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