Team:UC Berkeley/GatewayOverview

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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.

Lambda phage recombination in E. coli.
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 seven nucleotides in the core region of each site. Xis performs site-specific cutting and Int and IHF allow the cut fragments to recombine. 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.

Gateway LR reaction where gene of interest (flanked by attL sites) is transferred to destination vector containing 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

  1. http://www.bio.davidson.edu/courses/Molbio/MolStudents/spring2000/patton/gateway.htm
  2. http://tools.invitrogen.com/downloads/gateway-the-basics-seminar.html
  3. http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cloning/Gateway-Cloning.html|Gateway
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