Team:UC Berkeley/GatewayOverview

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Gateway Overview

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 is commonly used to facilitate the transfer of a single gene of interest from an entry clone to multiple destination vectors, as shown below. The efficiency and robustness of the Gateway mechanism are ideal for this application because the once the gene of interest is cloned and confirmed in the entry vector, subsequent transfers using Gateway need not be confirmed again. Thus, it is ideal for use in the first step of layered assembly where a part may be transferred to one or more of the double antibiotic vectors required for the subsequent assembly steps assembly.

A entry clone generated from any of the methods in the yellow boxes can be transferred to various different vectors using the Gateway method.
Image source: http://www.bio.davidson.edu/courses/Molbio/MolStudents/spring2000/patton/gateway.htm

Gateway Chemistry

The Natural Lambda Phage Recombination system

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. Int cooperatively binds with ihf (which is composed of A and B subunits) in order to catalyze both the integration and excision reactions in the natural lambda phage system shown below. Although int can independently catalyze both reactions, ihf greatly improves int's binding affinity for the att recombination sites by bending the DNA [6]. Although int performs both the forward and reverse reactions, the equilibrium heavily favors the reaction of attB and attP to produce attL and attR sites. Xis binds to the attR sire and serves to shift the equilibrium so that it favors the reverse reaction [5].

Lambda phage recombination in E. coli. Invitrogen adapted these components from this natural system to produce their Gateway cloning scheme.
Image source: http://tools.invitrogen.com/downloads/gateway-the-basics-seminar.html

Invitrogen's adaptation

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.

Directional, site-specific recombination between attL and attR sites.
Image source:https://commerce.invitrogen.com/index.cfm?fuseaction=iProtocol.unitSectionTree&treeNodeId=290D0521B5FFA1B782C62C0AB62FD7BC

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

Selection for Correct Clones

The above scheme employs both ccdB negative selection and antibiotic selection in order to yield >90% of colonies containing the desired expression clone. The entry and destination vectors contain different antibiotic resistances, so plating on the desired antibiotic (Ampicillin in case shown above) eliminates clones containing the entry vector or the by-product of the reaction. In addition, ccdB is a lethal gene, thereby eliminating colonies containing that contain the destination vector, which would otherwise survive on the antibiotic plate.

Animation source: http://www.bio.davidson.edu/courses/Molbio/MolStudents/spring2000/patton/gateway.htm

Gateway in vivo

Plasmid Based Gateway

We applied our ideas for facilitating the automation of synthetic biology to the first step of layered assembly--the LR gateway reaction used to move the gene of interest into one or more of the double-antibiotic assembly vectors. We began by attempting to reproduce a plasmid-based Gateway reaction, which was similar to that of Invitrogen's Gateway scheme. Although we were successful in having a completely in vivo version of the Gateway reaction, there were two major drawbacks: we had not eliminated the need for laborious and expensive mini-preps and we had relatively high background from ccdB mutations (the ccdB gene is relatively unstable because of its toxicity).

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
https://commerce.invitrogen.com/index.cfm?fuseaction=iProtocol.unitSectionTree&treeNodeId=290D0521B5FFA1B782C62C0AB62FD7BC
Cho, E et al. Interactions between Integrase and Excisionase in the Phage Lambda Excisive Nucleoprotein Complex. Journal of Bacteriology. September 2002; 184(18): 5200–5203. Available Online: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=135313 (Accessed: 28 October 2008).
Frumerie, C et al. Cooperative interactions between bacteriophage P2 integrase and its accessory factors IHF and Cox. Virology. 5 February 2005; 232(1):284-294. Available Online: http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WXR-4F29SN2-1&_user=4420&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_version=1&_urlVersion=0&_userid=4420&md5=0f9e41ba422140f157a2b1f1fb60b140 (Accessed: 28 October 2008).

@@ Line 12: / Line 12: @@
 ==Gateway Chemistry==
+===The Natural Lambda Phage Recombination system===
 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. Int cooperatively binds with ihf (which is composed of A and B subunits) in order to catalyze both the integration and excision reactions in the natural lambda phage system shown below. Although int can independently catalyze both reactions, ihf greatly improves int's binding affinity for the att recombination sites by bending the DNA [6]. Although int performs both the forward and reverse reactions, the equilibrium heavily favors the reaction of attB and attP to produce attL and attR sites. Xis binds to the attR sire and serves to shift the equilibrium so that it favors the reverse reaction [5].
 [[Image:LambdaRecombination.jpg|frame|center|Lambda phage recombination in ''E. coli''. Invitrogen adapted these components from this natural system to produce their Gateway cloning scheme. <br> Image source: http://tools.invitrogen.com/downloads/gateway-the-basics-seminar.html]]
+===Invitrogen's adaptation===
 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.
@@ Line 24: / Line 28: @@
 [[Image:LR reaction.jpg|frame|center|Gateway LR reaction where gene of interest (flanked by attL sites) is transferred to destination vector containing attR sites. <br> Image source: http://www.bio.davidson.edu/courses/Molbio/MolStudents/spring2000/patton/gateway.htm]]
+====Selection for Correct Clones====
 The above scheme employs both ccdB negative selection and antibiotic selection in order to yield >90% of colonies containing the desired expression clone. The entry and destination vectors contain different antibiotic resistances, so plating on the desired antibiotic (Ampicillin in case shown above) eliminates clones containing the entry vector or the by-product of the reaction. In addition, ccdB is a lethal gene, thereby eliminating colonies containing that contain the destination vector, which would otherwise survive on the antibiotic plate.