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

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Three concepts of conjugation

based antimicrobial

agents kill anti-kill

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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