Cloning MethodsRecombination cloning systems
GATEWAY Cloning Technology
The GATEWAY Cloning Technology is based on the site-specific recombination system used by phage l to integrate its DNA in the E. coli chromosome. Both organisms have specific recombination sites called attP in phage l site and attB in E. coli. The integration process (lysogeny) is catalyzed by 2 enzymes: the phage l encoded protein Int (Integrase) and the E. coli protein IHF (Integration Host Factor). Upon integration, the recombination between attB (25 nt) and attP (243 nt) sites generate attL (100 nt) and attR (168 nt) sites that flank the integrated phage l DNA (see Figure 1).
The process is reversible and the excision is again catalyzed Int and IHF in combination with the phage l protein Xis. The attL and attR sites surrounding the inserted phage DNA recombine site-specifically during the excision event to reform the attP site in phage l and the attB site in the E. coli chromosome.
The GATEWAY reactions are in vitro versions of the integration and excision reactions. To make the reactions directional two slightly different and specific site were developed, att1 and att2 for each recombination site. These sites react very specifically with each other. For instance in the BP Reaction attB1 only reacts with attP1 resulting in attL1 and attR1, and attB2 only with attP2 giving attL2 and attR2. The reverse reaction (LR Reaction) shows the same specificity.
The ultimate goal of the GATEWAY reactions is to make an expression clone. This is often a two step process:
|Step 1||Cloning the gene of interest into an Entry Vector using the BP Reaction.|
|Step 2||Subcloning the gene of interest from the Entry Clone (Step 1) into a Destination Vector using the LR Reaction producing the Expression Clone.|
Let's have a closer look at the LR Reaction of Step 2 (see also Figure 2). The gene of interest is cloned into an Entry Vector and flanked by the attL1 and attL2 recombination sites. The Entry Vector is transcriptionally silent and contains the gene for kanamycin resistance (Kmr). To produce the Expression Clone the gene has to be subcloned into a Destination Vector that contains all the sequence information necessary for expression, the gene for ampicillin resistance (Apr), and two recombination sites (attR1 and attR2) that flank a gene for negative selection, ccdB (the encoded protein is toxic for the standard E. coli strains).
The two plasmids are mixed and the LR CLONASE Enzyme Mix is added. The reaction is directional and specific, so that attL1 only reacts with attR1 and attL2 with attR2. The recombination yields two constructs: the intended Expression Clone and a by-product (labelled in Figure 2 as Donor Vector). The produced expression clone is under two forms of selection: the antibiotic resistance and the negative selection by the toxic ccdB protein. As a result high levels of positive clones (typicaly more than 99%) are obtained after transformation to a standard cloning or expression strain like DH5a or BL21 (DE3).
One of the main advantages of the GATEWAY Cloning Technology is that once you have made an Entry Clone the gene of interest can be easily subcloned into a wide variety of Destination Vectors using the LR Reaction (see Figure 3).
If you want to know more about the GATEWAY Cloning Technology go to the Invitrogen website.