molecular cloning
DESCRIPTION
Lecture 6. Molecular cloning. With thanks to David Tscharke @ RSB. Lecture overview. AKA gene cloning or DNA cloning The generation of identical copies of a piece of DNA Propagated in bacteria that originate from a single cell Was the first recombinant DNA technology - PowerPoint PPT PresentationTRANSCRIPT
Molecular cloning
Lecture 6
With thanks to David Tscharke @ RSB
Lecture overviewAKA gene cloning or DNA cloning
The generation of identical copies of a piece of DNA
-Propagated in bacteria that originate from a single cell
Was the first recombinant DNA technology
-Joining of DNA from one species to another such that both are propagated biologically
-Molecular Meccano, Molecular Lego…
Brings together a number of techniques in molecular biology
Nothing to do with reproductive cloning!
Cloning DNA into plasmidsBacterial culture Extract plasmid
Cut DNA with restriction enzymes - generate cohesive ends
+ligase
transformation
In v
ivo
In v
itro
Propagate in bacterial culture
“Insert”
Why clone in plasmids and bacteria
Amplificationup to 200x
Amplificationx billions!
Establish and maintainclonal purity
Why clone in plasmids and bacteria
extract
Express protein
Transfer to:- eukaryotic cells - plants- fungi- virus- etc…
Modify
Amplification in culture
Extractproduct
Expressin vitro
Move to new vector
Determinesequence
Cloning versus sub-cloningCloning is when the source of the ‘insert’ DNA is from genomic DNA, cDNA or another non-plasmid source
RE cut
RE cut
Or PCR
Cloning versus sub-cloningSubcloning is when the source of the ‘insert’ DNA is from another plasmid
RE cut
RE cut
Or PCR
Cloning is a marriage of techniquesCloning requiresEasy to extract and manipulate DNA ‘vector’ or host-Plasmid-Bacteriophage
Tools for cutting and joining DNA-Restriction enzymes-Ligase
Ability to move the new DNA back in vivo-Transformation-Selection and screening
Ability to identify clones of bacteria harbouring the desired recombinant plasmid-Colony PCR and sequencing
Easy to extract and manipulate DNA ‘vector’ or host-Plasmid-Bacteriophage
The essentials for a cloning vector
EcoRI
PstIBamHI
Unique restriction
sites
Antibiotic resistance
Origin of replication
The essentials for an overexpression vector
Antibiotic resistance
Origin of replication
Gene of interestRibosome binding site= Shine-Dalgarno sequence
promoter terminator
Antibiotic resistance
Antibiotic Target Mechanism of of action resistance
ampicillin murein-layer -lactamase bacterial cell wall
kanamycin ribosomal 30S aminophosphotransferase
subunit
tetracyclin ribosomal 30S efflux (pump)
subunit
chloramphenicol ribosomal 50S chloramphenicol
subunit acetyltransferase
For selection of bacteria containing the plasmid
Antibiotics and mechanism of resistance
ampicillin
-lactamase
kanamycin
APHs phosphorylate
Antibiotics and mechanism of resistance
chloramphenicol
tetracyclin
One or both OH groups acetylated bychloramphenicol acetyltransferase (CAT)
Exported by efflux pump (transmembrane protein)
Footnote to penicillin resistance-lactamase acts in the periplasmic space and is secreted intothe culture medium
“Satellites”
Cells without resistance gene can survive in the neighborhoodProblem occurs if -ampR is on high copy number plasmid-cells are allowed to grow for a long time (“stationary phase”)
Multiple cloning site
Cluster of unique restriction enzyme recognition sites used to insert foreign DNA-Abbreviated to MCS, also called a polylinker
23 restriction enzyme sites shown
Summary
Essential elements of a plasmid cloning vectorOri – origin of replication-e.g. ColE1 originSelection marker – antibiotic resistanceAt least one unique restriction site
A deluxe plasmid cloning vector has:Restriction sites clustered in an MCS
Cloning is a marriage of techniquesCloning requires
Tools for cutting and joining DNA-Restriction enzymes-Ligase
Ability to move the new DNA back in vivo-Transformation-Selection and screening
Ability to identify clones of bacteria harbouring the desired recombinant plasmid-Colony PCR and sequencing
Easy to extract and manipulate DNA ‘vector’ or host-Plasmid-Bacteriophage
SmaI
EcoRI
You need an enzyme to cut DNA
5’ GAATTC G AATTC
3’ CTTAAG CTTAA G
5’ CCCGGG CCC GGG
3’ GGGCCC GGG CCC
5’ CTGCAG CTGCA G
3’ GACGTC G ACGTC
5’ overhang5’ protruding5’ sticky
3’ overhang3’ protruding3’ sticky
blunt
PstI
Ends can be re-joined by ligase if the 5’ phosphate is intact
Ligase
Lodish Fig. 9-11
Ligase repairs brokenphosphodiester bonds-Uses ATP (one for each bond repaired)-Most common enzyme for joining DNA in vitro
Ligase
Ends that can be re-joined by ligase
Any blunt end to any other blunt end
Sticky ends as long as they are ‘cohesive’-Overhanging bases must be complementary
Ligated ends can be cut again if the recognition site is regenerated
Let’s play the ligation game!
Vector and insert must be cut with enzymes that produce ends
compatible for ligation
Several wells joined
+
Often need to purify the cut DNAPreparative gel electrophoresis
Restriction digest
cut fragments out
Purify (silica)
Temperature problem for ligationThe problemAssociation of DNA ends is best at low temperature-Also hybridisation of short sticky ends
But ligase works best at 37 oC
Solutions includeLong incubations at 16 oC (overnight)
Molecular ‘crowding agents’ and loads of ligase-Polyethylene glycol (PEG), long chain polymer-10x more ligase
Summary
Joining DNA in vitro
Digestion of plasmid DNA with restriction enzymes
Isolation of target DNA and digestion to generate compatible ends
Often gel purification of fragments
Ligation of the two DNA molecules
It’s trickier than it might appear at first
Cloning should be this simple
+ligase
Also need:-T7 promoter and RBS before gene of interest-T7 terminator after gene of interest
5’ GAATTC AATTC
3’ CTTAAG G
2 different restriction sites
NdeI
EcoRI
Ends not cohesive
EcoRI5’ CATATG CA
3’ GTATAC GTAT
NdeI
Plasmid re-ligates only with insert-Plasmid cannot ligate with plasmid-Insert cannot ligate with insert
Plasmid and insert must be cut with same restriction enzymes
In the plasmid:
What if…
The insert has internal NdeI or EcoRI sites Use different restriction sites-taking care that a RBS is at the right place….
Order a synthetic gene -only $0.5 per base pair
Use a restriction-free method
Cloning methods for the 21st century
Ligations can be tricky-Needs matching overhang -Ligase needs phosphorylated 5’ ends-blunt-ended ligations (i.e. no overhang) 10-fold less effective Restriction-free methods20 nucleotide overlaps are better than 1-4 nucleotide overlaps- But no restriction enzyme delivers large overlaps
SLIC “sequence and ligation independent cloning”
CPEC “circular polymerase extension cloning”
SLiCE “seamless ligation cloning extract”
SLIC Appl. Eviron. Microbiol. 2012 doi:10.1128/AEM.00844-12
Linearize vector-Cut with restriction enzyme
Combine linearized vector and insert-Add T4 DNA polymerase (2.5 min at room temperature)-Put on ice to stop digestion-10 min annealing (on ice)
Transform cells
T4 DNApol
3’ 5’
5’ 3’
Polymerase activityExonuclease activity
SLIC – why it works
T4 Pol exonuclease generates 5’ overhangs in plasmid and insert-Ends must be complementary for about 20 nucleotides-Spontaneous hybridisation
-Don’t worry about 5’ phosphorylation-No ligase -Segments of ssDNA acceptable
The polymerases, kinases and ligases present in E. coli do the rest to heal the vector!
Potential problemAnnealing at low temperature can lead to mis-hybridization
CPEC Nat. Protoc. 6, 242 (2011)
Use the strands from the insert as primers-“Overlap extension PCR”
Melting temperatures of all overlapping regions must have similar Tm
-Tm between 60 and 70 oC for stringent hybridization-15 – 35 bases overlap
Denatured linearized vector
Denatured insert
CPECAnneal
One round of overlap extension PCR
Transform cells-E. coli heals the nicks in the vector
Potential problemVector copy made by PCR-Even high-fidelity polymerases introduce ~1 error per 9000 bp
A vector can be linearized by PCRA single round of PCR makes a linear copy -Use high-fidelity polymerase (e.g. Phusion)
Errors do not propagate in a single round
Primers designed to introduce overlapping regions with the insert
SLiCE Nucl. Acids Res. 40, e55 (2012)
recombination system- Needs 20 nucleotide overlap- Ligates insert into vector even if the vector has some non-matching overhangs- Express necessary enzymes in a special E. coli strain and use cell-extract- Incubate linearized plasmid + insert for 30 min at 37 oC
Transform cells
E. coli enzymes do the rest
Recombination systemBacteriophage -Double-stranded DNA bacteriophage
Lives as Dr Jekyll and Mr Hyde1) Lysogenic phase-Viral DNA integrated in host chromosome (“recombination”)-Not killing E. coli-Replicated together with E. coli genome
2) Lytic phase-Phage replicates-Lyses E. coli cell after 45 min at 37 oC-Releasing ~100 progeny phages
The recombination system inserts phage DNA into E. coli genome
SLiCE is niceHigh fidelity-Clean insertion by phage recombination enzymes-Plasmid replicated by E. coli, not by PCR -> high fidelity
- Blunt ends are good: little accidental re-ligation of empty vectors
15 bp same in phage and E. coli DNA
SummarySLIC-Overhangs by exonuclease activity of T4 DNA polymerase
CPEC-Overlap extension by PCR
SLiCE recombination system
All need a linearized vector-By restriction enzyme-By overlap extension (PCR)
And an insert with matching ends-20 complementary bases
Anything that can happen will…
+ligase
x2 (or more)
&
& linear or circularmultimers
& multimers of insert
& either orientation