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Team:UC Berkeley/GatewayOverview
From 2008.igem.org
Contents |
Why Use Gateway?
The first step of the layered assembly scheme involves the transfer of biobrick parts from an entry vector to a double antibiotic assembly vector. Traditionally, this would require a fairly work-intensive protocol requiring digestion, gel purification, ligation, transformation, and plasmid isolation. In addition to being more time-consuming, the aforementioned procedure is also suboptimal because it is difficult to scale-up.
The [http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cloning/Gateway-Cloning.html| Gateway Cloning] approach developed by Invitrogen offers a more efficient and convenient alternative for parts transfer. Their procedure involves the enzyme-catalyzed exchange of parts flanked by specific recombination sites. Experimentally, it is a one-pot, room-temperature reaction where the plasmids, buffer, water and enzymes are added together. After the addition of another enzyme and a short incubation period to terminate the reaction, the entire mixture can be transformed directly. This one-pot approach with a fewer steps is much more suitable for large-scale experimentation.
Gateway Chemistry
In general, Gateway reactions involve the attB, attP, attL, and attR recombination sites and the integrase (Int), excisionase (Xis), and integration host factor (IHF) enzymes. Invitrogen adapted these components from the original lambda phage recombination system shown below.
Image source: http://tools.invitrogen.com/downloads/gateway-the-basics-seminar.html
In the Invitrogen scheme, the recombination sites are found in pairs flanking sequences that are intended for transfer. The recombination pairs are directional and specificity is given by ten nucleotides in the core region of each site, as shown below.
The schematic below depicts the LR reaction, during which the attL sites recombine with attR to yield attB and attP sites. The BP reaction proceeds in the opposite direction yielding attL and attR sites.
Image source: http://www.bio.davidson.edu/courses/Molbio/MolStudents/spring2000/patton/gateway.htm
Animation source: http://www.bio.davidson.edu/courses/Molbio/MolStudents/spring2000/patton/gateway.htm
Gateway in vivo
References
- http://www.bio.davidson.edu/courses/Molbio/MolStudents/spring2000/patton/gateway.htm
- http://tools.invitrogen.com/downloads/gateway-the-basics-seminar.html
- http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cloning/Gateway-Cloning.html|Gateway
