Post on 03-Jun-2018
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Cloning in bacteriaPresenter: Vito Baraka
(BSc,MSc Cand.)
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Introduction
DNA cloning involves separating a specific gene or DNA
segment from a larger chromosome, attaching it to a smallmolecule of carrier DNA, and then replicating this modifiedDNAboth an increase in cell number and the creation of multiplecopies of the cloned DNA in each cell. A cloneis an identicalcopy
First proposed by Peter Lobbane et al 1973 at the StanfordUniversity.Lobbanet al1973 "Biochemical Method for Inserting New Genetic Information intoDNA of Simian Virus 40: Circular SV40 DNA Molecules Containing Lambda PhageGenes and the Galactose Operon of Escherichia coli
Cloning was made possible by the discovery, isolation andapplication of restriction endonucleases by Werner et al(1970)
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Overview cloning bacteria
Key steps
1. Cutting DNA at preciselocations.
2. Cloning vector3. Joining two DNAfragments covalently
4. Transformation to a hostcell
5. Selecting or identifyinghost cells that containrecombinant DNA
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1.How can we cut DNA at specific sites?
Restriction endonuclease enzymestype II binds to DNA at a specific
sequence and make a double-stranded cut at or near thatsequence
Can form sticky or cohesive ends orBlunt ends
Most recognition sequences arepalindromic
eg
..live not on evil
The sticky ends are importantfor annealing. DNA ligase willeventually form a covalentbond btn the sugar-
phosphate
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2.Cloning vectors
Allow amplification of inserted DNA fragments
Developed fromnaturally occurring bacterialplasmids
Contain an origin of replication(ori )
Contain numerous restriction
sites
Contain genes that conferresistance to antibiotics, thusallowing selection of
bacterial colonies carrying theplasmid
Introduced into competentbacterialcells by transformation
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2.Cloning vectors cont..Expression vectors
Vectors that can yield the proteinproducts of the cloned genes.
For active gene expression:
i)strong promoter
ii)ribosome binding site near ATG
codon
The main function of an expressionvector is to yield the product of agene, therefore a strong promoter is
necessary.
The more mRNA is produced, themore protein product is made.
Oligohistidine regions likethis have a high affinity formetals like nickel, so
proteins that have suchregions can be purifiedusing nickel affinitychromatography
ATG
(His)6
MCS
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3. Joining two DNA fragments covalently
DNA LigaseDNA ligase ,much more efficient with
sticky ends, for blunt end not efficient.
Linkers: short, double-stranded, blunt-ended, DNA fragments.Contain a stickyend restriction site.
Adaptors.These are linkers with cohesiveends or a linker digested with RE,before ligation. By adding adaptors tothe ends of a DNA, sequences that areblunt can be converted into cohesiveends.
Homopolymer tailing (TdT). Terminaldeoxynucleotidyl transferase addssingle stranded tail in the presence ofone nucleotide producing sticky ends.
Fig1.Linkers
Fig 2.Adaptor
3 Li ti t
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3.Ligation cont..
What next ???
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4.Transformation a host cell
Most bacteria, including E. coli,only take up a limited amountof DNA.In GE bacteria aretreated to increase uptake.
Following treatment, cells aresaid to be competent
Treating growingE. coli
cells withsolutions (CaCl2 and MgCl2)the cells are madecompetent to take up DNA.
Incubate thawed cells with DNA,then heat-shock at 42C for30 seconds (DNA is taken upby cells).
Spread plate out on appropriateselection media
1: Mix competentcells and plasmidvector
2: Keep on icefor 30 min
3: Heat shock at42C 30 sec
4:Plate out cells on
Ampicillin
selective medium
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4.Transformation contOnly tranformed
E.Coli will grow onAmplicilinsupplemented media
Transformantionefficiency:
Can be calculated as
number of coloniesformed per mg of inputDNA.
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5:Selecting/Screening
Transformed cellswill grow
on selective media
Others will not!
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5.Selection/screening cont
Interrupting LacZ
Lac Z gene
Beta-galactosidase
X-gal
(colorless)Gal + X(Blue dye)blue colonies
pUC18
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Analysis of recombinants
For analysis ofrecombinants:
Colony PCR and resolve on
agarose
Perform restriction digestionanalysis
Sequencing
Immunoblotting forexpression vector
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Some cloning applications
Human insulin, Human growth hormone,
interferons, growth factors, blood clotting factors,Plasminogen activator, tumor necrosis factor,novel recombinant antibodies
Synthetic peptides as recombinant vaccines(Hep B, malaria, rabies, HIV/AIDS)
Gene Transfer in plants---increased yield,improved tolerance, herbicide res., diseaseresistance
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Aknowledgment
Steven and Danielle
Classmates
All IPMB staff
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...je vous remercie de votre attention