Team:Berkeley Wetlab/Project Overview

From 2009.igem.org

(Difference between revisions)

Latest revision as of 06:23, 21 October 2009

What is Cell Surface Display?

Cell surface display is a system for exposing proteins/peptides to the extracellular environment by anchoring them to the outermembrane of a cell. This is done by fusing a protein or peptide of interest to a protein domain that naturally inserts itself into the outer membrane. Genetic devices for cell surface display are generally composed of three basic components:

Passenger domain: the protein or peptide exposed to the extracellular environment.
Displayer domain: the domain that anchors the passenger to the outer membrane.
Structural Spacer Element: a link between the passenger and the displayer.

The Problem

Certain functions cannot be engineered into E. coli without a cell surface display system. For example, the presence of large insoluble molecules incapable of passing through the cell membrane can only be detected extracellularly. Cell surface display promises many advantages over conventional cytoplasmic protein expression systems. It offers the possibility of creating and screening proteins for directed evolution. Cell surface display also enables researchers to display proteins on the surface of cells thereby making them freely accessible to their substrates without having to deal with transport problems. 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 choose 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 many display systems in a combinatorial fashion to come up with basic design principles for cell surface display. We tested a few passengers with many different displayers and spacers in order to find combinations and patterns that produce functional devices. This approach requires the construction of a very large number of parts, so we developed a high throughput automated assembly method to facilitate our project. The data generated by our project should allow future investigators to estimate the number of combinations that must be constructed in order to find functional display, and should help investigators chose subsets of combinations within the developed design space that are most likely to yield success.

Passengers

Passengers are typically proteins/peptides that would not naturally reside on the outermembrane of E.coli, but are put there by a cell surface display device. They are the functional units of cell surface display devices. We chose to examine a subset of passengers that would be essential to display for bacterial systems of different applications. We also chose passengers whose functional display could be assayed in vivo and high throughput. To read more about the passengers we used and our success in dispalying them, visit our parts page.

Displayers

Displayers can be divided into two classes, N terminal and C terminal, based on which terminus of the displayer its passenger is fused to. We built cell surface display systems using many different N and C terminal displayers. Most of the displayers we examined were autotransporters, proteins that form a pore in the outermembrane and then pull their N terminus through this pore, but many were common outermembrane proteins that have exposed extracellular termini. To read more about the displayers we used, 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.

References

Jose, J et al. The Autodisplay Story, from Discovery to Biotechnical and Biomedical Applications. Microbiol Mol Biol. December 2007; 71(4): 600–619. Available Online: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2168652/?tool=pubmed (Accessed: 20 October 2009).

Retrieved from "http://2009.igem.org/Team:Berkeley_Wetlab/Project_Overview"

@@ Line 1: / Line 1: @@
+__NOTOC__
 {{newtemplateBerkeley}}
-===What is Cell Surface Display?===
+===<center>What is Cell Surface Display?</center>===
-[[Image:schematic.jpg|center]]<br>
+[[Image:Berkeleydisplay2.png|900px|center]]
-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.
+Cell surface display is a system for exposing proteins/peptides to the extracellular environment by anchoring them to the outermembrane of a cell. This is done by fusing a protein or peptide of interest to a protein domain that naturally inserts itself into the outer membrane. Genetic devices for cell surface display are generally composed of three basic components: <br>
+# <font color=#E9AB17>'''Passenger domain'''</font>: the protein or peptide exposed to the extracellular environment.
+# <font color=#C47451>'''Displayer domain'''</font>: the domain that anchors the passenger to the outer membrane.
+# <font color=#7E587E>'''Structural Spacer Element'''</font>: a link between the passenger and the displayer.
 ===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.
+Certain functions cannot be engineered into ''E. coli'' without a cell surface display system.  For example, the presence of large insoluble molecules incapable of passing through the cell membrane can only be detected extracellularly. Cell surface display promises many advantages over conventional cytoplasmic protein expression systems. It offers the possibility of creating and screening proteins for directed evolution. Cell surface display also enables researchers to display proteins on the surface of cells thereby making them freely accessible to their substrates without having to deal with transport problems.  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 choose such a combination rationally.
 ===Our Goal===
@@ Line 12: / Line 16: @@
 ===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.
+We made many display systems in a combinatorial fashion to come up with basic design principles for cell surface display. We tested a few passengers with many different displayers and spacers in order to find combinations and patterns that produce functional devices. This approach requires the construction of a very large number of parts, so we developed a high throughput automated assembly method to facilitate our project. The data generated by our project should allow future investigators to estimate the number of combinations that must be constructed in order to find functional display, and should help investigators chose subsets of combinations within the developed 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.
+{|style="background-color:#ECD872;"
+|Passengers are typically proteins/peptides that would not naturally reside on the outermembrane of E.coli, but are put there by a cell surface display device. They are the functional units of cell surface display devices. We chose to examine a subset of passengers that would be essential to display for bacterial systems of different applications. We also chose passengers whose functional display could be assayed in vivo and high throughput. To read more about the passengers we used and our success in dispalying them, visit our [[Team:Berkeley_Wetlab/Assay_Protocols | parts]] page.
+|}
 =====Displayers=====
-Displayers are the segments of a cell surface display device that anchors a Passenger to the outer membrane of E.coli. Displayers can be divided into two classes, N terminal and C terminal, based on which terminus of the displayer its 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 many were common outermembrane proteins that have exposed extracellular termini. To read more about the displayers we used, visit our [[Team:Berkeley_Wetlab/Assay_Protocols | parts]] page.
+{|style="background-color:#F9966B;"
+|Displayers can be divided into two classes, N terminal and C terminal, based on which terminus of the displayer its passenger is fused to.  We built cell surface display systems using many different N and C terminal displayers. Most of the displayers we examined were autotransporters, proteins that form a pore in the outermembrane and then pull their N terminus through this pore, but many were common outermembrane proteins that have exposed extracellular termini. To read more about the displayers we used, visit our [[Team:Berkeley_Wetlab/Assay_Protocols | 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 [[Team:Berkeley_Wetlab/Assay_Protocols | parts]] page.
+{|style="background-color:#D2B9D3;"
+|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 [[Team:Berkeley_Wetlab/Assay_Protocols | parts]] page.
+|}
+===References===
+Jose, J et al. The Autodisplay Story, from Discovery to Biotechnical and Biomedical Applications. Microbiol Mol Biol. December 2007; 71(4): 600–619. Available Online: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2168652/?tool=pubmed (Accessed: 20 October 2009).