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E. coli is your friend
Kathrin EngelMolecular Biochemistry
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E. coli
11/29/1857 – 02/15/1911
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• procaryots enterobacteriaceae (greek: entero = intestine)
• part of the intestinal flora, symbiosis
• gram negative, rod-shaped, eubacterium
• facultative anaerobic O2 consumption or anaerobic fermentation
• exercises the intestinal immune system production of IgA by host
• produces vitamin K
• facultative pathogen pathogen if extraintestinal
E. coli
indicates water pollution by faecesbiosynthesis of insulin and amino acids because E.coli is resident in the human intestinal flora there are no allergic reactions to E.coli-produced molecules
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facultative pathogen
• sepsis• urinary tract infection• meningitis• wound infection• peritonitis• cholecystitis• cholangitis
obligatory pathogen
• diarrhea • enteritis • enterocolitis
Pathogenicity of E. coli
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• separation of cells after duplication of a DNA molecule
• generation time at 37°C in complete medium 20 – 30 minin minimal medium 60 – 90 min
• cultivation on agar plates (Robert Koch 1880)
• grows easily in a simple nutrient broth in culture bottles
Reproduction of E. coli
37°C
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• LB (Luria-Bertani) medium
• liquid and solid medium must be autoclaved
• antibiotics and amino acids are degraded by autoclaving
• add antibiotics only prior to use of liquid medium
• add antibiotics only below 50°C to solid medium
• plates should not be stored longer than 1 month
Growth and culture of E.coli in the lab
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Macromolecules of an E. coli cell
11
80> 1,000
3 x 104
3 x 104
4 x 105
103
RNA16S rRNA23S rRNAtRNAmRNA
12 - 4DNA
> 3,000106proteins
typesnmacromolecule
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• 4.65 x 106 bp encode 4,288 protein coding genes
• circular and closed nucleoid
• superhelical structure– IHF binds AT-rich regions and causes bending
– HU protein stabilizes bending and crossing of DNA strains
– DNA Topoisomerases: type II (gyrase) superhelical turns
type I relaxation of superhelical turns for replication or transcription
The genome of E. coli
gyrase inhibitors = antibiotics; inhibition of gyrase leads to an extension of the nucleoid DNA loses its compact structure and expands bacterial cell bursts
origin
terminus
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• naturally in bacteria (0 to 20 copies)
• circular dsDNA molecules
• separately from the main bacterial chromosome
• replicate independently from the bacterial chromosome
• horizontal gene transfer within a population of microbes
• selective advantage:– ampicillin resistance
– cloacin production
– multi drug resistance
– enterotoxin production
Plasmids in nature
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• F plasmids– carry information for own transfer from cell to cell (conjugation)
• R plasmids– encode resistance to antibiotics
• degradative plasmids– carry specific sets for degradation of unusual chemicals
• cryptic plasmids– carry no functional coding gene
Types of plasmids
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Exchange of genes
• donor contains F plasmid (fertility)• F plasmid can be isolated from the chromosome (F+ cells)
integrated into chromosome (hfr cells)
bacterial chromosome
F+ (donor) F- (recipient) conjugation tubereplication of
F factor
two F+ cells
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Characteristics of a plasmid
circular DNA
cloning site
antibiotic resistance gene
origin of replication
name
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• different numbers of plasmid molecules in one cell
• pBR322 20 – 30 copies
• pUC 200 – 300 copies
• low copy plasmids can get lost
High- and low-copy plasmids
pBR322
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Prominent expression vectors
cloning site
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• mammalian expression vector:– used for amplification of plasmid
– isolation of plasmid-DNA
– transfection of mammalian cells portein production in mammalian cells
– promotor upstream the target gene is a mammalian promotor
– protein produced = ß lactamase
• bacterial expression vectors:– used for expression of proteins in bacteria
– bacterial promotor upstream the target gene
bacterial and mammalian expression vectors
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• target genes expressed under T7 promotor
• exclusively for expression of cloned genes
• high expression levels of genes that are not expressed efficiently in other systems
• not recognized by E.coli RNA polymerase
• genes are “off” in non-expression hosts no plasmid instability
• transfer of plasmids into expression hosts containing chromosomal copy of T7 pol
• expression induced by IPTG or lactose
• 2 components: bacteriophage T7 RNA polymerase, plasmid vector with bacteriophage T7 promotor upstream the gene to be expressed
The pET system
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• isopropyl-ß-D-thiogalactopyranosid
• inducer of the lac operon
• binds lac repressor
• is not metabolized by E.coli
• used to produce recombinant proteins by expression of cloned genes
• cloned gene under lac promotor
IPTG
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plasmids as tools for gene cloning
INSERT
VECTOR PLASMID
LIGATION
TRANSFORMATION IN E.COLI
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Transformation of bacterial cells
CaCl2
bacterial cell
competentbacterial cell
42°C(30‘‘-1‘)
transformedbacterial cell
• uptake of DNA into a cell• chemically induced competence
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• transformation of competent cells with 1 ng control plasmid containing an antibiotic resistance gene
• plate onto LB-agar plates containing relevant antibiotics
• compare number of colonies obtained with control plasmids with those of interest
• transformation of cells with 20 µl buffer or water
• plate on LB-agar plate containing relevant antibiotics
• absence of colonies indicates an active antibiotic
Transformation efficiency and antibiotic activity
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• untransformed cells no growth on medium with antibiotics
• E.coli with plasmid growth
Selection I
• untransformed cells no growth on medium with antibiotics
• E.coli with plasmid growth
• untransformed cells no growth on medium with antibiotics
• E.coli with plasmid growth
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Selectable markers
• agar plate contains antibiotic and X-gal
• LacZ gene on the plasmid encodes ß-galactosidase
• ß-galactosidase metabolizes X-gal
• colonies containing antibiotic resistance gene grow
• w/o insert with insert
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Selection II
• E.coli with religated plasmid growth on medium containing antibiotics
• blue colonies due to active ßgalactosidase
• E.coli with plasmid and insert growth on medium containing antibiotics
• white colonies due to disrupted ßgalactosidase gene
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• Mini (2 ml), Midi (50-200 ml), Maxi (>400 ml)
• harvesting of cells by centrifugation
• lyses of cells by basic pH (NaOH)
• neutralization by acetate
• on column washing
• elution of purified plasmid DNA with buffer or water
Preparation of plasmids
37°C
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• cut with restriction enzyme
• agarose gel analysis:– size and conformation of nucleic acids in a sample
– approximate quantification of DNA
– separation and extraction of DNA fragments for recloning
• sequencing
Identification of plasmid DNA
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advantages
• low costs
• time efficient
• high yield
• many systems with differentlyregulated promotors
disadvantages
• no posttranslationalmodification
• inclusion bodies
Bacterial expression systems
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• “molecular farming”
• gene technology production of proteins from recombinant DNA
• expression vectors with poly linker site, marker and origin of replication
• initiation of transcription is regulated by distinct RNAP binding sites (promotor)
• translation dependent on binding sites for ribosomes (Shine-Dalgarno)
• E. coli regulation sequences need to be ahead of cloned gene
Expression of heterologous proteins in E. coli
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• expression in inclusion bodies:
– higher amounts of protein– decreased proteolytic
degradation– easy to purify– refolding and purification steps time-consuming, loss of protein
• expression in membranes:
– lower expression level than in IB– “correct folding“ of the protein– lipid environment– challenge: protein purification
Expression of protein in E.coli
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• in bacteria cytoplasmic aggregates of proteins
• usually sites of viral multiplication that consist of viral capsid proteins
• in human cells neuronal i.b. in disorders like Parkinson’s disease
• do not only contain misfolded protein
• results of expression of genes from an organism in another
• eucaryotic cDNA expressed as recombinant gene in procaryots risks the formation of inactive aggregates of proteins due to a foreign microenvironment (pH, osmolarity, folding mechanisms, processing systems)
inclusion bodies
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different protein species
separation of cells fromsupernatant
diluted, low proteinyield
cell disruption
isolationrenaturation
renaturation problems, low ionic strength
cell disruption
lipids, proteases, high protein yield
separation of unsoluble material (cell membranes, cell walls,…)
chromatography
intracellular proteins inclusion bodies extracellular proteins
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• cell suspensions are pressed through very narrow spaces
purification of proteins from inclusion bodies
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cellbankampoulle
shaking flask100 – 500 ml
prefermenter50 l
production fermenter5,000 l
biomass harvesting
washing
cell breakage
harvest inclusion bodies
washing
purified inclusion bodies
Industrial isolation of inclusion bodies
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cell disruption
• chemical (EDTA, lyzozyme, hypotonic solutions, Triton-X 100)
• freezing and thawing
• mortar and pestille
• vibration mills
• ultrasonics
• french press (small volume)
• Manton-Gaulin homogenator (V > 1 l)
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• unphysiological environment inactivation, denaturation, proteolysis
• changes in pH buffer
• prevention of oxidation DTT, ß-mercaptoethanol (mM)
• complexation of metall ions EDTA
• low ionic strength NaCl, KCl (50 – 100 mM)
• aggregation of proteins Triton-X 100 (0.02%)
protection of proteins
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• once from pancreas of cattle and pig– immunological reactions
– 50 pigs per diabetic person per year
• 1982 production in genetically engineered bacteria (NovoNordisk)
• today production in bacteria and yeast
Production of human insulin I
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CUT
LIGATION
TRANSFORMATION
FERMENTATION
PURIFICATION
Production of human insulin II
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• characteristics and „content“ of a plasmid
• transformation efficiency and antibiotic activity
• advantages and disadvantages of E.coli as an expression system
• purification of different protein species
Just to sum up…
E. coli is your friend