Team:Washington/Accomplishments

From 2009.igem.org

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[[image:Target_structure.png |300px]]
 
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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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This part is composed of:
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='''University of Washington 2009 iGEM Team Accomplishments'''=
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BBa_K215000
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=== Demonstrated that at least one new BioBrick Part of our own design works as expected ===
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* Protein Expression Vector
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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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BBa_K215001
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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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* Fusion protein sequence
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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]
 +
=== Characterized existing BioBrick Parts and documented this information on the Registry ===
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*[https://2009.igem.org/Team:Washington/Project/Display#Experiments Find these in our wiki]
 +
*[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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This part is used in:
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=== Synthesized and BioBricked type I secretion system from ''Erwinia chrysanthemi'' ===
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BBa_K215010
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*[https://2009.igem.org/Team:Washington/Project/Secretion Find these in our wiki]
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* GFP fusion protein
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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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BBa_K215011
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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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* BBa_K215090 (organic phosphotriesterase) fusion protein
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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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BBa_K215091
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Current Status: Complete
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=== 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.  [https://2009.igem.org/Team:Washington/Notebook Find this in our wiki]
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Future directions:
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=== 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.  [https://2009.igem.org/Team:Washington/Future#Custom_Display_Vector Find this in our wiki]
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* Make more universal, to work with all proteins
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=== Worked on computational design of novel biotin-binding protein using Rosetta and Fold-It ===
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* Optimize secretion-tag and nano-tag for fusion proteins
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* [https://2009.igem.org/Team:Washington/Future#Fold-It Find this on our wiki]
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[[image:Secretion_structure.png |300px]]
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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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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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='''BioBricks'''=
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=== Target Vector ===
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This part is composed of:
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<table><tr><td>
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BBa_K215100
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[[image:Target_structure.png |300px]]
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* Gene encoding prtD
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</td><td>
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BBa_K215101
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* Gene encoding prtE
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BBa_K215102
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* Gene encoding prtF
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BBa_K215103
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* BBa_K21500 + BBa_K21501 prtD + prtE
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BBa_K215104
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* BBa_K25103 + BBa_K25102 prtD + prtE + prtF
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BBa_K215105
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* BBa_K215104 + BBa_B0014 in PSB1AK3 prtDEF + Terminator
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BBa_K215106
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* BBa_K215104 + BBa_B0014 in PSB3T5 prtDEF + Terminator
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BBa_K215108
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* BBa_J23114 + BBa_K215106 Medium Promoter + prtDEF-Terminator
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BBa_K215109
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* BBa_J23113 + BBa_K215106 Weak Promoter + prtDEF-Terminator
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Current Status: In progress
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Future directions:
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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" />
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*This part is composed of:
 +
**[http://partsregistry.org/wiki/index.php/Part:BBa_K215000 BBa_K215000]: Protein Expression Vector
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**[http://partsregistry.org/wiki/index.php/Part:BBa_K215001 BBa_K215001]: Fusion protein sequence
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- Optimize RBS's
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*This part is used in:
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**[http://partsregistry.org/wiki/index.php/Part:BBa_K215010 BBa_K215010]: GFP 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
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- Move secretion sequence into new plasmid, not TetR
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*Current Status: Complete
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- Try variety of cell lines.
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</td></tr></table>
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----
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[[image:Display_structure.png |300px]]
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=== Secretion System ===
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<table><tr><td>
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[[image:Secretion_structure.png |300px]]
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</td><td>
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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].
 +
<br clear="all" />
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*This part is composed of
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**[http://partsregistry.org/wiki/index.php/Part:BBa_K215100 BBa_K215100]: Gene encoding prtD
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**[http://partsregistry.org/wiki/index.php/Part:BBa_K215101 BBa_K215101]: Gene encoding prtE
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**[http://partsregistry.org/wiki/index.php/Part:BBa_K215102 BBa_K215102]: Gene encoding prtF
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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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**[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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**[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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**[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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**[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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**[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)
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BBa_K215200
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*Current Status: In progress
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</td></tr></table>
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BBa_K215201
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----
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BBa_K215210
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=== Display System ===
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BBa_K215211
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<table><tr><td>
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[[image:Display_structure.png |300px]]
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BBa_K215250
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</td><td>
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BBa_K215251
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BBa_K215260
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BBa_K215261
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Current Status: In progress
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Future Directions:
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- Use new CDS display vector to display streptavidin
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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" />
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*This Part is composed of:
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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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**[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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**[http://partsregistry.org/wiki/index.php/Part:BBa_K215251 BBa_K215251]: Coding Sequence for fusion protein scaffold, 5 trans-membrane OMPA
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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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*This Part is used in:
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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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**[http://partsregistry.org/wiki/index.php/Part:BBa_K215211 BBa_K215211]: Display system (5 trans-membrane) in protein expression vector
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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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**[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
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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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*Current Status: In progress
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!align="center"|[[Team:Washington/Accomplishments/Biobricks|Biobricks Submitted]]
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</td></tr></table>
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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)
 
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</div>
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{{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

  • [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. Find this in our wiki
  • [http://partsregistry.org/wiki/index.php?title=Part:BBa_K215091 BBa_K215091] Expresses OpdA, a nerve agent-degrading enzyme. Find this in our wiki
  • [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). Find this in our wiki

Characterized existing BioBrick Parts and documented this information on the Registry

  • Find these in our wiki
  • [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
  • [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
  • [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
  • [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

Synthesized and BioBricked type I secretion system from Erwinia chrysanthemi

  • Find these in our wiki
  • [http://partsregistry.org/wiki/index.php/Part:BBa_K215107 BBa_K215107], Type I secretion system from Erwinia Chrysanthemi, constitutive expression, low copy, STRONG PROMOTER
  • [http://partsregistry.org/wiki/index.php/Part:BBa_K215108 BBa_K215108], Type I secretion system from Erwinia Chrysanthemi, constitutive expression, low copy, MEDIUM PROMOTER
  • [http://partsregistry.org/wiki/index.php/Part:BBa_K215109 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

[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.

  • This part is composed of:
    • [http://partsregistry.org/wiki/index.php/Part:BBa_K215000 BBa_K215000]: Protein Expression Vector
    • [http://partsregistry.org/wiki/index.php/Part:BBa_K215001 BBa_K215001]: Fusion protein sequence
  • This part is used in:
    • [http://partsregistry.org/wiki/index.php/Part:BBa_K215010 BBa_K215010]: GFP fusion protein
    • [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

Secretion System

Secretion structure.png

[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].

  • This part is composed of
    • [http://partsregistry.org/wiki/index.php/Part:BBa_K215100 BBa_K215100]: Gene encoding prtD
    • [http://partsregistry.org/wiki/index.php/Part:BBa_K215101 BBa_K215101]: Gene encoding prtE
    • [http://partsregistry.org/wiki/index.php/Part:BBa_K215102 BBa_K215102]: Gene encoding prtF
    • [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
    • [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
    • [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
    • [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
    • [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)
    • [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

Display System

Display structure.png

[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.

  • This Part is composed of:
    • [http://partsregistry.org/wiki/index.php/Part:BBa_K215201 BBa_K215201]: Display Vector with 5 trans-membrane OMPA
    • [http://partsregistry.org/wiki/index.php/Part:BBa_K215250 BBa_K215250]: Coding Sequence for fusion protein scaffold, 1 trans-membrane OMPA
    • [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:
    • [http://partsregistry.org/wiki/index.php/Part:BBa_K215210 BBa_K215210]: Display system (1 trans-membrane) in protein expression vector
    • [http://partsregistry.org/wiki/index.php/Part:BBa_K215211 BBa_K215211]: Display system (5 trans-membrane) in protein expression vector
    • [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
    • [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


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