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Gateway cloning

The Gateway technology provides a fast and efficient route for cloning. This technology relies on the use of modified versions of the recombinases from the bacteriophage lambda. This virus inserts its genome into the host DNA using these enzymes. The Gateway cloning system uses them to achieve high efficiency, site specific recombination. The second useful characteristic of this system is its recognition sites for the clonase enzymes called att sites, and the use of different types of vectors.

BP and LR reactions

BP and LR reactions are the way different DNA segments are moved from one place to another within the constructs. During the BP reaction, attB and attP sites are recombined. This reaction swaps the DNA between strands, creating an attL and an attR site. During the LR reaction, attL and attR sites are similarly recombined, yielding attB and attP sites. There is a limited number of att sites, annotated with numbers. This means that there is a limited number of combinations. Each site only recombines within the group: for example, attL1 sites will only recombine with attR1 sites, yielding attB1 and attP1 sites. The orientation of these sites is also specific, and the gene construct highly predictable. The desired construct can be built by selecting for the proper att ends, the reactions, and the vectors.

Vector types

The recyclable att sites are ideally suited for creating vector libraries that can be used to efficiently combine different circuit parts.

• Expression vector (AmpR): The expression clone is the final product of a gateway reaction. It is the complete plasmid, ready for transformation.

• Donor vector (ccdB, KanR): The donor vector provides the backbone for the creation of entry clones. Donor vectors are typically denoted with names starting with pDONR. These plasmids carry attB or attP sites flanking the ccdB gene, as well as the kanamycin resistance gene.

• Entry vector (KanR): The entry vectors are created from the donor vector and a DNA sequence flanked with the matching att sites. These sequences of interest are usually produced by PCR, with primers that contain the att sites. Entry vectors contain attL or attR sites flanking the sequence of interest, as well as the kanamycin resistance gene. The entry vectors are ideally suited for a library of circuit parts.

• Destination Vector (ccdB, AmpR): The destination vector provides the backbone for expression clones. Destination vectors are typically denoted with names starting with pDEST. These plasmids contain the ccdB gene flanked by attL or attR sites, as well as an ampicillin resistance gene. Destination vectors also contain the origins of replications for specific hosts and additional DNA motifs.

Selection

The selection of the vectors of interest in the different steps of the Gateway cloning procedure is very important. For selection, the Gateway system relies on two antibiotic resistances and the ccdB gene for positive selection. The antibiotic resistance enables transformed cells to grow on a medium containing antibiotics that kill untransformed cells. ccdB encodes a bacteriotoxin that prevents DNA replication. All of the backbones for Gateway clones (pDEST and pDONR) carry this gene. To amplify the backbones, special competent cells carrying the ccdA gene that inhibits ccdB are used. However, these competent cells should never be used to amplify the Gateway construct, as it would nullify the positive selection of ccdB.

The Gateway cloning system. This cloning method is based on site-specific recombination. The att sequences originate from a phage that infects bacteria by inserting its DNA into a region of the bacterial genome that has an att site. These sites consist of a 7 nucleotide overhang followed by 3 A-T base pairs. These overhangs act as recognition sites for the clonase enzymes. There are four types of att sites: attB, attP, attL and attR. The BP cloning mix contains several enzymes that bind to the attB and attP sites. A restriction enzyme in the BP reaction cuts all attB and attP sites. The fragments are then rearranged by another enzyme, yielding attL and attR sites. The LR reaction is basically the reverse process. The LR clonase mix contains a restriction enzyme that recognizes the attL and attR sites and cuts them. Another enzyme recombines the fragments to form attB and attP again. Each BP or LR reaction can be performed in a single tube and cloning efficiency is much higher than traditional methods! In addition, there can be several sets of each att site, each named with a number. For example, attB3, which would only recombine with attP3 and attL2 with attR2. Figure 1. The Gateway Cloning System

Referred from:

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Cloning techniques
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Position vector
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ccdB