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Prof. Marjanca Starčič Erjavec, PhD Molecular Genetics and Microbiology Group
Department of Biology Biotechnical Faculty Ljubljana, Slovenia
nuclein
Johannes Friedrich Miescher (1844 – 1895)
Miescher‘s lab at the University of Tübingen
1869
Smooth colony S. pneumoniae – with capsule
Rough colony S. pneumoniae – without capsule
Frederick Griffith (1879 - 1941)
1928 TRANSFORMATION
1944
Performed experiments with the cell extract of S. pneumoniae.
Proof: DNA is the hereditary molecule .
Oswald T. Avery (1877-1955)
Colin MacLeod (1909 – 1972)
Maclyn McCarty (1911 – 2005)
Avery-MacLeod-McCarty Assay
centrifugation, heat inactivation, homogenization, filtration
bacterial filtrate
RNase protease DNase
pneumococci without capsule –
non-pathogenic
pneumococci with capsule and without capsule
pneumococci without capsule –
non-pathogenic
1952 A biological proof that the genes are made of DNA.
Alfred Hershey (1908 – 1997)
Martha C. Chase (1927 – 2003)
Hershey-Chase experiment
Labeling of bacteriophages Labeled bacteriophages infect cells Detection of radioactivity
Phage proteins labeled with radioactive sulphur
Radioactivity is outside of the cell
Radioactivity is IN the cell Phage DNA is labeled with radioactive phosphorus
1953
James D. Watson (1928–)
3D-model of DNA
Francis Crick (1916–2004)
genetic code
(Nirenberg, later also Ochoa and Khorana)
Marshall W. Nirenberg (1927-2010)
1961
Werner Arber (1929 – )
1965
Restriction-modification systems in bacteria
Research into restriction of phages
RESTRICTION ENZYMES
1968: EcoB (Linn & Arber),
EcoK (Meselson, Yuan)
DNA-ligase
1967
Martin Gellert (1929 -)
Succeed to form covalent circles of phage l linear DNA using an E. coli extract
Hamilton O. Smith (1931 – )
1970
Endonuclaese R
Haemophilus influenzae
HindII 5‘-GTY^RAC-3‘
Daniel Nathans (1928 – 1999)
1971
Applications of restriction enzymes
Paul N. Berg (1926 – )
1971 Cut DNA molecules with EcoRI to generate complementary sequences on the vector and the fragment
Fragment DNA to be cloned – insert from phage l
Vector molecule – DNA of SV40 virus
Join insert from phage l
with SV40 virus vector
Recombinant DNA molecule
Stanley N. Cohen (1935 – )
Herbert W. Boyer (1936 – )
1972
Cohen – plasmids,
their transfer - Abr
Boyer – restriction enzymes (EcoRI) and religation
corned beef
hot pastrami
Cut DNA with EcoRI to generate complementary sequences on the vector and the fragment
Recombinant DNA molecule
Recombinant DNA molecule
Fragment DNA of the R6-5 plasmid – insert
Join insert and vector
Vector molecule – DNA of pSC101 plasmid
Bacteria
Bacterial chromosome
Insertion of rDNA into bacteria for replication
MOLECULAR CLONING
Molecular cloning (recombinant DNA technology, genetic engineering) includes isolation of a certain DNA fragment and its amplification to a large number of copies.
INSERT + VECTOR
Plasmid DNA isolation Digestion with restriction enzyme
Chromosomal DNA isolation Digestion with restriction enzyme
Chromosomal DNA
Plasmid DNA
Ligation of insert into the vector
Trasnformation into the host cell
Replication of host cells
Molecular cloning of inserts obtained with DNA isolation
Molecular cloning of inserts obtained with PCR
Primer 2
Primer 1
Replication of host cells
Ligation of insert into the vector
Trasnformation into the host cell
Gene to be cloned
Vectors and host cells
VECTOR INSERT SIZE HOST CELLS
Plasmid, phagmid
10 kb Bacteria, mammalian cells
Virus 5–100 kb Bacteria, insect and mammalian cells
Cosmid 30–40 kb Bacteria
BAC 100 kb Bacteria
YAC 1 Mb Yeasts
Properties of a good plasmid vector
1. A small size. 2. A replication region, which allows the multiplication of the
plasmid in a host cell until the desired number of copies. 3. A polyclonal site (polylinker). 4. A selection marker (a gene that allows selection, e.g. a gene
coding for an antibiotic resistance). 5. A differentiation marker (a gene that allows differentiation of
cells harboring a plasmid vector with an insert from those without an insert, e.g. gene for -complementation).
Vector plasmid pUC19
Vector plasmid pUC19 - polylinker
-complementation
Special vectors
• “shuttle” vectors • expression vectors • vectors for site-specific mutagenesis • promoter-probe vectors • etc.
Standard enzymes:
• restrictase: cuts dsDNA at specific palindromic sequences
• ligase: introduction of phosohodiester bonds
• polymerase: elongation of DNA strands
• RNase: digestion of RNA
But also:
• phosphatase: removes phosphates
• polynucleotide kinase: binds phosphates; e.g. radioactive labelling
• reverse transcriptase: makes cDNA on RNA templates
• exonuclease: removes nucleotides from the end of the strand
• methylase: addition of Met into the DNA
Enzymes in molecular cloning
restrictase
ligase
EcoRI
homodimer 6 bp in the recognition site Mg2+ ions as cofactors first restrictase, used for molecular cloning
EcoRI
5‘ TTACGCGAGAATTCGCTCATTG 3‘ 3‘ AATGCGCTCTTAAGCGAGTAAC 5‘
5‘ TTACGCGAG AATTCGCTCATTG 3‘ 3‘ AATGCGCTCTTAA GCGAGTAAC 5‘
• Multiple resistant bacteria pose one of greatest risks for human health.
• Since 1987 decline in the number of new approved antibiotics.
Number of death cases today and in 2050
The Review on Antimicrobial Resistance, Chaired by Jim O’Neill
WHO list of bacteria for which new antibiotics are urgently needed 27 February 2017 News Release GENEVA
WHO priority pathogens list for R&D of new antibiotics Priority 1: CRITICAL • Acinetobacter baumannii, carbapenem-resistant • Pseudomonas aeruginosa, carbapenem-resistant • Enterobacteriaceae, carbapenem-resistant, ESBL-producing Priority 2: HIGH • Enterococcus faecium, vancomycin-resistant • Staphylococcus aureus, methicillin-resistant, vancomycin-intermediate and
resistant • Helicobacter pylori, clarithromycin-resistant • Campylobacter spp., fluoroquinolone-resistant • Salmonellae, fluoroquinolone-resistant • Neisseria gonorrhoeae, cephalosporin-resistant, fluoroquinolone-resistant Priority 3: MEDIUM • Streptococcus pneumoniae, penicillin-non-susceptible • Haemophilus influenzae, ampicillin-resistant • Shigella spp., fluoroquinolone-resistant
WHO list of bacteria for which new antibiotics are urgently needed 27 February 2017 News Release GENEVA
WHO priority pathogens list for R&D of new antibiotics Priority 1: CRITICAL • Acinetobacter baumannii, carbapenem-resistant • Pseudomonas aeruginosa, carbapenem-resistant • Enterobacteriaceae, carbapenem-resistant, ESBL-producing Priority 2: HIGH • Enterococcus faecium, vancomycin-resistant • Staphylococcus aureus, methicillin-resistant, vancomycin-intermediate and
resistant • Helicobacter pylori, clarithromycin-resistant • Campylobacter spp., fluoroquinolone-resistant • Salmonellae, fluoroquinolone-resistant • Neisseria gonorrhoeae, cephalosporin-resistant, fluoroquinolone-resistant Priority 3: MEDIUM • Streptococcus pneumoniae, penicillin-non-susceptible • Haemophilus influenzae, ampicillin-resistant • Shigella spp., fluoroquinolone-resistant
E. coli
https://www.newsweek.com/2019/05/31/death-antibiotics-running-out-effective-drugs-fight-superbug-army-1423712.html
DNA transfer between two bacterial cells that are in direct contact
Conjugation / conjugal transfer
Main positive regulator - TraJ
Bacterial conjugation
• direct contact with the F-pilus and formation of a mating pore;
• mobilization of the DNA transfer (cut at a specific site oriT of F-plasmid);
• transfer of a single linear DNA molecule with the 5 'end from the donor into the recipient cell and simultaneous synthesis of the missing DNA strand in the donor and the recipient;
• transferred linear DNA molecule (double-stranded) is circularized and thus a functional plasmid is formed
Three concepts of conjugation
based antimicrobial
agents
Filu
tow
icz
et a
l. B
acte
rial
co
nju
gati
on
-bas
ed a
nti
mic
rob
ial a
gen
ts. P
lasm
id, 2
00
8; 6
0: 3
8–4
4.
Three concepts of conjugation
based antimicrobial
agents kill anti-kill
Filu
tow
icz
et a
l. B
acte
rial
co
nju
gati
on
-bas
ed a
nti
mic
rob
ial a
gen
ts. P
lasm
id, 2
00
8; 6
0: 3
8–4
4.
Based on the probiotic E. coli strain Nissle 1917, EcN, (MUTAFLOR), the conjugative plasmid pOX38 and bacteriocin colicin E7 a novel conjugation-based antimicrobial agent was constructed with the techniques of molecular biology.
pOX38 (55 kb)
tra
cat
Construction of the novel strain with the conjugation-
based antimicrobial agent
Strain with the conjugation-based antimicrobial system (strain ŽP)
The plasmid is in conjugation transferred into the target recipient cell. In the recipient cell the ColE7 is synthesised and the target cell is killed.
pOX38 with the ColE7 activity gene
chromosome with ColE7 immunity gene
Killing effect of ŽP
Real time PCR – revealing traJ gene expression
traJ expression was higher at 4 h than at 24 h
Mating ŽP × K12 TG1
Mating control strain × K12 TG1 Conjugation
in CFU rec. CFU tc. Conjug.
freq. CFU rec. CFU tc. Conjug.
freq.
Liquid medium
3.9×107 (3.1×106) 0 n. a.
6.1×107 (7.4×106)
6.4×104 (7.2×103)
1.0×10–3 (2.7×10–4)
Planktonic growth
1.1×108 (1.7×107) 0 n. a.
1.6×108 (8.5×107)
1.3×105 (3.4×104)
8.0×10–4 (2.2×10–4)
Biofilm 3.7×107 (4.9×106) 0 n. a.
3.1×107 (4.6×106)
7.1×103 (2.3×103)
2.3×10–4 (6.2×10–5)
Preformed biofilm
2.7×107 (2.0×106) 0 n. a.
5.4×107 (8.1×106)
4.9×105 (5.8×104)
9.2×10–3 (2.9×10–3)
Conjugative assays of the ŽP strain
CFU data
The CFU of recipients, donors and transconjugants in conjugation mixture. E. coli K-12 TG1 (pXen lux+ Apr) per se and in conjugation mixture with either the control donor strain N4i pOX38:Cm or the killer donor strain N4i pOX38a:Cm .
Bioluminescence assays
The bioluminescence (relative light units) of the recipients per se and in conjugation mixtures. E. coli K-12 TG1 (pXen lux+) per se (filled circles) and in conjugation mixture with either the control donor strain N4i pOX38:Cm (filled triangles) or the killer donor strain N4i pOX38a:Cm (filled squares). The means and the standard deviations of at least three independent experiments preformed in 6 to 12 technical repeats are shown. *Significant differences between variants at P ≤ 005.
https://www.economist.com/business/2016/01/09/cutting-remarks
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