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.