1. 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

4. 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

5. Annealing intermolecolare
METODI DI CLONAGGIO: Polymerase Incomplete Primer Extension (PIPE)
CLONAGGIO
I-PIPE PCR product 5’ 3’ 5’
Mix
Annealing intermolecolare
V-PIPE PCR product
trasformazione
Le cellule vengono trasformate solamente dal plasmide chiuso (ricombinato)

7. METODI DI CLONAGGIO: Polymerase Incomplete Primer Extension (PIPE)
MUTAGENESI

8. METODI DI CLONAGGIO: Polymerase Incomplete Primer Extension (PIPE)
MUTAGENESI

10. Gateway® Cloning Technology
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.

11. 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.

12. The Gateway® Reactions
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
As we discussed in the introductory section, the entry clone is the door to the Gateway® system. Once you have cloned your DNA fragment into a Gateway® entry vector, you can easily transfer it into a destination vector to generate the expression clone.

13. Different ways to generate the entry clone
PCR Product +
TOPO-Activated
Entry Vector L1 L2
Gene +
attB
PCR Product B2
Gene B1
Donor Vector P2
ccdB P1
BP Cloning
TOPO® Cloning
BP Clonase™
TOPO® L1
Gene L2
Entry Clone
Ligase
You can clone PCR products to make entry clones in three different ways:
By using a Gateway® BP cloning reaction
By using directional TOPO® or TOPO® TA Cloning
By using restriction enzymes and ligation reaction Or
and 5. You can use pre-made or customized entry clones
4. Pre-made entry clone
5. Custom-made entry clone
Restriction/Ligase Cloning +
digested DNA Fragment
Gene B1
digested Entry Vector L2 L1 L1
ORF L2
ORF Collection

14. 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
Your restriction strategy must be carefully planned. However, Gateway will make subsequent subcloning much easier.
Available vectors are: pENTR™ 1A, pENTR™ 2B, pENTR™ 3C, pENTR™ 4, pENTR™ 11.
Corresponding plasmid sequences, multiple cloning regions and restriction enzyme tables for all Invitrogen’s vectors can be found on the web site.

15. Pre-existing ORF collection
Invitrogen’s Ultimate™ ORF collection
16,272 human ORFs (Oct 2006 release)
Amber stop codons
Sequence verified
Ready to use in LR reactions
The Ultimate™ ORF collection contains over 16,000 human and 2,000 mouse open reading frames (ORFs). Each ORF has been cloned into a Gateway® donor vector, pDONR™221, using the BP recombination method. The ORF contains the particular gene of interest from the start to stop codon. To ensure integrity of the specific gene sequence, each ORF is fully sequenced from start to stop, and is guaranteed down to the amino acid sequence.
To express C-terminal tagged proteins in mammalian cells, the amber stop codon can be suppressed using the Tag-On-Demand™ technology.

16. BP Cloning – The Reaction+ +
BP Clonase™
90-99% correct clones
on Kan plates
To perform the BP Cloning reaction, you will combine the PCR product, which is flanked by attB sequences, with a pDONR™ vector and add BP Clonase™ II. The reaction is incubated at room temperature for an hour and then transformed into standard competent E. coli cells, such as DH5α, TOP10 or Mach1™.
The next day, you will have >90% correct clones in kanamycin resistant colonies. Efficiency is high because of the specificity of the recombination reaction (i.e., attB1 x attP1, attB2 x attP2), and negative selection is conferred by the presence of ccdB.
Let’s take a look at the attB sequences.

17. pDESTTM17

18. 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 Specific
Primer Sequence
The attB sequences are 21 bp in length. Of the four types of att sites, attB is the shortest. The addition of specific attB sequences to each primer confers directionality to the PCR product. Site-specific changes were made in the attB2 sequence to confer specificity in the recombination reaction. The attB sites were also engineered to eliminate stop codons. The 4G-residues added to 5'-end of each attB sequence improve the efficiency of the recombinase reaction. Reading frame is determined in attB1 by the two lysine-encoding codons (AAA AAA). If you clone in frame with these codons, you will stay in frame in all subsequent reactions. There is a similar rule for the construction of C-terminal fusion proteins: your sequence needs to be in frame with: TTT GTA.

19. PRIMERS PER RICOMBINAZIONE NEL pDONOR FORWARD:
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

20. 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.

21. PROTEINE DI FUSIONE CON TAG N-TERMINALI
TEV site: Glu-Asn-Leu-Tyr-Phe-Gln/Gly
Plasmide di espressione
Target
pDEST17
pTH34
pTH30
pETG20A
pETG30A
pDESTHis-MBP
pETG60A
6-His
GB1
Z-Tag
Trx
GST
MBP
NusA

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

23. TOPO Adapted Gateway Entry Vector
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.
The key to TOPO® Cloning is DNA topoisomerase I. The biological role of the enzyme is to cleave and rejoin DNA during replication. Vaccinia virus topoisomerase I specifically recognizes the pentameric sequence 5´-(C/T)CCTT-3´ and forms a covalent bond with the phosphate group of the 3´ thymidine. It cleaves one DNA strand, enabling the DNA to unwind. The enzyme then re-ligates the ends of the cleaved strand and releases itself from the DNA.
To harness the religation activity of topoisomerase, TOPO® vectors are provided linearized with topoisomerase I covalently bound to each 3´ phosphate group. This enables the vectors to readily ligate DNA sequences with compatible ends. Vectors with protruding T- overhangs on both the 5’ and 3’ ends are available to clone PCR products (up to 5 kb) produced by Taq polymerase. This is called TOPO® TA cloning.
An example of a TOPO®-adapted entry vector is our pCR8/GW/TOPO® TA plasmid, with attL1 and attL2 flanking sites. This is one of the few entry vectors with a resistance different from kanamycin, making it compatible with a variety of destination vectors bearing ampicillin or kanamycin resistance.

24. TOPO® Cloning – Directional TOPO®
Directional joining of double-stranded DNA using TOPO®-charged oligonucleotides occurs by adding a 3 single-stranded end (overhang) to the incoming DNA. This single-stranded overhang is identical to the 5 end of the TOPO®-charged DNA fragment. At Invitrogen, this idea has been modified by adding a 4 nucleotide overhang sequence to the TOPO®-charged DNA.
In this system, PCR products (up to 5 kb) are directionally cloned by adding four bases to the forward primer (CACC). The overhang in the cloning vector (GTGG) invades the 5 end of the PCR product, anneals to the added bases, and stabilizes the PCR product in the correct orientation. Inserts can be cloned in the correct orientation with high efficiency.

25. METODI DI CLONAGGIO: TOPO CLONING_____________________
Mappa e sequenza del polilynker
del plasmide pENTR/SD/D-TOPO

26. 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

27. 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.

28. Primers and PCR products
Gateway technology
Primers and PCR products
Gateway cloning
Entry vectors (pDONR)
E. coli
P. pastoris
Insect cells
SFV mammalian
Mammalian cells
pDest14
pDest17
pDest24
pDest42 ….
pPICZaC-GW
pPIC3.5K-Dest1
pPIC9K-Dest1
pDest8
pDest10
pDest-SFV1
pDest12.2
pDest26
pDest27 28

29. Gateway: LR Reaction

30. The Gateway

31. 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. 31

32. 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.
Bressanone, 2004 32