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Team:Berkeley Wetlab/Project Overview
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What is Cell Surface Display?
Cell surface display is the ___ 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 protein or peptide of interest, referred to as the passenger, to a protein domain that naturally inserts itself into the outer membrane, referred to as the displayer. Genetic devices for cell surface display are generally composed of three basic components: a passenger domain, which will be displayed to the extracellular environment, a displayer domain, which will anchor the passenger to the outer membrane, and a structural spacer element, which links these two regions.
The Problem
Certain functions cannot be engineered into E. coli without a cell surface display system. However, success in building a functional cell surface display system currently relies 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 Goal
To create basic design principles for cell surface display which can serve as guidelines for future iGEM teams (and others) attempting to build systems that involve cell surface display.
Our Approach
- We made passengers representative of the major types of protiens/peptides people have tried to display in the past. Made many display systems in a combinatorial fashion to come up stemployed a combinatorial method, testing many variations of display and spacers for each passenger, in order to come . 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 are the segments of a cell surface display devices which anchor Passengers to the outer membrane of E.coli. Displayers can be divided into two classes, N terminal and C terminal, depending on which terminus of the displayer the passenger is fused to. We built cell surface display systems using many different displayers. Most of them were autotransporters, proteins that form a pore in the outermembrane and then pull their N terminus through this pore, but we also examined several outermembrane proteins which have a exposed extracellular termini. To read more about displayers visit our parts page.
Spacers
Structural spacer regions are common in natural display systems. However, their function is unclear, so most people engineer cell surface display devices without spacer regions. We decided to examine the effect that spacer elements have on engineered cell surface display devices. To read more about the spacer parts we built visit our parts page.
