Team:Washington/Accomplishments

From 2009.igem.org

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''Accomplished''
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='''University of Washington 2009 iGEM Team Accomplishments'''=
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*Create Protein Expression Vector for IPP system
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**BBa_K215000, Strong laq promoter with strong RBS
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*Demonstrate Stable, Functional, His-Tagged Fusion protein from expression vector
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**[https://2009.igem.org/Team:Washington/Project/Target]See Project Page: Target
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*Synthesize and BioBrick type I secretion system from Erwinia Chrysanthemi
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**BBa_K215107, prtDEF from Erwinia Chrysanthemi under constitutive expression in low copy plasmid
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*Develop assay for looking for secreted proteins
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**[https://2009.igem.org/Team:Washington/Project/Secretion]See Project Page: Secretion
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*Extensively characterize Legacy Surface Display BioBricks in registry[https://2009.igem.org/Team:Washington/Project/]
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**BBa_J36848
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**BBa_J36849
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**BBa_J36850
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**BBa_J36851
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*Develop new modular surface display system
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*Computationally design novel Biotin-binding protein using Rosetta
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*Submit at least one new Biobrick functioning as designed
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**Target Expression Vector
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**OPDA [https://2009.igem.org/Team:Washington/Project/Opda]
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*Gain real-world synthetic biology experience
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=== Demonstrated that at least one new BioBrick Part of our own design works as expected ===
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*[http://partsregistry.org/wiki/index.php?title=Part:BBa_K215000 BBa_K215000] Target Expression Vector - expresses a favorite protein fused to streptavidin-binding Nano-Tag, secretion tag that directs export of the protein, and a His tag - all tags are removable by incubation with TEV protease. [https://2009.igem.org/Team:Washington/Project/Target Find this in our wiki]
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*[http://partsregistry.org/wiki/index.php?title=Part:BBa_K215091 BBa_K215091] Expresses OpdA, a nerve agent-degrading enzyme. [https://2009.igem.org/Team:Washington/Project/Target#BioBricking_and_Characterization_of_OpdA Find this in our wiki]
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*[http://partsregistry.org/wiki/index.php?title=Part:BBa_K215000 BBa_K215000] Protein Expression Vector - when IPTG-induced, expresses a protein with strong promoter and RBS (a good combination of BioBricks not previously found in the Registry). [https://2009.igem.org/Team:Washington/Project/Target#Expression_Vector_Construction Find this in our wiki]
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''To Do''
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=== Characterized existing BioBrick Parts and documented this information on the Registry ===
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*Test and optimize computationally designed biotin binding molecule.  
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*[https://2009.igem.org/Team:Washington/Project/Display#Experiments Find these in our wiki]
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*Demonstrate selective secretion of proteins harboring secretion tag
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*[http://partsregistry.org/wiki/index.php/Part:BBa_J36848 BBa_J36848] 2006 Harvard iGEM Streptavidin Cell Surface Display Construct - monomeric streptavidin, one OmpA transmembrane helix
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*[http://partsregistry.org/wiki/index.php/Part:BBa_J36849 BBa_J36849] 2006 Harvard iGEM Streptavidin Cell Surface Display Construct - single chain dimeric streptavidin, one OmpA transmembrane helix
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*[http://partsregistry.org/wiki/index.php/Part:BBa_J36850 BBa_J36850] 2006 Harvard iGEM Streptavidin Cell Surface Display Construct - monomeric streptavidin, five OmpA transmembrane helices
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*[http://partsregistry.org/wiki/index.php/Part:BBa_J36851 BBa_J36851] 2006 Harvard iGEM Streptavidin Cell Surface Display Construct - single chain dimeric streptavidin, five OmpA transmembrane helices
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== Target Vector ==
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=== Synthesized and BioBricked type I secretion system from ''Erwinia chrysanthemi'' ===
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------------------------------------------------------------------------------------------------------------
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*[https://2009.igem.org/Team:Washington/Project/Secretion Find these in our wiki]
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*[http://partsregistry.org/wiki/index.php/Part:BBa_K215107 BBa_K215107], Type I secretion system from Erwinia Chrysanthemi, constitutive expression, low copy, STRONG PROMOTER
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*[http://partsregistry.org/wiki/index.php/Part:BBa_K215108 BBa_K215108], Type I secretion system from Erwinia Chrysanthemi, constitutive expression, low copy, MEDIUM PROMOTER
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*[http://partsregistry.org/wiki/index.php/Part:BBa_K215109 BBa_K215109], Type I secretion system from Erwinia Chrysanthemi: constitutive expression, low copy, WEAK PROMOTER
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=== Developed assay to quantify secreted protein ===
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*This assay assess for the presence of secreted His-tagged protein in the growth media by running media over a nickel column (His-tagged proteins will be retained), wash column, and elute protein.  Eluted protein is quantified by comparing to a calculated standard curve.  [https://2009.igem.org/Team:Washington/Notebook Find this in our wiki]
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=== Designed new modular surface display system ===
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*This BioBrick is designed to express any protein as a cell surface display protein, using the well-characterized Lpp-OmpA cell surface display system.  Generated BioBrick, functionality not confirmed.  [https://2009.igem.org/Team:Washington/Future#Custom_Display_Vector Find this in our wiki]
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[[image:Target_structure.png |300px| left]]
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=== Worked on computational design of novel biotin-binding protein using Rosetta and Fold-It ===
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* [https://2009.igem.org/Team:Washington/Future#Fold-It Find this on our wiki]
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BBa_K215002 Is a BioBrick part that was made to work in conjunction with the Secretion System (BBa_K215107). When used it creates a fusion protein, that contains the prtB secretion signal recognized by the secretion system. It also fuses to the protein a nano-tag that is able to bind Streptavidin, TEV protease site (for isolating your protein from the fusion) and Histidine Epitote Tags for easy protein purification.
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=== Gained real-world synthetic biology experience ===
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*We learned about the power of synthetic biology to address real-world problems, gained experience in designing and constructing our own molecular biology projects, and discovered that proficiency in troubleshooting is a must with synthetic biology.  [https://2009.igem.org/Team:Washington/Team Find us in our wiki]
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='''BioBricks'''=
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=== Target Vector ===
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<table><tr><td>
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[[image:Target_structure.png |300px]]
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</td><td>
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[http://partsregistry.org/wiki/index.php/Part:BBa_K215002 BBa_K215002] Is a BioBrick part that was designed to work in conjunction with the Secretion System ([http://partsregistry.org/wiki/index.php/Part:BBa_K215107 BBa_K215107]). Expression of protein from this vector creates a fusion protein that contains the prtB secretion signal recognized by the secretion system. The fusion protein also contains: a Nano-Tag to the protein that is able to bind Streptavidin, Histidine Epitope Tags for easy protein purification, and TEV protease sites for removing all tags from the expressed protein.
<br clear="all" />
<br clear="all" />
*This part is composed of:
*This part is composed of:
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**BBa_K215000: Protein Expression Vector
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**[http://partsregistry.org/wiki/index.php/Part:BBa_K215000 BBa_K215000]: Protein Expression Vector
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**BBa_K215001: Fusion protein sequence
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**[http://partsregistry.org/wiki/index.php/Part:BBa_K215001 BBa_K215001]: Fusion protein sequence
*This part is used in:
*This part is used in:
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**BBa_K215010: GFP fusion protein
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**[http://partsregistry.org/wiki/index.php/Part:BBa_K215010 BBa_K215010]: GFP fusion protein
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**BBa_K215011: BBa_K215090 (organic phosphotriesterase) fusion protein
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**[http://partsregistry.org/wiki/index.php/Part:BBa_K215011 BBa_K215011]: [http://partsregistry.org/wiki/index.php/Part:BBa_K215090 BBa_K215090] (organic phosphotriesterase) fusion protein
*Current Status: Complete
*Current Status: Complete
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*Future directions:
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</td></tr></table>
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**Make more universal, to work with all proteins
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----
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**Optimize secretion-tag and nano-tag for fusion proteins
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<br clear="all" /><br clear="all" />
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=== Secretion System ===
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------------------------------------------------------------------------------------------------------------
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<table><tr><td>
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[[image:Secretion_structure.png |300px]]
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<br clear="all" /><br clear="all" />
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</td><td>
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[[image:Secretion_structure.png |300px | left]]
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[http://partsregistry.org/wiki/index.php/Part:BBa_K215107 BBa_K215107] Is the BioBrick version of the Type 1 secretion system from ''Erwinia Chrysanthemi''. It directs the secretion of proteins that contain the prtB C-terminus Epitote tag. To create a fusion protein with the prtB tag see [http://partsregistry.org/wiki/index.php/Part:BBa_K215002 BBa_K215002].  
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BBa_K215107 Is a BioBrick version of the Type 1 secretion system of Erwinia Chrysanthemi. It secretes proteins that contain the prtB C-terminus Epitote tag. To create a fusion protein with the prtB tag see BBa_K215002.  
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<br clear="all" />
<br clear="all" />
*This part is composed of
*This part is composed of
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**BBa_K215100: Gene encoding prtD
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**[http://partsregistry.org/wiki/index.php/Part:BBa_K215100 BBa_K215100]: Gene encoding prtD
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**BBa_K215101: Gene encoding prtE
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**[http://partsregistry.org/wiki/index.php/Part:BBa_K215101 BBa_K215101]: Gene encoding prtE
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**BBa_K215102: Gene encoding prtF
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**[http://partsregistry.org/wiki/index.php/Part:BBa_K215102 BBa_K215102]: Gene encoding prtF
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**BBa_K215103: BBa_K21500 + BBa_K21501 prtD + prtE
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**[http://partsregistry.org/wiki/index.php/Part:BBa_K215103 BBa_K215103]: [http://partsregistry.org/wiki/index.php/Part:BBa_K21500 BBa_K21500] + [http://partsregistry.org/wiki/index.php/Part:BBa_K21501 BBa_K21501] prtD + prtE
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**BBa_K215104: BBa_K25103 + BBa_K25102 prtD + prtE + prtF
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**[http://partsregistry.org/wiki/index.php/Part:BBa_K215104 BBa_K215104]: [http://partsregistry.org/wiki/index.php/Part:BBa_K25103 BBa_K25103] + [http://partsregistry.org/wiki/index.php/Part:BBa_K25102 BBa_K25102] prtD + prtE + prtF
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**BBa_K215105: BBa_K215104 + BBa_B0014 in PSB1AK3 prtDEF + Terminator
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**[http://partsregistry.org/wiki/index.php/Part:BBa_K215105 BBa_K215105]: [http://partsregistry.org/wiki/index.php/Part:BBa_K215104 BBa_K215104] + [http://partsregistry.org/wiki/index.php/Part:BBa_B0014 BBa_B0014] in PSB1AK3 prtDEF + Terminator
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**BBa_K215106: BBa_K215104 + BBa_B0014 in PSB3T5 prtDEF + Terminator
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**[http://partsregistry.org/wiki/index.php/Part:BBa_K215106 BBa_K215106]: [http://partsregistry.org/wiki/index.php/Part:BBa_K215104 BBa_K215104] + [http://partsregistry.org/wiki/index.php/Part:BBa_B0014 BBa_B0014] in PSB3T5 prtDEF + Terminator
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**BBa_K215108: BBa_J23114 + BBa_K215106 (Medium Promoter + prtDEF-Terminator)
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**[http://partsregistry.org/wiki/index.php/Part:BBa_K215108 BBa_K215108]: [http://partsregistry.org/wiki/index.php/Part:BBa_J23114 BBa_J23114] + [http://partsregistry.org/wiki/index.php/Part:BBa_K215106 BBa_K215106] (Medium Promoter + prtDEF-Terminator)
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**BBa_K215109: BBa_J23113 + BBa_K215106 (Weak Promoter + prtDEF-Terminator)
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**[http://partsregistry.org/wiki/index.php/Part:BBa_K215109 BBa_K215109]: [http://partsregistry.org/wiki/index.php/Part:BBa_J23113 BBa_J23113] + [http://partsregistry.org/wiki/index.php/Part:BBa_K215106 BBa_K215106] (Weak Promoter + prtDEF-Terminator)
*Current Status: In progress
*Current Status: In progress
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</td></tr></table>
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*Future directions:
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----
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**Optimize RBS's
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**Move secretion sequence into new plasmid, not TetR
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**Try variety of cell lines.
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<br clear="all" /><br clear="all" />
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=== Display System ===
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------------------------------------------------------------------------------------------------------------
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<br clear="all" /><br clear="all" />
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<table><tr><td>
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[[image:Display_structure.png |300px | left]]
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[[image:Display_structure.png |300px]]
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</td><td>
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BBa_K215200 is a Display vector that will allow for the presentation of any protein onto the surface of a cell by fusing the protein to Outer Membrane Protein A (OMPA). The fusion protein contains a GS peptide linker to space the displayed protein away from the outer membrane and also contains a TEV protease site to allow for purification of the displayed protein.  
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[http://partsregistry.org/wiki/index.php/Part:BBa_K215200 BBa_K215200] is a Display vector that will allow for the presentation of any protein on the cell surface by fusing the protein to the transmembrane region of Outer Membrane Protein A (OmpA). The fusion protein contains a GS peptide linker to space the displayed protein away from the outer membrane and also contains a TEV protease site to allow for purification of the displayed protein.  
<br clear="all" />
<br clear="all" />
*This Part is composed of:
*This Part is composed of:
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**BBa_K215201: Display Vector with 5 trans-membrane OMPA
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**[http://partsregistry.org/wiki/index.php/Part:BBa_K215201 BBa_K215201]: Display Vector with 5 trans-membrane OMPA
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**BBa_K215250: Coding Sequence for fusion protein scaffold, 1 trans-membrane OMPA
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**[http://partsregistry.org/wiki/index.php/Part:BBa_K215250 BBa_K215250]: Coding Sequence for fusion protein scaffold, 1 trans-membrane OMPA
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**BBa_K215251: Coding Sequence for fusion protein scaffold, 5 trans-membrane OMPA
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**[http://partsregistry.org/wiki/index.php/Part:BBa_K215251 BBa_K215251]: Coding Sequence for fusion protein scaffold, 5 trans-membrane OMPA
*This Part is used in:
*This Part is used in:
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**BBa_K215210: Display system (1 trans-membrane) in protein expression vector
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**[http://partsregistry.org/wiki/index.php/Part:BBa_K215210 BBa_K215210]: Display system (1 trans-membrane) in protein expression vector
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**BBa_K215211: Display system (5 trans-membrane) in protein expression vector
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**[http://partsregistry.org/wiki/index.php/Part:BBa_K215211 BBa_K215211]: Display system (5 trans-membrane) in protein expression vector
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**BBa_K215260: BBa_K215210 fused to Streptavidin
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**[http://partsregistry.org/wiki/index.php/Part:BBa_K215260 BBa_K215260]: [http://partsregistry.org/wiki/index.php/Part:BBa_K215210 BBa_K215210] fused to Streptavidin
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**BBa_K215261: BBa_K215211 fused to Streptavidin
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**[http://partsregistry.org/wiki/index.php/Part:BBa_K215261 BBa_K215261]: [http://partsregistry.org/wiki/index.php/Part:BBa_K215211 BBa_K215211] fused to Streptavidin
*Current Status: In progress
*Current Status: In progress
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</td></tr></table>
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*Future Directions:
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**Use new CDS display vector to display streptavidin
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**Design monomeric protein to bind peptide sequence
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**Utilize other proteins on CDS to test for activity of other proteins
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{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"
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!align="center"|[[Team:Washington/Accomplishments/Biobricks|Biobricks Submitted]]
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!align="center"|[[Team:Washington/Accomplishments/Status|Current Status Report]]
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!align="center"|[[Team:Washington/Accomplishments/Future|Future Directions]]
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|}
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Quick overview of what was done and the status of everyting.  Maybe 3 paragraphs (display, secretion, conclusions)
 
{{Template:Team:Washington/Templates/Footer}}
{{Template:Team:Washington/Templates/Footer}}

Latest revision as of 20:22, 20 October 2009

Uw title logo.png



University of Washington 2009 iGEM Team Accomplishments

Demonstrated that at least one new BioBrick Part of our own design works as expected

  • BBa_K215000 Target Expression Vector - expresses a favorite protein fused to streptavidin-binding Nano-Tag, secretion tag that directs export of the protein, and a His tag - all tags are removable by incubation with TEV protease. Find this in our wiki
  • BBa_K215091 Expresses OpdA, a nerve agent-degrading enzyme. Find this in our wiki
  • BBa_K215000 Protein Expression Vector - when IPTG-induced, expresses a protein with strong promoter and RBS (a good combination of BioBricks not previously found in the Registry). Find this in our wiki

Characterized existing BioBrick Parts and documented this information on the Registry

  • Find these in our wiki
  • BBa_J36848 2006 Harvard iGEM Streptavidin Cell Surface Display Construct - monomeric streptavidin, one OmpA transmembrane helix
  • BBa_J36849 2006 Harvard iGEM Streptavidin Cell Surface Display Construct - single chain dimeric streptavidin, one OmpA transmembrane helix
  • BBa_J36850 2006 Harvard iGEM Streptavidin Cell Surface Display Construct - monomeric streptavidin, five OmpA transmembrane helices
  • BBa_J36851 2006 Harvard iGEM Streptavidin Cell Surface Display Construct - single chain dimeric streptavidin, five OmpA transmembrane helices

Synthesized and BioBricked type I secretion system from Erwinia chrysanthemi

  • Find these in our wiki
  • BBa_K215107, Type I secretion system from Erwinia Chrysanthemi, constitutive expression, low copy, STRONG PROMOTER
  • BBa_K215108, Type I secretion system from Erwinia Chrysanthemi, constitutive expression, low copy, MEDIUM PROMOTER
  • BBa_K215109, Type I secretion system from Erwinia Chrysanthemi: constitutive expression, low copy, WEAK PROMOTER

Developed assay to quantify secreted protein

  • This assay assess for the presence of secreted His-tagged protein in the growth media by running media over a nickel column (His-tagged proteins will be retained), wash column, and elute protein. Eluted protein is quantified by comparing to a calculated standard curve. Find this in our wiki

Designed new modular surface display system

  • This BioBrick is designed to express any protein as a cell surface display protein, using the well-characterized Lpp-OmpA cell surface display system. Generated BioBrick, functionality not confirmed. Find this in our wiki

Worked on computational design of novel biotin-binding protein using Rosetta and Fold-It

Gained real-world synthetic biology experience

  • We learned about the power of synthetic biology to address real-world problems, gained experience in designing and constructing our own molecular biology projects, and discovered that proficiency in troubleshooting is a must with synthetic biology. Find us in our wiki

BioBricks

Target Vector

Target structure.png

BBa_K215002 Is a BioBrick part that was designed to work in conjunction with the Secretion System (BBa_K215107). Expression of protein from this vector creates a fusion protein that contains the prtB secretion signal recognized by the secretion system. The fusion protein also contains: a Nano-Tag to the protein that is able to bind Streptavidin, Histidine Epitope Tags for easy protein purification, and TEV protease sites for removing all tags from the expressed protein.

  • Current Status: Complete

Secretion System

Secretion structure.png

BBa_K215107 Is the BioBrick version of the Type 1 secretion system from Erwinia Chrysanthemi. It directs the secretion of proteins that contain the prtB C-terminus Epitote tag. To create a fusion protein with the prtB tag see BBa_K215002.

  • Current Status: In progress

Display System

Display structure.png

BBa_K215200 is a Display vector that will allow for the presentation of any protein on the cell surface by fusing the protein to the transmembrane region of Outer Membrane Protein A (OmpA). The fusion protein contains a GS peptide linker to space the displayed protein away from the outer membrane and also contains a TEV protease site to allow for purification of the displayed protein.

  • This Part is composed of:
    • BBa_K215201: Display Vector with 5 trans-membrane OMPA
    • BBa_K215250: Coding Sequence for fusion protein scaffold, 1 trans-membrane OMPA
    • BBa_K215251: Coding Sequence for fusion protein scaffold, 5 trans-membrane OMPA


  • Current Status: In progress


Retrieved from "http://2009.igem.org/Team:Washington/Accomplishments"