{"id":1809,"date":"2021-06-05T16:45:56","date_gmt":"2021-06-05T16:45:56","guid":{"rendered":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/?page_id=1809"},"modified":"2021-08-02T12:09:55","modified_gmt":"2021-08-02T12:09:55","slug":"recombination-based-cloning-gateway","status":"publish","type":"page","link":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/services\/strategy-and-construct-design\/recombination-based-cloning-gateway\/","title":{"rendered":"Recombination-based cloning: Gateway"},"content":{"rendered":"\n
\n
\n\n

The Gateway cloning technology<\/strong> is based on the site-specific recombination system used by phage \u03bb to integrate its DNA in the E. coli<\/em> chromosome. Both organisms have specific recombination sites, which are called att<\/em>P in the phage \u03bb  and att<\/em>B in E. coli<\/em>. The integration process (lysogeny) is catalyzed by 2 enzymes: the phage \u03bb encoded protein Int (Integrase) and the E. coli<\/em> protein IHF (Integration Host Factor). Upon integration, the recombination between att<\/em>B (25 nt) and att<\/em>P (243 nt) sites generate att<\/em>L (100 nt) and att<\/em>R (168 nt) sites that flank the integrated phage \u03bb DNA.<\/p>\n\n\n\n

The process is reversible and the excision is again catalyzed by Int and IHF in combination with the phage\u00a0\u03bb protein Xis (excisionase). The\u00a0att<\/em>L and\u00a0att<\/em>R sites surrounding the inserted phage DNA recombine site-specifically during the excision event to reform the\u00a0att<\/em>P site in phage\u00a0\u03bb and the\u00a0att<\/em>B site in the\u00a0E. coli<\/em>\u00a0chromosome.<\/p>\n\n\n\n

The Gateway cloning<\/strong> reactions<\/strong> are in vitro<\/em> versions of the integration and excision reactions. To make the reactions directional, two slightly different and specific sites were developed (called att<\/em>1 and att<\/em>2) for each recombination site. These sites react very specifically with each other. For example, in the BP <\/strong>r<\/strong>eaction<\/strong>,<\/strong> att<\/em>B1 only reacts with att<\/em>P1, resulting in att<\/em>L1 and att<\/em>R1, and att<\/em>B2 only reacts with att<\/em>P2, leading to att<\/em>L2 and att<\/em>R2. The reverse reaction (LR Reaction<\/strong>) shows the same specificity.<\/p>\n\n\n\n

Gateway cloning<\/strong> is generally a 2-step process: in the first step an entry clone<\/strong> is generated using the BP reaction<\/strong> and in a second step the gene of interest is moved to a destination vector<\/strong> using the LR reaction<\/strong>.<\/p>\n\n\n\n

Once you have generated a PCR fragment containing your gene of interest flanked by att<\/em>B1 and att<\/em>B2 sites, you can mix this with a Gateway entry vector<\/strong> and the BP Clonase\u00ae enzymes, leading to an entry clone in which the gene of interest is flanked by the att<\/em>L1 and att<\/em>L2 sites. The Gateway destination vectors<\/strong> contain a ccdB<\/em> cassette (the encoded protein is toxic for most E. coli<\/em> strains) flanked by the attR1<\/em> and attR2<\/em> sites. Upon mixing the entry clone and the destination vector with the LR Clonase enzymes\u00ae, you\u2019ll generate an expression construct in which your gene of interest is flanked by the att<\/em>B1 and att<\/em>B2 sites. Negative selection by the toxic ccdB protein and different antibiotic resistances between the entry clone and the destination vector will lead to a very high level of positive clones after transformation into an E. coli<\/em> strain.<\/p>\n\n\n\n

The multitude of destination vectors that are available provides a lot of flexibility to generate many different expression constructs in parallel. However, there can be some disadvantages as well to Gateway cloning, such as the long N-terminal overhangs that are created with some destination vectors. If you desire to remove these overhangs fully from the mature protein, it might be advisable to introduce an extra protease cleavage site behind these overhangs (e.g. by including this into your PCR primers).<\/p>\n\n\n\n

More information about Gateway cloning and available Gateway vectors, can be found on the Thermo Fischer Scientific website<\/a>.<\/p>\n\n<\/div>\n<\/div>\n\n\n

\n
\n\n
\"diagram\"<\/a>
Overview of Gateway cloning.<\/figcaption><\/figure>\n\n<\/div>\n<\/div>\n<\/div>\n","protected":false},"excerpt":{"rendered":"","protected":false},"author":1,"featured_media":0,"parent":971,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"template-title-left-aligned.php","meta":{"_acf_changed":false,"footnotes":""},"embl_taxonomy":[],"class_list":["post-1809","page","type-page","status-publish","hentry"],"acf":[],"embl_taxonomy_terms":[],"_links":{"self":[{"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/pages\/1809","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/comments?post=1809"}],"version-history":[{"count":4,"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/pages\/1809\/revisions"}],"predecessor-version":[{"id":2138,"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/pages\/1809\/revisions\/2138"}],"up":[{"embeddable":true,"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/pages\/971"}],"wp:attachment":[{"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/media?parent=1809"}],"wp:term":[{"taxonomy":"embl_taxonomy","embeddable":true,"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/embl_taxonomy?post=1809"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}