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Team:Berkeley Wetlab/Project Overview
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We employed a combinatorial method, testing many variations of display and spacers for each passenger. Because this requires the construction of a very large number of parts, we developed a high throughput automated assembly method. The data generated by this method allows future investigators to estimate the number of combinations that must be constructed in order to find functional display, and helps investigators chose subsets of combinations within the design space that are most likely to yield success. | We employed a combinatorial method, testing many variations of display and spacers for each passenger. Because this requires the construction of a very large number of parts, we developed a high throughput automated assembly method. The data generated by this method allows future investigators to estimate the number of combinations that must be constructed in order to find functional display, and helps investigators chose subsets of combinations within the design space that are most likely to yield success. | ||
=====Passengers===== | =====Passengers===== | ||
| - | In choosing which passengers to examine, we looked at systems in which cell surface display could lead to some novel functionality of the passenger. Because we are concerned functional display of the passenger, passengers must be able to be assayed in vivo. Additionally, due to he very large number of parts constructed, all assays must be amenable to high throughput. | + | In choosing which passengers to examine, we looked at systems in which cell surface display could lead to some novel functionality of the passenger. Because we are concerned with functional display of the passenger, passengers must be able to be assayed in vivo. Additionally, due to he very large number of parts constructed, all assays must be amenable to high throughput. |
=====Displayers===== | =====Displayers===== | ||
Revision as of 23:01, 18 October 2009
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Contents |
What is Cell Surface Display?
Cell surface display requires that a protein of interest be exposed to the extracellular environment but remain anchored to the outer membrane. This is done by the fusion of a domain of interest, referred to as the passenger, to a protein, referred to as the displayer, that naturally inserts itself into the outermembrane. Genetic devices for cell surface display can be thought of as composed of three basic components: the passenger domain that will be displayed to the extracellular environment, the displayer domain which will anchor the passenger to the outer membrane, and the structural spacer elements that link these two regions.
The Problem
Functions can be engineered into e. coli that would not be possible without the localization of the passenger protein provided by cell surface display. Successes in this space, however, rely on a trial and error approach that is not guided by design principles. While it is almost certain that for a given passenger, a combination of displayer and structural spacers exists that leads to functional display, it is not clear what this combination is or how to chose such a combination rationally.
Our Approach
We employed a combinatorial method, testing many variations of display and spacers for each passenger. Because this requires the construction of a very large number of parts, we developed a high throughput automated assembly method. The data generated by this method allows future investigators to estimate the number of combinations that must be constructed in order to find functional display, and helps investigators chose subsets of combinations within the design space that are most likely to yield success.
Passengers
In choosing which passengers to examine, we looked at systems in which cell surface display could lead to some novel functionality of the passenger. Because we are concerned with functional display of the passenger, passengers must be able to be assayed in vivo. Additionally, due to he very large number of parts constructed, all assays must be amenable to high throughput.
Displayers
Displayers can be divided into two classes, N terminal and C terminal, depending on which terminus of the displayer the passenger must be fused to. The majority of the displayers we examined are autotransporters, proteins that form a pore in the outermembrane and then pull their N terminus through this pore. We also examined several outermembrane proteins which have an exposed, extracellular terminus.
Linkers
In natural display systems, the displayer domain and the passenger domain are always separated by some sort of spacer region. We examined the effect of including different structural proteins as spacer elements separating the passenger from the displayer domain.
