paternity tests)
̶ Microsatellite polymorphism – tandem repeat of 2-5bp length
̶ Single Nucleotide Polymorphism (SNP)
̶ SNPs, VNTRs RFLPs are also used in physical mapping
̶ For large genomes we need additional markers
- STSs (Sequence Tagged Sites) – the unique sequence of 100-500 bp
- ESTs (Expressed Sequence tags) – identification in cDNA libraries
̶ Digestion of gDNA using restriction enzymes - physical mapping method
Clark and Pazdernik, 2016
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Genetic markers
Clark and Pazdernik, 2016
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Physical mapping techniques
̶ FISH (Fluorescence in-situ hybridization) – the location of a
specific DNA sample on chromosomes in metaphase relative to
banding (chromosome painting)
̶ radiation hybrid mapping – large segments of the cloned genome
may contain two fragments from different parts of the genome
Clark and Pazdernik, 2016
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Number of genes x Genome
Organismus Velikost genomu
(Mbp)
Počet protein-kódujcích
genů
Wheat 17 000 95 000
Rice 520 45 000
Paris Japonica (Pieris japonský) 149 000 26 000
Trichomonas vaginalis 160 46 000
Encephalozoon intestinalis 2.25 1833
Marbled lungfish 130 000 ?
Human 3200 21 850
Nematode 97 20 493
Fruit fly 180 13 600
Streptomyces coelicolor 8.7 7800
E. coli 4.6 4300
Mycoplasma genitalium 0.58 470
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DNA libraries
̶ Used for:
- finding new genes
- genome sequencing
- comparison of genes from different organisms
̶ Basic steps in creating a library:
- isolation of chromosomal DNA
- cleavage of DNA with a restriction enzyme
- linearization of the vector
- insertion of fragments into the vector
- transformation into E. coli
Clark and Pazdernik, 2016
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̶ The vector contains the sequence necessary for
transcription and translation
̶ Constructed from complementary DNA (cDNA)
̶ Identification of new genes, splicing variants
Clark and Pazdernik, 2016
Eukaryotic expression libraries
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Medical genomics
̶ The largest application of genomic data in disease diagnosis
̶ Genetic testing – determination of the presence of a gene
associated with the disease:
- muscular dystrophy (dystrophin gene)
- cystic fibrosis (CFTR gene)
- Huntington's disease (HTT gene)
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Medical genomics
̶ To identify causal mutations, it is more advantageous to sequence the exome (2%) than the genome
̶ Currently, more than 3,000 diseases have been identified using genomics and pedigree analysis
- the so-called Mendelian disease (a mutation in one gene leads to the disease)
̶ Many diseases are polygenic (contribution of multiple genes to the development of the disease)
- Crohn's disease
- autoimmune disease
- psychiatric disorders (schizophrenia, AD, mild cognitive impairment)
̶ Within these diseases, the use of GWAS (genome-wide association study)
- analysis of single point polymorphisms (SNPs)
- frequency lower than 1%
- influence of genotype and environment on disease development
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Gene expression – WGAs, ChIP
̶ WGAs (whole-genome tiling arrays
cover all genome
̶ Firstly, in Arabidopsis (25-mer
oligonucleotides)
̶ Discovery of new genes, splicing
variants
̶ ChIP (chromatin immunoprecipitation):
- analysis of DNA regions of individual
transcription factors
- DNA analysis of regions associated
with histone PTMs
Clark and Pazdernik, 2016
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Gene expression – RT-qPCR
Using of specific fluorescence probes
Hydrolysis probe
TaqMan
Hybridization probes
Molecular beacons, FRET
Using intercalating dye
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Gene expression - RNAseq
̶ Advantages of the RNAseq method:
- does not depend on probes (more correct quantification of given RNA molecules)
- large dynamic range
- detection of alternative splicing and the possibility of their quantification
- the possibility of analysis without knowledge of the genome sequence
- the possibility of analysis from one cell
Clark and Pazdernik, 2016
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MetaRibo-Seq
Fremin et al. 2020
̶ Riboseq – translation arrest and subsequent
sequencing of the translatome
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Metagenomics
̶ A study of the genetic material contained in a sample
̶ ShotGun approach X sequencing of specific phylogenetic regions
(16S, 18S, ITS, mcrA)
Johnson et al. 2019
Microbiome Microbiota Metagenome
Microorganisms (and
their genes) living
in a specific
environment
Microorganisms
(by type) living
in a specific
environment
The genes
of microorganisms
in a specific
environment
Gohl et al. 2016
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Monitoring of gene expression
̶ A whole range of details about a gene obtained using reporter
genes
- adding a reporter gene behind the promoter
- adding a reporter gene behind the CDS
̶ Using the following genes:
- lacZ gene (b-galactosidase)
- phoA gene (alkaline phosphatase)
- lux/luc gene (luciferase)
- gfp gene (Green Fluorescent Protein)
Clark and Pazdernik, 2016
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Analysis of methylome
̶ Analysis of gDNA methylation sites
̶ Methylation usually silences transposon elements
̶ Silencing of one copy of the X chromosome in
females
̶ Analysis using the bisulfite method
- the addition of sodium sulfite leads to the
conversion of non-methylated cytosines to uracil
- subsequent sequencing without and with the
addition of sulfite leads to the detection of
methylation sites
̶ 3rd generation sequencers (Nanopores, PacBIO)
are able to directly read cytosine methylation
Clark and Pazdernik, 2016
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GENE TECHNOLOGIES
Gene Cloning Strategies
Restriction endonucleases, plasmids, and cloning vectors, optimization of gene expression, expression in foreign hosts
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Restriction enzymes
̶ Bacterial enzymes binding to a specific sequence and cleaving both strands
̶ Protection of bacteria from foreign DNA (viruses)
̶ Sensitive to DNA methylation
̶ Two basic types:
Type I - cleaves the DNA strand 1000 or more bases from the recognized
sequence
Type II - cleaves the DNA strand at the location of the recognized sequence
(blunt, sticky ends)
̶ The number of bases recognized = the degree
of DNA fragmentation
̶ Joining fragments - ligase (T4 ligase)
Clark and Pazdernik, 2016
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Restriction enzymes (structure)
Pingoud and Jeltsch, 2001
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Fragmentase
̶ Used for DNA fragmentation in NGS
̶ A mixture of endonucleases (NEas) cleaving one strand and then the opposite one
̶ A mixture of two enzymes (DNase I and SD (strand-displacement) polymerase)
Ignatov et al. 2019
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Cloning vectors
̶ Specialized plasmids (other elements) carrying foreign DNA for study/manipulation
̶ Currently, we also use artificial chromosomes and viruses
̶ Basic properties of cloning vectors:
- small size (easy handling and isolation)
- easy transfer between cells by transformation
- easy isolation from the host organism
- easy detection and selection
- occurrence in a larger number of copies (ori site)
- multiple cloning sites for insertion of cloned DNA
- method confirming the presence of inserted DNA in the vector
Clark and Pazdernik, 2016
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Cloning vectors
̶ DNA insertion control options
- insertional inactivation (ATB resistance gene)
- ccdB gene (death gene interfering with DNA
gyrase activity) https://link.springer.com/article/10.1007/BF00280310)
- alpha complementation (b-galactosidase)
̶ Yeast vectors based on a 2m circle
- ori site from two organisms, the Cen sequence
- selection based on AA synthesis
Clark and Pazdernik, 2016
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Virus vectors
̶ Bacteriophage vectors
- modified to carry non-viral DNA in the capsid
- connection of cos sequences = formation of a
replication form (RF) replicated by a rolling circle
- an insert with a size of 37 to 52 kb can be used
- use of helper viruses to package DNA into virus
capsid
̶ Cosmids
- a highly modified lambda vector having only cos
sites
- the necessity of packaging by helper phage
Clark and Pazdernik, 2016
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̶ Used for handling large pieces of DNA (150 – 2000 kb)
̶ Include
- yeast artificial chromosomes (YACs)
- bacterial artificial chromosomes (BACs)
- P1 bacteriophage artificial chromosomes (PACs)
̶ YACs contain a centromere and telomeres for permanent
maintenance in yeast
̶ BACs are circularized and propagated in bacteria (ori site
and resistance gene)
Trends in Biotechnology 2000, DOI: (10.1016/S0167-7799(00)01438-4)
Artificial chromosomes
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DNA transformation
̶ Transformation is the process by which foreign DNA is
introduced into a cell.
̶ Competent E. coli cells:
- the use of calcium ions and thermal shock to increase the
permeability of the cell wall and membrane
- use of electroporation to open the cell wall and membrane
̶ Competent yeast:
- a combination of lithium acetate, single-stranded carrier
DNA and polyethylene glycol (PEG)
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Cloning strategies
̶ TOPO Cloning (Thermo)
- use of topoisomerase I
- Vaccinia virus topoisomerase I specifically recognizes the sequence 5'-(C/T)CCTT-3’
- topoisomerase is covalently attached to the 3' end of the vector
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Cloning strategies
̶ TA cloning
- using the property of Taq DNA polymerase to add A to
the 3' end
- pMiniT 2.0 (toxic mini-genes) (NEB)
- pGEM-Teasy (blue-white selection) (Promega)
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̶ GATEWAY cloning vectors (Invitrogen-Thermo)
- use of phage lambda integrase and excisionase enzymes
- use of ENTRY and DESTINATION vectors
- the BP reaction removes the gene of interest from attR sites and
inserts it into attL sites.
- the LR reaction removes the gene of interest from attL sites and
inserts it into attR sites
Clark and Pazdernik, 2016
Cloning strategies
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https://www.youtube.com/watch?v=tlVbf5fXhp4
̶ In 2009 Dr. Daniel Gibson and colleagues at the J.
Craig Venter Institute developed a new method to
easily assemble multiple linear DNA fragments
̶ Advantages
I. There is no need for specific restriction sites.
II. Join any fragments regardless of order.
III. The reaction takes place in one tube.
̶ Gibson's Mix consists of three different enzymes
I. T5 Exonuclease
II. Phusion DNA Polymerase
III. Taq DNA ligase
Cloning strategies
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Expression vectors
̶ The most commonly used lacUV promoter (modified lac promoter)
- RNA polymerase binding site
- lacI repressor site
- transcription start site
- transcription termination site
̶ Another frequently used promoter is the lambda left promoter (PL)
- lambda repressor binding site
- most frequent activation by increased temperature (42°C)
̶ Expression systems also use a promoter binding only bacteriophage T7 RNA polymerase
- E. coli strains carrying T7 RNA polymerase after inducer control
̶ Expression vectors often contain sequences for various tags (6xHis, Myc, FLAG, S-tag, MBP)
Clark and Pazdernik, 2016
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Bacterial Expression Vectors
̶ pET, prSET E. coli T7 expression vectors
- expression in BL21(DE3)pLysS cells
̶ pMAL expression vectors
- carry maltose-binding protein (MBP)
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Yeast Expression Vectors
̶ Inducible AOX promotor (methanol)
̶ Possibility of intra- and extracellular expression
̶ Expression in yeasts P. pastoris and S. cerevisiae
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̶ Special plasmids (expression vectors) are used to increase proteins
expression
- strong promoter, adequate ori site, selection marker for antibiotic
̶ Expression of eukaryotic proteins is more problematic
- promoter modification, absence of splicing, low rate of translation
- weak interaction of the ribosome with the RBS site, mRNA instability,
limited amount of tRNA
̶ The necessity of using specially modified vectors
Clark and Pazdernik, 2016
Expression in Bacteria
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E. Coli OrigamiTM 2
Exprese Oncostatinu M (OSM): A (37°C), B (18°C). C-kontrola bez IPTG,
I-lyzát, P-pelet, S-solubilní frakce (Nguyen et al., 2019, SciRep)
̶ They carry a mutation in the gene thioredoxin reductase (trxB) and glutathione reductase (gor)
̶ Increase in the formation of disulfide bonds in the cytoplasm of E. coli
̶ Suitable for proteins requiring the formation of S-S bridges for proper composition
Berkmen, 2012
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Translational Expression Vectors
̶ Designed for protein expression (pET, pRSET)
- maximum translation initialization
- consensus RBS site
- ATG codon at an optimal distance of 8 bases from the RBS
- cloning site directly in the ATG codon (Nco I)
̶ The possibility of further complications in protein folding
Clark and Pazdernik, 2016
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Codons Effect
̶ Protein expression in other organisms (eukaryotic in bacteria)
̶ Different organisms prefer different codons for a given AA
- optimization of the codons used in gene synthesis
- up to a 10-fold increase in production
- delivery of tRNA carrying rare codons to the organism
- E. coli ROSETTA – seven tRNAs for rare codons
(AGA, AGG, AUA, CUA, GGA, CCC, and CGG)
Clark and Pazdernik, 2016
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Toxic effect of overexpression
Lactose operon Arabinose operon
Clark and Pazdernik, 2016
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Autoinduction Medium
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Inclussion Bodies
̶ Misfolded proteins accumulate in inclusion
bodies
̶ Molecular chaperones – they help with proper
packing
̶ Possible secretion of proteins into the
periplasm or medium
̶ Proteins can be solubilized from inclusion
bodies with a chaotropic agent and renaturation
Clark and Pazdernik, 2016
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Secretion of Proteins
̶ Possible expression into the periplasm or medium
̶ Secretion controlled by a hydrophobic sequence at the N-terminus cleaved by signal peptidase
- possible addition of a signal sequence to the protein (risk of inclusion bodies)
- possible fusion with a naturally secreted protein (maltose-binding protein in E. coli)
- possible secretion in gram-positive bacteria (Bacillus)
- use of a special Type I secretion system (hemolysin secretion, E. coli) or Type II (Endotoxin A,
Pseudomonas)
- use of autotransport proteins
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Clark and Pazdernik, 2016
Secretion system of type I and II
Autotransporter proteins
Secretion of Proteins
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Protein glycosylation
̶ A whole range of proteins in higher organisms is glycosylated
̶ Glycosylation is necessary for proper function - e.g. membrane proteins
̶ The bacterium carries out O-glycosylation (N-glycosylation was also discovered in the genus
Campylobacter)
̶ Eukaryotic organisms mostly have N-glycosylation
̶ Insect cells are the solution for the expression of glycosylated proteins
- a different pattern of glycosylation compared to mammals
- the solution is modified insect cells with a mammalian glycosylation pathway
̶ A change in the glycosylation pattern can affect the properties of the protein
- recombinant human erythropoietin contains an extra N-glycosylation site (Asn-Xxx-Ser/Thr)
- lower affinity to the receptor, but a longer half-life prolongs the overall clinical activity
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Clark and Pazdernik, 2016
Protein glycosylation
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Protein Expression in Eukaryotic Cells
̶ A number of eukaryotic proteins are more efficiently
expressed in eukaryotic cells
̶ Possibility of post-translational modifications
- chemical modifications forming new amino acids
- formation of disulfide bridges
- glycosylation
- addition of functional groups (fatty acids, acetylation,
phosphorylation, methylation, sulfurization)
- cleavage of pre-cursor proteins required for
secretion, assembly, and/or activation
Clark and Pazdernik, 2016
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Yeasts
̶ A whole range of advantages
- easy cultivation on a small and large scale
- the yeast S. cerevisiae is considered a safe organism
- yeasts secrete very few of their own proteins – an advantage in
secreting the expressed protein
- DNA can be easily transformed (chemically, enzymatically,
electroporation)
- characterization of a whole series of promoters for targeted
expression
- capable of a whole range of post-translational modifications
characteristic of eukaryotic organisms
- glycosylation takes place only in secreted proteins
̶ Frequent secretion of recombinant proteins by the signal
sequence of the mating factor a gene
̶ The signal peptidase recognizes the Lys-Arg sequence
Clark and Pazdernik, 2016
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Yeasts
̶ Currently expressed in the yeast S. cerevisiae and P. pastoris
- insulin
- clotting factor VIIIa
- various growth factors
- viral proteins for the production of vaccines or diagnostics (HIV,
HBV, HCV)
̶ The most common expression problems in yeast
- loss of expression plasmids in large-scale cultivations
- secreted proteins remain between the PM and the cell wall
- hyper-glycosylation of secreted proteins occurs (solution by strain
modification) Sheng et al. 2017
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GENE TECHNOLOGIES
Technologies in Immunology
Antibodies (structure, function), targeted antibody design, monoclonal antibodies, ELISA, vaccines (design and production,
identification of potential new antigens, DNA vaccines)
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Introduction
̶ The surrounding environment is full of infectious microorganisms and virusesOchrana
organismu pomocí buněk imunitního systému
̶ Protection of the body by the cells of the immune system
̶ Antigens - mostly proteins on the surface of microorganisms = activation of immune response
̶ Antibodies - recognize and bind to antigens = produced by B-cells of the adaptive immune
system
̶ Antibodies mostly secreted into the lymph, some bind to surface = B-cell receptors
̶ Massive proliferation of B-cells producing antibodies recognizing a given antigen
̶ Immune system records all successfully used antibodies = faster and more massive response
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Introduction
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Antigen, antibody, epitope
̶ Antigen - a foreign molecule that activates the immune
system
̶ Strongest immune responses = glycoproteins and lipoproteins
̶ Very often polysaccharides on the surface of microorganisms
serve as antigens
̶ DNA can also serve as an antigen
̶ The animal immune system is based on specific (acquired)
immunity divided into:
- humoral immunity (mediated by immunoglobulins)
- cell-mediated immunity (T-lymphocytes = TH and TC)
̶ Antibody = binding to whole proteins
̶ T-lymphocytes = binding to protein fragments
̶ Epitope - region of protein recognized by antibody
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T-lymphocytes
̶ recognize only antigens expressed on the surface of other cells, mainly macrophages, virus-infected
cells or B-lymphocytes
̶ T-lymphocytes recognise these cells via class I and II major histocompatibility complex (MHCs) receptor
proteins
̶ Class I activates TH cells and class II activates TC cells
̶ MHC receptors are encoded by a family of genes specific to each individual
̶ MHC receptors are also called major histocompatibility complexes HLA
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T-lymphocytes
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Structure and Function of Immunoglobulins
̶ Antibodies divided into 5 basic classes
̶ The most abundant are IgG in serum
̶ Only IgG antibodies cross the placenta
̶ IgA - secretory antibodies important in
suppressing respiratory and
gastrointestinal infections
̶ IgM - 10 binding sites = coating
microorganisms and stimulating cells
̶ IgE - on the surface of mast cells,
stimulation of allergic response by
histamine release
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Structure and Function of Immunoglobulins
̶ IgG antibody consists of two light and two heavy
chains
̶ Light chains encoded by one of two gene loci k or l
̶ Each of the light and heavy chains consists of one to
four constant regions and one variable region
̶ The variable regions form the so-called paratopeantigen
binding
̶ We have millions of different variable regions
̶ In the Pant region, antibodies can be divided
chemically (by papain) into Fc and two Fab fragments
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Diversity of Antibodies
̶ There are an almost infinite number of antigens = an almost
infinite number of antibodies are needed
̶ Genetic problem concerning the number of genes encoding
each antibody
̶ The entire human genome would encode only a few million
antibodies
̶ The immune system generates a large number of sequences
from a relatively small number of genes in the process of
V(D)J recombination
̶ The immune system assembles genes for antibodies from
collections of short DNA segments
̶ V(D)J recombination occurs in the bone marrow during B-cell
development and is initiated by RAG1 and RAG2 proteins
followed by NHEJ
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V(D)J Recombination
Smith et al. 2019
Backhaus et al. 2018
Chr. 2 Chr. 14
https://youtu.be/QTOBSFJWogE
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Monoclonal Antibodies
̶ Antibodies find wide clinical use
̶ Need for one specific antibody against an antigen
̶ One antigen has many epitopes = polyclonal antibodies
̶ Polyclonal antibodies = mixture of antibodies with different degrees
of specificity and binding
̶ Monoclonal antibody = one specific antibody from one B-cell
̶ Viability of B-cells outside the body is very low = fusion with
myeloma cells
̶ The resulting cell is called a hybridoma = a forever living cell
producing the targeted antibody
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Use of Antibodies
ELISA
Rapid tests FACS (Fluorescence-activated cell sorting)
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"Humanization" of Monoclonal Antibodies
̶ Human immune system recognises mouse antibodies
̶ Several solutions:
- Replacing the C-region with a human variant of the antibody
- Replacement of V-regions not involved in antigen recognition with a human
variant
- Complementarity Determining Region (CDR) - hypervariable region
recognizing Ag
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Herceptin and Casirivimab
̶ Monoclonal antibody recognises the epidermal growth factor receptor type 2 (HER2)
̶ In breast cancer patients, HER2 overproduction is associated with resistance to chemotherapy
̶ Binding of antibodies to the receptor prevents its internalization = better efficacy of chemotherapy
̶ Casirivimab - a monoclonal antibody that recognizes the SARS-CoV-2 coronavirus spike protein
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Nanobodies
̶ Antibodies from camels, alpacas and llamas have only heavy-chain
antibodies (hcAb)
̶ The antigen is bound by the terminal variable region of the heavy
chain called the VHH (12-15 kDa)
̶ Recombinant antibodies containing only this part are called
nanobodies (Nb)
̶ The VHH region has a very high affinity for the antigen
̶ Nanobodies can cross into the brain
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Vaccines
̶ The immune system remembers foreign antigens - immune memory
̶ Special memory B-cells mediate immune memory
̶ Vaccines consist of derived infectious agents that can no longer cause disease but are still
antigenic
Vaccines:
- Attenuated = pathogens still alive but no longer producing disease-causing toxins or proteins
- Subunits = effective against only one component of the pathogen, often requires the use of
adjuvants
- multivalent = targets several proteins from one or more viruses
̶ Vaccines from attenuated microorganisms usually induce best immune response
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Vaccines
Attenuated vaccines
Subunit vaccines
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Search for Suitable Antigens and Adjuvants
̶ Reverse vaccinology = sequential cloning of pathogen genes and expression of proteins used for
immunization (vaccine for Neisserie meningitidis serogroup B)
Adjuvant Composition Vaccines
Aluminum One or more of the following: amorphous aluminum
hydroxyphosphate sulfate (AAHS), aluminum hydroxide,
aluminum phosphate,
potassium aluminum sulfate (Alum)
Anthrax, DT, DTaP (Daptacel), DTaP (Infanrix), DTaP-IPV
(Kinrix), DTaP-IPV (Quadracel), DTaP-HepB-IPV (Pediarix),
DTaP –IPV/Hib (Pentacel), Hep A (Havrix), Hep A (Vaqta),
Hep B (Engerix-B), Hep B (Recombivax), HepA/Hep B
(Twinrix), HIB (PedvaxHIB), HPV (Gardasil 9), Japanese
encephalitis (Ixiaro), MenB (Bexsero, Trumenba),
Pneumococcal (Prevnar 13), Td (Tenivac), Td (Mass
Biologics), Tdap (Adacel), Tdap (Boostrix)
AS04 Monophosphoryl lipid A (MPL) + aluminum salt Cervarix
MF59 Oil in water emulsion composed of squalene Fluad
AS01B Monophosphoryl lipid A (MPL) and QS-21, a natural
compound extracted from the Chilean soapbark tree,
combined in a liposomal formulation
Shingrix
CpG 1018 Cytosine phosphoguanine (CpG), a synthetic form of DNA
that mimics bacterial and viral genetic material
Heplisav-B
No adjuvant ActHIB, chickenpox, live zoster (Zostavax), measles, mumps
& rubella (MMR), meningococcal (Menactra, Menveo),
rotavirus, seasonal influenza (except Fluad), single antigen
polio (IPOL), yellow fever
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Search for Suitable Antigens and Adjuvants
Reverse vaccinology Differential fluorescence induction (DFI) In-vivo induced antigen technology (IVIAT)
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Adenovirus vaccines
Bermejo et al, 2020
https://sputnikvaccine.com/about-vaccine/
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mRNA vaccines
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mRNA vaccines
Versteeg et al, 2019