METODI DI CLONAGGIO: Polymerase Incomplete Primer Extension (PIPE) Method

Non richiede digestioni con enzimi di restrizione

Non necessita reazioni di ligasi

Veloce

Utilizzabile per clonaggio di frammenti di DNA in plasmidi e per mutagenesi

METODI DI CLONAGGIO: Polymerase Incomplete Primer Extension (PIPE)

Protocollo:• V-PIPE PCR: amplificazione del vettore• I-PIPE PCR: amplificazione dell’inserto • Mix prodotti di PCR V-PIPE e I-PIPE• Trasformazione di cellule competenti

Gene ccdB is lethal to most strains of E. coli. 'Empty' destination vectors are therefore selected against upon transformation of E. coli cells with the recombination reaction

I-PIPE PCR product

V-PIPE PCR product

5’5’

MixAnnealing intermolecolare

trasformazione

Le cellule vengono trasformate solamente dal plasmide chiuso (ricombinato)

5’5’

3’

3’

METODI DI CLONAGGIO: Polymerase Incomplete Primer Extension (PIPE)

CLONAGGIO

METODI DI CLONAGGIO: Polymerase Incomplete Primer Extension (PIPE)

MUTAGENESI

METODI DI CLONAGGIO: Polymerase Incomplete Primer Extension (PIPE)

MUTAGENESI

This cloning technology is based on the nature of site-specific recombination: the integrative and excisive recombination reactions between chromosomes of Bacteriophage lambda and E. coli bacterium.

These biochemical reaction processes, performed in vitro, are the basis of the Gateway® Cloning Technology.

Gateway® Cloning Technology

The integrative recombination is catalyzed by Int (integrase) and IHF (Integration Host Factor). The recombination between attB and attP sites results in attL and attR sites that flank the integrated lambda DNA. Three proteins (IHF, Int and Xis) are required for the excisive recombination. The attL and attR sites located at both sides of the inserted phage genome DNA recombine site-specifically during the excision event, reforming the attP site in lambda and the attB site in the E. coli chromosome.

The Gateway® Reactions

LR clonase

++BP clonase

attL1 attL2

EntryClone

KanR

ExpressionClone

attB1 attB2

AmpR

attR1 attR2

ccdB

DestinationVector

AmpR

attP1 attP2

ccdB

DonorVector

KanR

LR Clonase= enzyme mix of Int, IHF, Xis catalyising in vitro recombination between an Entry clone (containing a gene of interest flanked by attL sites) and a Destination vector (containing attR sites) to generate your expresion clone.

BP Clonase= enzyme mix of Int and IHP catalyising the in vitro recombination of PCR products or Dna segments from clones (containing attB sites) and a Donor vector (containing attP sites) to generate the Entry clones

Different ways to generate the entry clone

2. TOPO® Cloning

TOPO®BP Clonase™

1. BP Cloning

PCR Product

+TOPO-ActivatedEntry Vector

L1 L2

Gene+attB PCR Product

B2GeneB1Donor Vector

P2ccdBP1

Entry Clone

L2GeneL1

+digested DNA Fragment

GeneB1digested Entry Vector

L2L1

4. Pre-made entry clone5. Custom-made entry clone

Ligase

3. Restriction/Ligase Cloning

ORF Collection

L2ORFL1

3. Restriction/Ligase cloning• Use when there are convenient sites to cut

insert out of another plasmid

• Must cut out ccdB gene by using one of four RE sites flanking the ccdB

• Reading frame of insert must be considered, as well as downstream expression elements

• Various reading frames of pENTR vectors are available

pENTR™ 1A Vector in reading frame 0

pENTR™ 2B Vector in reading frame +1

pENTR™ 3C Vector in reading frame +2

pENTR™ 4 Vector in reading frame 0; modified polylinker from 1A

pENTR™ 11 Contains both an E. coli (SD) and eukoryotic (Kozak) ribosome binding site

4. Pre-existing ORF collection16,272 human ORFs (Oct 2006

release)

Amber stop codons

Sequence verified

Ready to use in LR reactions

http://orf.invitrogen.com/cgi-bin/ORF_Browser

Invitrogen’s Ultimate™ ORF collection

1.BP Cloning – The Reaction

90-99% correct cloneson Kan plates

geneattB1 attB2

+attP1 attP2

ccdB

D o norVector

K anR

+

gene

attL1 attL2

EntryC lone

K anRccdBattR1 attR2

BP Clonase™

pDESTTM17

1.BP Cloning - Primer Design for PCR• GGGG and the attB1 sequence must be added to the 5’-primer

(sense)

• GGGG and the attB2 sequence must be added to the 3’-primer (antisense)

attB1

5’ – GGGGACAAGTTTGTACAAAAAAGCAGGCTNNN…

attB2

5’ – GGGGACCACTTTGTACAAGAAAGCTGGGTNNN…

Gene SpecificPrimer Sequence

PRIMERS PER RICOMBINAZIONE NEL pDONOR

FORWARD:

a) Per esprimere la proteina nativa o con tag C-terminale

b) Per esprimere la proteina con tag N-terminale

REVERSE:

a) Per esprimere la proteina nativa o con tag N-terminale

b) Per esprimere la proteina con tag C-terminale

The vector pDEST17 allows the addition of a polyhistidine tag to the target gene sequence. An inherent result of the cloning technology used is the presence of 21 supplementary amino acids at the N-terminus of the recombinant protein compared to the native protein. The majority of these additional amino acids are due to the recombination sequence attB1 and the histidine tag. A target sequence for the TEV protease is therefore added systematically to our current constructions, between the ORF and the attB1 recombination site. The elimination of 20 out of the 21 supplementary amino acids after digestion of the recombinant protein.

6-His

6-His

6-His

6-His

6-His

6-His

6-His

GB1

Z-Tag

Trx

GST

MBP

NusA

Target

TEV site: Glu-Asn-Leu-Tyr-Phe-Gln/Gly

pDEST17

pTH34

pTH30

pETG20A

pETG30A

pDESTHis-MBP

pETG60A

PROTEINE DI FUSIONE CON TAG N-TERMINALI

Plasmide di espressione

pDEST: Plasmidi di espressione (pET-derived) Possibilità di tag N- o C- terminali

TOPO Adapted Gateway Entry VectorTOPO Adapted Gateway Entry Vector

http://tools.invitrogen.com/downloads/gateway-entry-options-seminar.html

All entry clones have attL's on both sides of their gene of interest. • Necessary in the Gateway system because L's cut to form sticky ends by the Gateway recombination proteins.• These sticky ends will then match up with the sticky ends on a destination vector which contains attR restriction sites.

2. TOPO® Cloning – Directional TOPO®

METODI DI CLONAGGIO: TOPO CLONING_____________________

Mappa e sequenza del polilynker del plasmide pENTR/SD/D-TOPO

CACC facilitate directional incorporation into the pENTR/D-TOPO vector (obtained from Invitrogen).

topoisomerase molecules (TOPO) catalyze ligation of target and vector sequences

Once flanked by attL recombination sites, the sequence can be recombined with attR sites using the LR clonase reaction mix (Invitrogen). This reaction transfers the target sequence into a desired destination vector

Destination vectors contain a gene (ccdB) that is lethal to most strains of E. coli. 'Empty' destination vectors are therefore selected against upon transformation of E. coli cells with the recombination reaction

The Gateway® Technology is a universal cloning method based on the site-specific recombination properties of bacteriophage lambda and provides a rapid and highly efficient way to move DNA sequences into multiple vector

systems for functional analysis and protein expression.

Primers and PCR products

Gateway cloning

Entry vectors (pDONR)

E. coli P. pastoris Insect cells SFV mammalian Mammalian cells

pDest14pDest17pDest24pDest42….

pPICZC-GWpPIC3.5K-Dest1pPIC9K-Dest1

pDest8pDest10

pDest-SFV1 pDest12.2pDest26pDest27

Gateway technology

Gateway: LR ReactionGateway: LR Reaction

http://www.bio.davidson.edu/courses/Molbio/MolStudents/spring2000/patton/gateway.htm

The GatewayThe Gateway

http://www.bio.davidson.edu/courses/Molbio/MolStudents/spring2000/patton/gateway.htm

Gateway technology

Advantages

1) No restriction analysis of ORF prior to cloning.

2) No restriction digestion of the vector.

3) Fast, parallel sub-cloning into different expression vectors.

4) ~100% sub-cloning efficiency (no background).

5) Flexibility, automation.

6) Recombination sites may serve as linkers.

Disadvantages

1) Number of available expression vectors.

2) Mandatory recombination sites.

3) Very long primers containing the attB sites and also other sequences (TAGs) can be inefficient for the PCR reaction.

Bressanone, 2004

Gateway technology

Comments on disadvantages

1) number of available expression vectors :

a) Invitrogen provides a conversion cassette to make your favorite expression vector(s) a Gateway expression vector.

b) as a consequence, there is a growing list of new vectors.

2) mandatory recombination sites: attb1/2-encoded peptide would hamper crystallogenesis:

a) protease cleavage site (Tev…) can be added at primer synthesis.

b) short tags (His6) can be encoded by primers.