Cloning Paste Vectors
21
Cloning – pasting into vectors and transformation
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Plasmid Vectors
Transcript of Cloning Paste Vectors
Cloning – pasting into vectors and transformation
Cloning
Fragment of DNA
Vector
Recombinant vector
Clone(s)
Host
Vectors• Carriers of DNA – transfer and replicate inserted DNA– Plasmid – circular dsDNA
• In nature: contain non-essential genes• In the lab: highly modified and engineered to be easier to manipulate
and contain useful components
Plasmid vectors• Circular
Plasmids can be used as vectors• Useful restriction sites for cloning into vectors are– Unique – only one recognition sequence in the whole plasmid– Clustered – many different recognition sequences close
together in a multiple cloning site (polylinker)
Plasmid vectors• Circular• Multiple
cloning site
EcoRI BamHI HindIII KpnI
5’ …ACGGAATTCAAAGGATCCTTTAAGCTTAAAGGTACCACG… 3’3’ …TGCCTTAAGTTTCCTAGGAAATTCGAATTTCCATGGTGC… 5’
EcoRI BamHI HindIII KpnI
Ligating DNA into a plasmid• The DNA ends must be compatible
5’ …ACGGAATTCAAAGGATCCTTTAAGCTTAAAGGTACCACG… 3’3’ …TGCCTTAAGTTTCCTAGGAAATTCGAATTTCCATGGTGC… 5’
EcoRI BamHI HindIII KpnI
5’ …ACGG3’ …TGCCTTAA
EcoRI
Ligase
5’ AATTC3’ G
G 3’CTTAA 5’
AATTCAAAGGATCCTTTAAGCTTAAAGGTACCACG… 3’GTTTCCTAGGAAATTCGAATTTCCATGGTGC… 5’
5’ …ACGG3’ …TGCCTTAA
AATTCAAAGGATCCTTTAAGCTTAAAGGTACCACG… 3’GTTTCCTAGGAAATTCGAATTTCCATGGTGC… 5’
AATTC G
G CTTAA
Ligating a PCR product with a plasmid
• Because PCR primers are incorporated into the product, you can use primers to “add” sequences to products
GCCCCACCACGCCTCATGTG
5’ TGGGCGCCCCACCACGCCTCATGTGTGAC……GCCTGCAGGACAGGGGACAGATGACCA 3’
3’ ACCCGCGGGGTGGTGCGGAGTACACACTG……CGGACGTCCTGTCCCCTGTCTACTGGT 5’
CGTCCTGTGGGGTGTCT5’GAATTC
PCR
GAATTCCTTAAG
GAATTCCTTAAG
EcoRI
AATTC G
GCTTAA
CTTAAG 5’
Ligase
DNA ligase• Compatible ends come together, sealed by DNA ligase– In nature: joins Okazaki fragments
• DNA ligase forms covalent bond to seal backboneNH
N
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NH2N
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OH
5 ’ … G T A G3 ’ … C A T C
C G T … 3 ’G C A … 5 ’
5 ’ … T C A G3 ’ … A G T C C T A G
A T C C G T … 3 ’ G C A … 5 ’
5 ’ … A A A G3 ’ … T T T C C T A G
G A T C C G T … 3 ’ G C A … 5 ’
Ligase only joins ‘compatible ends’• Sticky ends are compatible if the overhangs are
fully complementaryLigase
Ligase
Ligase
• Blunt ends are always compatible (but not very ‘sticky’)
5 ’ … A A A G3 ’ … T T T C C T A G
G A T C C G T … 3 ’ G C A … 5 ’
5 ’ … T C A G3 ’ … A G T C C T A G
A T C C G T … 3 ’ G C A … 5 ’
5 ’ … G T A G3 ’ … C A T C
C G T … 3 ’G C A … 5 ’
Transformation• Plasmids are transformed into “competent” bacteria
(able to take up DNA from the environment)
Plasmid vectors• Circular• Multiple
cloning site• Small Each colony arises from a single bacterial cell –
therefore all cells in a colony are genetically identical – these cells in each colony are a “clone”
Competent cellsRecovery &
growth
Heat or electricity
Plasmid replication in the host• If there is just one plasmid in a cell… how does it share?
Plasmid vectors• Circular• Multiple
cloning site• Small• ORIoriV
MCS
V for vegetative – allows many
copies
• Therefore plasmid needs an origin of replication (ori)
Selecting for transformants• Not all bacteria will be transformed
Competent cells
• How to tell the difference between transformants and non-transformants?– Kill the non-
transformants!
Transformant
Non-transformant
Selecting for transformants• Ampicillin (an antibiotic) kills bacteria• β-lactamase breaks down ampicillin
Media containing ampicillin
Transformant: resistant
Non-transformant:
sensitive
Selecting for transformants• Selectable markers – genes that can produce easily
observed characteristicsPlasmid vectors• Circular• Multiple
cloning site• Small• ORI• Selectable
marker(s)
ampR
• E.g. the ampR gene can be transcribed and translated by the host to produce β-lactamase
Selecting for plasmids with inserts
• How do we ensure the plasmid contains an inserted piece of DNA (recombinant)?
Plasmid vectors• Circular• Multiple cloning site• Small• ORI• Selectable marker(s)• Screenable marker
ampR
oriV
MCS (within lacZ gene)
lacZ
Competent cells
• lacZ gene (screenable marker) encodes β-galactosidase
X-gal
Blue compound
Insertional inactivation Media containing
ampicillin & X-gal
Reco
mbi
nant
Non
-rec
ombi
nant
Beta-galactosidase crystal image from Wikimedia Commons
Selecting for plasmids with inserts
• Colony PCRChecking for inserts in plasmids
Pick colony Replica plateAdd to PCR tube and
amplify
• Restriction digestionChecking for inserts in plasmids
Pick colony Replica plate
Grow small-scale culture
Purify plasmid
DNA
• SequencingChecking for inserts in plasmids
Sequence DNA
Pick colony Replica plate
Grow small-scale culture
Purify plasmid
DNA
Two real-life vectors
Plasmid vectors• Circular• Multiple cloning site• Small• ORI• Selectable/screenable
marker(s)
http://www.fermentas.com/templates/files/tiny_mce/family_images/sd004_fam.gif and http://www.fermentas.com/templates/files/tiny_mce/family_images/sd005_fam.gif
Cloning
Selected fragment of DNA
+ vector
= recombinant vectors
= clone(s)
+ host
Learning objectives– Determine and manipulate sequences in relation to molecular
operations on DNA (e.g. digestion and ligation)– Identify and explain the components of cloning vectors and
extrapolate the function(s) and action(s) of these components
