Team:Berkeley Wetlab/Cell Surface Display Parts

From 2009.igem.org

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==<center>Passengers</center>==
==<center>Passengers</center>==
<font color="#41383C" size="2">Follow any of the links below to see assay information for each of the passengers we made.</font>
<font color="#41383C" size="2">Follow any of the links below to see assay information for each of the passengers we made.</font>
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{| cellpadding="25" cellspacing="0"
{| cellpadding="25" cellspacing="0"
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| <html>
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| {{linkedImage|Mgfp5button.png|Team:Berkeley_Wetlab/Passenger:_MGFP}}
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<div align="left">
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[[Team:Berkeley_Wetlab/Passenger: MGFP | '''MGFP-5''']]<br>
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<a href="https://2009.igem.org/Team:Berkeley_Wetlab/Passenger:_MGFP">
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'''The Sticky Protein'''<br>
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  <img width="175" src="https://static.igem.org/mediawiki/2009/6/63/Mgfp5button.png">
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Displaying this sticky peptide would allow you to make an underwater bio-glue!
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</a>
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|| {{linkedImage|Cellulasebutton.png|Team:Berkeley_Wetlab/Passenger:_Cellulases}}
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</div>
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[[Team:Berkeley_Wetlab/Passenger: Cellulases | '''Cellulase''']]<br>
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</html><br>
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'''The Enzyme'''<br>
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[[Team:Berkeley_Wetlab/Passenger: MGFP | '''The Sticky Protein''']]<br>
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Displaying enzymes that degrade cellulose would allow consolidated bioprocessing for biofuel production!<br>
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A protein used by mussels to stick to rocks.  An underwater bio-glue!
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|| {{linkedImage|Scfvbutton.png|Team:Berkeley_Wetlab/Passenger:_TypeIII_Needle_scFv}}
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|| <html>
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[[Team:Berkeley_Wetlab/Passenger: TypeIII Needle scFv | '''Type III Needle scFv''']]<br>
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<div align="left">
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'''The Antibody Fragment'''<br>
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<a href="https://2009.igem.org/Team:Berkeley_Wetlab/Passenger:_Cellulases">
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Displaying antibody fragments would allow easy pathogen detection!<br>
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  <img width="175" src="https://static.igem.org/mediawiki/2009/d/df/Cellulasebutton.png">
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</a>
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</div>
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</html><br>
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[[Team:Berkeley_Wetlab/Passenger: Cellulases | '''The Enzyme''']]<br>
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Enzymes that degrade cellulose!<br>
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|| <html>
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<div align="left">
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<a href="https://2009.igem.org/Team:Berkeley_Wetlab/Passenger:_TypeIII_Needle_scFv">
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  <img width="175" src="https://static.igem.org/mediawiki/2009/d/d8/Scfvbutton.png">
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</a>
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</div>
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</html><br>
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[[Team:Berkeley_Wetlab/Passenger: TypeIII Needle scFv | '''The scFv''']]<br>
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An antibody fragment that binds a motif common to enteropathogenic bacteria!<br>
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|-
|-
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| <html>
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| {{linkedImage|Ag4.png|Team:Berkeley_Wetlab/Passenger:_Streptavidin}}
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<div align="left">
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[[Team:Berkeley_Wetlab/Passenger: Streptavidin | '''Streptavidin Binding Tag''']]<br>
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<a href="https://2009.igem.org/Team:Berkeley_Wetlab/Passenger:_Streptavidin">
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'''The Tag'''<br>
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  <img width="175" src="https://static.igem.org/mediawiki/2009/2/22/Streptagbutton.png">
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A tag on the surface of E.coli would allow you to purify bacteria preferentially!<br>
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</a>
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|| {{linkedImage|Leucinezipeprsbutton.png|Team:Berkeley_Wetlab/Passenger:_Leucine_Zippers}}
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</div>
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[[Team:Berkeley_Wetlab/Passenger: Leucine Zippers | '''Leucine Zippers''']]<br>
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</html><br>
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'''The Heterodimeric Binding Peptide'''<br>
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[[Team:Berkeley_Wetlab/Passenger: Streptavidin | '''The Tag''']]<br>
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Displaying heterodimeric binding peptides would allow you to make heterogeneous bacteria systems!<br>
-
A tag that binds the protein streptavidin!<br>
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|| {{linkedImage|Streptagbutton.png|Team:Berkeley_Wetlab/Passenger:_Ag4_Peptide}}
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|| <html>
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[[Team:Berkeley_Wetlab/Passenger: Ag4 Peptide | '''Ag4 ''']]<br>
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<div align="left">
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'''The Silver Binding Peptide'''<br>
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<a href="https://2009.igem.org/Team:Berkeley_Wetlab/Passenger:_Leucine_Zippers">
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The metal head of our set! Displaying a heavy metal binding peptide would promote bioremediation of heavy metals and aid in synthesis of biomaterials. <br>
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  <img width="175" src="https://static.igem.org/mediawiki/2009/d/df/Leucinezipeprsbutton.png">
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</a>
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</div>
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</html><br>
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-
[[Team:Berkeley_Wetlab/Passenger: Leucine Zippers | '''The Heterodimeric Binding Peptide''']]<br>
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A structural motif that can allow different cell types to recognize and to bind each other!<br>
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|| <html>
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<div align="left">
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<a href="https://2009.igem.org/Team:Berkeley_Wetlab/Passenger:_Ag4_Peptide">
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  <img width="175" src="https://static.igem.org/mediawiki/2009/a/aa/Ag4.png">
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</a>
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</div>
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</html><br>
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[[Team:Berkeley_Wetlab/Passenger: Ag4 Peptide | '''The Other Binding Peptide ''']]<br>
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A peptide that reduces silver ions to form a silver precipitate!<br>
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|}
|}
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Spacer elements are present in most natural display systems, which suggests that they are important components of cell surface display. However, the precise role of spacers in the functionality of outer membrane proteins has not been extensively characterized and most people engineer cell surface display devices without structural spacers.  We decided to build a few cell surface display systems with spacer elements in order to begin characterizing their role within engineered cell surface display devices. We introduced five different spacer domains to our surface display system: INP-repeats, beta roll, beta helix, Gly-Ser repeats and GFP-LVA. These elements are further described below. <br>
Spacer elements are present in most natural display systems, which suggests that they are important components of cell surface display. However, the precise role of spacers in the functionality of outer membrane proteins has not been extensively characterized and most people engineer cell surface display devices without structural spacers.  We decided to build a few cell surface display systems with spacer elements in order to begin characterizing their role within engineered cell surface display devices. We introduced five different spacer domains to our surface display system: INP-repeats, beta roll, beta helix, Gly-Ser repeats and GFP-LVA. These elements are further described below. <br>
-
{|style="background-color:#D2B9D3;" cellpadding="30"; cellspacing="20";
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{|style="background-color:#D2B9D3;" cellpadding="10" cellspacing="0" border="1"
| '''INP-Repeats'''
| '''INP-Repeats'''
||Synthetic spacer region composed of repeated portions of ice nucleation protein (INP) sequence.
||Synthetic spacer region composed of repeated portions of ice nucleation protein (INP) sequence.
|-
|-
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|<font color=#7E587E>'''Beta Roll'''</font>
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|'''Beta Roll'''
||Protein tertiary structure that is natively found in autotransporters and other outermembrane proteins.  
||Protein tertiary structure that is natively found in autotransporters and other outermembrane proteins.  
|-
|-
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|<font color=#7E587E>'''Beta Helix''' </font>
+
|'''Beta Helix'''
||Protein helical structures stabilized by hydrogen bonds and protein-protein interactions. The resulting structure contains two to three faces formed by the association of parallel beta strands.  
||Protein helical structures stabilized by hydrogen bonds and protein-protein interactions. The resulting structure contains two to three faces formed by the association of parallel beta strands.  
|-
|-
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|<font color=#7E587E> '''Gly-Ser Repeats'''</font>
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| '''Gly-Ser Repeats'''
|| Synthetic spacer made from fourteen repeated glycine and serine residues. This spacer is essentially devoid of secondary structure, it is simply a long flexible linker.  We made in order to differentiate between the importance of a natural spacer with secondary/tertiary structure and a long linker between the displayer and its passenger.
|| Synthetic spacer made from fourteen repeated glycine and serine residues. This spacer is essentially devoid of secondary structure, it is simply a long flexible linker.  We made in order to differentiate between the importance of a natural spacer with secondary/tertiary structure and a long linker between the displayer and its passenger.
|-
|-
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|<font color=#7E587E>'''GFP-LVA''' </font>
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|'''GFP-LVA'''
||Modified green fluorescent protein
||Modified green fluorescent protein
|}
|}
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In constructing our parts, we looked into a broad range of autotransporters, some well characterized and others putative, to explore the spectrum of display machinery and to establish the functionality of novel autotransporters for cell surface display.  
In constructing our parts, we looked into a broad range of autotransporters, some well characterized and others putative, to explore the spectrum of display machinery and to establish the functionality of novel autotransporters for cell surface display.  
-
1. azo1653 AtD (putative) - organism Azoarcus sp. (strain BH72)<br>
+
{|style="background-color:#F9966B;" border="1;"
 +
|'''Azo1653 AtD (putative)'''
 +
||Organism: Azoarcus sp. (strain BH72)<br>
Autotransporter type: AT-1 family
Autotransporter type: AT-1 family
-
 
+
|-
-
2. OprF AtD - organism Pseudomonas fluorescens<br>
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|'''OprF AtD'''
 +
||Organism: Pseudomonas fluorescens<br>
Structure: an 8-stranded beta barrel in the outermembrane
Structure: an 8-stranded beta barrel in the outermembrane
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+
|-
-
3. Cl02365 AtD (putative) - organism Neisseria meningitidis<br>
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|'''Cl02365 AtD (putative)'''
 +
||Organism: Neisseria meningitidis<br>
Autotransporter type: AT-1 family
Autotransporter type: AT-1 family
-
 
+
|-
-
4. VtaA11 AtD - organism Haemophilus parasuis<br>
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|'''VtaA11 AtD'''
 +
||Organism: Haemophilus parasuis<br>
Autotransporter type: AT-2 family
Autotransporter type: AT-2 family
-
 
+
|-
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5. Hag AtD - organism Moraxella catarrhalis<br>
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|'''Hag AtD'''
-
Autotransporter type: dimeric family
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||Organism: Moraxella catarrhalis<br>
 +
Autotransporter type: dimeric family<br>
Structure: 200kDa protein with 10-stranded beta barrel<br>
Structure: 200kDa protein with 10-stranded beta barrel<br>
-
[[Image:Hag autotransporter.jpg|predicted 2D structure of Hag AtD|150px|right]]
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[[Image:Hag autotransporter.jpg|predicted 2D structure of Hag AtD|150px]]
-
 
+
|-
-
6. Pcryo_1225 AtD (putative) - organism Psychrobacter cryohalolentis<br>
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|'''Pcryo_1225 AtD (putative)'''
-
 
+
||Organism: Psychrobacter cryohalolentis<br>
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7. Hia AtD - organism Haemophilus influenzae <br>
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|-
 +
|''' Hia AtD'''
 +
||Organism: Haemophilus influenzae <br>
Autotransporter domain: trimeric family<br>
Autotransporter domain: trimeric family<br>
Structure: modular segments containing repeats of structurally distinct domains<br>
Structure: modular segments containing repeats of structurally distinct domains<br>
-
[[Image:Hia ATD.jpg|150px|right]]
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[[Image:Hia ATD.jpg|150px]]
-
 
+
|-
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8. upaG_short - organism Escherichia Coli<br>
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|'''upaG_short'''
 +
||Organism: Escherichia Coli<br>
Autotransporter type: trimeric family
Autotransporter type: trimeric family
-
 
+
|-
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9. espP(beta) - organism Escherichia coli<br>
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|'''espP(beta)'''
 +
||Organism: Escherichia coli<br>
Structure: 12-stranded beta barrel<br>
Structure: 12-stranded beta barrel<br>
-
[[Image:EspP ATD.jpg|170px|right]]
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[[Image:EspP ATD.jpg|170px]]
-
 
+
|-
-
10. ehaB - organism Escherichia coli<br>
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|'''ehaB'''
 +
||Organism: Escherichia coli<br>
Features: primary sequence alone is sufficient for crossing the bacterial membrane
Features: primary sequence alone is sufficient for crossing the bacterial membrane
-
 
+
|-
-
11. TshA - organism Escherichia coli<br>
+
|'''TshA'''
 +
||Organism: Escherichia coli<br>
Autotransporter type: serine protease subfamily (because of the 7AA serine protease motif)<br>
Autotransporter type: serine protease subfamily (because of the 7AA serine protease motif)<br>
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+
|-
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12. VirG(IcsA) - organism Shigella flexneri<br>
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| '''VirG(IcsA)'''
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+
||Organism: Shigella flexneri
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13. YuaQ AtD (putative) - organism Escherichia coli<br>
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|-
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Features: bears sequence similarity to the confirmed autotransporters AIDA and Ag43<br>
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|'''YuaQ AtD (putative)'''
-
 
+
||Organism: Escherichia coli<br>
-
14. AIDA-I - organism Escherichia Coli
+
Features: bears sequence similarity to the confirmed autotransporters AIDA and Ag43
 +
|-
 +
|'''AIDA-I'''
 +
||Organism: Escherichia Coli<br>
Features: identified to be similar to IgA1, the first autotransporter used for surface display. Occurs naturally in the host organism, E. coli, and is a robust tool for surface display
Features: identified to be similar to IgA1, the first autotransporter used for surface display. Occurs naturally in the host organism, E. coli, and is a robust tool for surface display
-
 
+
|-
-
15. Ag43_short - organism Escherichia Coli MG1655<br>
+
|'''Ag43_short'''
 +
||Organism: Escherichia Coli MG1655<br>
Features: expression of Ag43 is evenly distributed around the bacterial cell<br>
Features: expression of Ag43 is evenly distributed around the bacterial cell<br>
Structure: 14 antiparallel beta strands each composed of about 12AA residues
Structure: 14 antiparallel beta strands each composed of about 12AA residues
-
 
+
|-
-
16. eCPX (circularly permuted OmpX)- organism Escherichia Coli<br>
+
|'''eCPX (circularly permuted OmpX)'''
 +
||Organism: Escherichia Coli<br>
Features: protein is an enhanced CPX variant located in the outermembrane that joins the N- and C-termini of OmpX.<br>
Features: protein is an enhanced CPX variant located in the outermembrane that joins the N- and C-termini of OmpX.<br>
-
[[Image:Ecpx image using molecular operating environment.jpg|120px|right]]
+
[[Image:Ecpx image using molecular operating environment.jpg|120px]]
-
 
+
|-
-
17. CPG_L2 (circularly permuted OmpG)- organism Escherichia Coli<br>
+
|'''CPG_L2 (circularly permuted OmpG)'''
 +
||Organism: Escherichia Coli<br>
Features: protein is circularly permuted with its backbone opening in loop 2, allowing both the N- and C- termini to be present in the extracellular space.
Features: protein is circularly permuted with its backbone opening in loop 2, allowing both the N- and C- termini to be present in the extracellular space.
-
 
+
|-
-
18. CPG_L6 (circularly permuted OmpG)- organism Escherichia Coli<br>
+
|'''CPG_L6 (circularly permuted OmpG)'''
 +
||Organism: Escherichia Coli<br>
Features: protein is circularly permuted with its backbone opening in loop 6, allowing both the N- and C- termini to be present in the extracellular space.
Features: protein is circularly permuted with its backbone opening in loop 6, allowing both the N- and C- termini to be present in the extracellular space.
-
 
+
|}
Some of these proteins are putative autotransporters that have sequence homology to confirmed autotransporters. We chose these proteins because we wanted to test their functionality and expand the range of displayers available for surface display.
Some of these proteins are putative autotransporters that have sequence homology to confirmed autotransporters. We chose these proteins because we wanted to test their functionality and expand the range of displayers available for surface display.

Latest revision as of 02:17, 22 October 2009



Cell Surface Display Parts


Schematic.jpg

Passengers

Follow any of the links below to see assay information for each of the passengers we made.

Mgfp5button.png

MGFP-5
The Sticky Protein
Displaying this sticky peptide would allow you to make an underwater bio-glue!

Cellulasebutton.png

Cellulase
The Enzyme
Displaying enzymes that degrade cellulose would allow consolidated bioprocessing for biofuel production!

Scfvbutton.png

Type III Needle scFv
The Antibody Fragment
Displaying antibody fragments would allow easy pathogen detection!

Ag4.png

Streptavidin Binding Tag
The Tag
A tag on the surface of E.coli would allow you to purify bacteria preferentially!

Leucinezipeprsbutton.png

Leucine Zippers
The Heterodimeric Binding Peptide
Displaying heterodimeric binding peptides would allow you to make heterogeneous bacteria systems!

Streptagbutton.png

Ag4
The Silver Binding Peptide
The metal head of our set! Displaying a heavy metal binding peptide would promote bioremediation of heavy metals and aid in synthesis of biomaterials.

Spacers

Spacer elements are present in most natural display systems, which suggests that they are important components of cell surface display. However, the precise role of spacers in the functionality of outer membrane proteins has not been extensively characterized and most people engineer cell surface display devices without structural spacers. We decided to build a few cell surface display systems with spacer elements in order to begin characterizing their role within engineered cell surface display devices. We introduced five different spacer domains to our surface display system: INP-repeats, beta roll, beta helix, Gly-Ser repeats and GFP-LVA. These elements are further described below.

INP-Repeats Synthetic spacer region composed of repeated portions of ice nucleation protein (INP) sequence.
Beta Roll Protein tertiary structure that is natively found in autotransporters and other outermembrane proteins.
Beta Helix Protein helical structures stabilized by hydrogen bonds and protein-protein interactions. The resulting structure contains two to three faces formed by the association of parallel beta strands.
Gly-Ser Repeats Synthetic spacer made from fourteen repeated glycine and serine residues. This spacer is essentially devoid of secondary structure, it is simply a long flexible linker. We made in order to differentiate between the importance of a natural spacer with secondary/tertiary structure and a long linker between the displayer and its passenger.
GFP-LVA Modified green fluorescent protein

Displayers

A displayer is defined as an outmembrane protein that carries another protein to the extracellular space of the cell.

For successful cell surface display of proteins, there must be an effective protein localization mechanism. Gram-negative bacteria such as E. Coli have two membranes, which present a problem for transporting proteins synthesized in the cytoplasm to the outside of the cell. Various transport schemes exist in gram-negative bacteria to effectively localize proteins to the outermembrane. The most common schemes are TypeI, TypeIII, and TypeV secretion.

In our display systems, we chose a class of outermembrane proteins called autotransporters that localizes proteins via the TypeV secretion mechanism. Over 700 autotransporters have been sequenced, many of which are used to export virulence factors to the outside of the cell. We decided to harvest this localization system for cell surface display because the outermembrane protein (aka displayer) spontaneously inserts into the outermembrane and pulls the protein it is covalently linked to (aka passenger) into the extracellular space. Moreover, autotransporters are capable of pulling through large proteins, such as enzymes and single-chain variable fragments. We have removed the native passengers in our autotransporter constructs and fused heterologous passengers of interest to the N-terminus of these autotransporters.

Autotransporter secretion.png

As depicted in the diagram above, autotransporter transport begins with localization to the periplasm via the Sec secretion pathway. The translocated protein remains unfolded in the periplasm until it inserts into the outermembrane by forming a beta barrel with its C-terminal 250-300 amino acyl residues. The N-terminus of the protein (containing our passenger of interest) is then pulled through the barrel to the outside of the cell. Passengers of displayers are often cleaved for extracellular secretion. In our systems, however, we removed the signal sequence that signals for peptide cleavage so our passengers remain attached to the transmembrane displayer protein.

In constructing our parts, we looked into a broad range of autotransporters, some well characterized and others putative, to explore the spectrum of display machinery and to establish the functionality of novel autotransporters for cell surface display.

Azo1653 AtD (putative) Organism: Azoarcus sp. (strain BH72)

Autotransporter type: AT-1 family

OprF AtD Organism: Pseudomonas fluorescens

Structure: an 8-stranded beta barrel in the outermembrane

Cl02365 AtD (putative) Organism: Neisseria meningitidis

Autotransporter type: AT-1 family

VtaA11 AtD Organism: Haemophilus parasuis

Autotransporter type: AT-2 family

Hag AtD Organism: Moraxella catarrhalis

Autotransporter type: dimeric family
Structure: 200kDa protein with 10-stranded beta barrel
predicted 2D structure of Hag AtD

Pcryo_1225 AtD (putative) Organism: Psychrobacter cryohalolentis
Hia AtD Organism: Haemophilus influenzae

Autotransporter domain: trimeric family
Structure: modular segments containing repeats of structurally distinct domains
Hia ATD.jpg

upaG_short Organism: Escherichia Coli

Autotransporter type: trimeric family

espP(beta) Organism: Escherichia coli

Structure: 12-stranded beta barrel
EspP ATD.jpg

ehaB Organism: Escherichia coli

Features: primary sequence alone is sufficient for crossing the bacterial membrane

TshA Organism: Escherichia coli

Autotransporter type: serine protease subfamily (because of the 7AA serine protease motif)

VirG(IcsA) Organism: Shigella flexneri
YuaQ AtD (putative) Organism: Escherichia coli

Features: bears sequence similarity to the confirmed autotransporters AIDA and Ag43

AIDA-I Organism: Escherichia Coli

Features: identified to be similar to IgA1, the first autotransporter used for surface display. Occurs naturally in the host organism, E. coli, and is a robust tool for surface display

Ag43_short Organism: Escherichia Coli MG1655

Features: expression of Ag43 is evenly distributed around the bacterial cell
Structure: 14 antiparallel beta strands each composed of about 12AA residues

eCPX (circularly permuted OmpX) Organism: Escherichia Coli

Features: protein is an enhanced CPX variant located in the outermembrane that joins the N- and C-termini of OmpX.
Ecpx image using molecular operating environment.jpg

CPG_L2 (circularly permuted OmpG) Organism: Escherichia Coli

Features: protein is circularly permuted with its backbone opening in loop 2, allowing both the N- and C- termini to be present in the extracellular space.

CPG_L6 (circularly permuted OmpG) Organism: Escherichia Coli

Features: protein is circularly permuted with its backbone opening in loop 6, allowing both the N- and C- termini to be present in the extracellular space.

Some of these proteins are putative autotransporters that have sequence homology to confirmed autotransporters. We chose these proteins because we wanted to test their functionality and expand the range of displayers available for surface display.

References

Pina, S et al. Trimeric Autotransporters of Haemophilus parasuis: Generation of an Extensive Passenger Domain Repertoire Specific for Pathogenic Strains. J Bacteriol. January 2009; 191(2): 576–587. Available Online: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2620822/ (Accessed: 20 October 2009).

Kostakioti, M et al. Functional analysis of the Tsh autotransporter from an avian pathogenic Escherichia coli strain. Infect Immun. October 2004;72(10):5548-54. Available Online: http://www.ncbi.nlm.nih.gov/pubmed/15385451?ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum (Accessed: 20 October 2009).

http://www.uniprot.org/uniprot/?query=H81868&sort=score

http://pfam.sanger.ac.uk/family?acc=PF05736

http://www.genome.jp/dbget-bin/www_bget?azo:azo1653

http://www.genome.jp/dbget-bin/www_bget?pcr:Pcryo_1225

http://www.uniprot.org/uniprot/Q9JMS3