1. Gateway Cloning
13 November 2013

2. Cloning method based on site specific recombination
E. coli Phage lambda

3. Cloning method based on site specific recombination
2 enzymes only
Clone of larger inserts (>5kb )
Clone up to 4DNA fragments at once
Widely adopted (>1500 refs). “Open access”
Highly efficient

4. Cloning method based on site specific recombination
2 enzymes only
Clone of larger inserts (>5kb )
Clone up to 4DNA fragments at once
Widely adopted (>1500 refs). “Open access”
Highly efficient

5. Cloning method based on site specific recombination
2 enzymes only
Clone of larger inserts (>5kb )
Clone up to 4DNA fragments at once
Widely adopted (>1500 refs). “Open access”
Highly efficient

6. Cloning method based on site specific recombination
2 enzymes only
Clone of larger inserts (>5kb )
Clone up to 4DNA fragments at once
Widely adopted (>1500 refs). “Open access”
Highly efficient
Typical recombination
Expected # colonies per
Number of
recombining
fragments
10 L reaction ef
ficiency (%) 2 10 3 - 5 80
100 4 70 90 30 1 6

7. Phage l
attP
cos
232 bp
attB
E. coli x
21 bp
attL
Lysogen
attR
96 bp
157 bp
Integration
(Int, IHF)
Excision
(Int, IHF, Xis)
Relies on five sets of specific and non cross-reacting att sequences.
4 classes: attB, attP, attL, and attR.
The specificity is given by the 7 nucleotides of the core region
Bacteriophage lambda att site recombination is a well-characterized phenomenon.
In bacteria, there is a stretch of DNA called attB, (B stands for bacteria), and in the phage there is a stretch of DNA called attP (P stands for phage). When the phage infects a bacterium, the injected lambda DNA recombines with the corresponding bacterial DNA via the att sites in the presence of integration-specific enzymes. When an attB site recombines with an attP site, the outcome is integration of the phage DNA into the bacterial genome. Once integrated, the hybrid recombination sites are called attL and attR (L stands for left, R stands for right). These recombination reactions (“LR” and “BP”) are the basis of the Gateway® Cloning System
If the phage undergoes the lytic phase, phage DNA can excise itself from the bacterial DNA. In the presence of a different set of recombination and excision enzymes, the attL site recombines with the attR site, resulting in phage DNA separation from the bacterial genome.

8. Entry clone + Destination Clone = Your vector
With transposition sites
KanR
AmpR
CamR
Entry clone + Destination Clone = Your vector

9. + + Create an Entry Clone (1.5 days )
Amplify DNA of interest with primers with attB overhangs.
Choose of primers critical for:
Number/position of DNA fragments to be inserted
Direction of insertion
Clean DNA fragment and perform a BP reaction with a Donor vector
OR: use restriction enzymes
Transform and plate cells (Dh5α) and screen colonies (M13 primers)
Create your vector (1.5 days )
Perform a LR reaction with the entry vector(s), destination vector
Transform and plate cells (Dh5α) and screen colonies +
BP Clonase™
1-2h RT
LR Clonase™ +
4/6h-O.N. RT

10. 2-fragment MultiSite Gateway® Pro
PCR Fragments
attB1
attB5r
attB5
attB2 X X X X
BP reactions
pDONRs
attP1
attP5r
attP5
attP2
attL5
attL2
Entry Clones X
attL1
attR5 X
In MultiSite Gateway® and MultiSite Gateway® Pro, entry clones are also constructed via BP recombination but in order for them to have the correct configuration in the final LR assembly reaction, a combination of flanking attL and attR sites is used. This is facilitated by the modular nature of the att sites. A different set of pDONR vectors is required.
In this example a 2-fragment recombination using MultiSite Gateway® Pro is shown. Here, by reversing the “standard”’ orientation of the attB5 site to an attB5r site in the PCR fragment, and by doing the same with its cognate attP5 counterpart in the donor vector, an attR5, instead of an attL5 is generated in one of the entry clones. The second entry clone bears a standard attL5 sequence that allows pairing with attR5 and the generation of an attB5 via LR recombination. This concept is key to the function of the MultiSite Gateway® system.
To perform the LR reaction the entry vectors are mixed with an appropriate destination vector and LR Clonase™ II Plus. The reaction is incubated for 16 hours at room temperature and an aliquot is used to transform E. coli competent cells. Recombinants are selected using the destination vector’s antibiotic resistance.
Destination Vectors
attR1
attR2
LR reaction
Expression clones
attB1
attB5
attB2
Invitrogen Proprietary & Confidential

11. 3-fragment MultiSite Gateway® Pro
PCR Fragments
attB1
attB4
attB4r
attB3r
attB3
attB2 X X X X X X
pDONRs
attP1
attP4
attP4r
attP3r
attP3
attP2
BP reactions
attL1
attL4
attL3
attL2
Entry clones
attR4
attR3
The same rationale is applied to a 3-fragment recombination scheme using MultiSite Gateway® Pro. However, a different arrangement of the att sequences and a different set of pDONR vectors is required as shown in this slide.
Destination vector
attR1
CmR
ccdB
attR2
LR reaction
Expression clone
attB1
attB4
attB3
attB2
Invitrogen Proprietary & Confidential

12. 4-fragment MultiSite Gateway® Pro
PCR Fragments
attB1
attB5r
attB5
attB4
attB4r
attB3r
attB3
attB2 X X X X X X X X
pDONRs
attP1
attP5r
attP5
attP4
attP4r
attP3r
attP3
attP2
BP reactions
attL5
attL4
attL3
attL2
Entry Clones X X X
attL1
attR5
attR4
attR3 X
And the same applies to a 4-fragment recombination scheme using MultiSite Gateway® Pro.
Destination Vectors
attR1
attR2
LR reaction
Expression clones
attB1
attB5
attB4
attB3
attB2
Invitrogen Proprietary & Confidential

13. att sites with 27 bp

14. Exemple of a pDEST clone

15. Advantages Disadvantages for us
Big library of clones created and available for free
Zebrafish and drosophila labs
FPs in N or C terminal
Localization signals
Cleavage signals (expl 2A peptide)
poly A tails
Promoters
Genes of interest (expl cell ablation (NTR, KillerRed)
Specific cassettes (brainbow, CRISPR)
MCS
Support pages
Lawson lab
Wiki page
Specific transposition systems
Library of destination vectors would need to be build

16. Advantages Disadvantages for us
Big library of clones created and available for free
Zebrafish and drosophila labs
FPs in N or C terminal
Localization signals
Cleavage signals (expl 2A peptide)
poly A tails
Promoters
Genes of interest (expl cell ablation (NTR, KillerRed)
Specific cassettes (brainbow, CRISPR)
MCS
Support pages
Lawson lab
Wiki page
Specific transposition systems
Library of destination vectors would need to be build
€€€: BP clonase – 20 (up to 40) reactions 222€
100 (up to 200) reactions 940€
LR clonase - 20 (up to 40) reactions 221€
100 (up to 200) reactions 919€

17. 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
Invitrogen Proprietary & Confidential