"
Team:Washington/Accomplishments
From 2009.igem.org
(Difference between revisions)
(→Synthesized and BioBricked type I secretion system from Erwinia chrysanthemi) |
|||
| (90 intermediate revisions not shown) | |||
| Line 1: | Line 1: | ||
__NOTOC__ | __NOTOC__ | ||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
{{Template:Team:Washington/Templates/Header}} | {{Template:Team:Washington/Templates/Header}} | ||
| - | |||
| - | |||
| - | + | ='''University of Washington 2009 iGEM Team Accomplishments'''= | |
| - | BBa_K215000 | + | === Demonstrated that at least one new BioBrick Part of our own design works as expected === |
| - | -Protein Expression Vector | + | *[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] |
| - | + | *[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] | |
| - | - | + | *[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 === | ||
| + | *[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 | ||
| + | *[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'' === | |
| - | + | *[https://2009.igem.org/Team:Washington/Project/Secretion 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. [https://2009.igem.org/Team:Washington/Notebook 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. [https://2009.igem.org/Team:Washington/Future#Custom_Display_Vector Find this in our wiki] | ||
| - | - | + | === Worked on computational design of novel biotin-binding protein using Rosetta and Fold-It === |
| + | * [https://2009.igem.org/Team:Washington/Future#Fold-It Find this on our wiki] | ||
| - | - | + | === 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. [https://2009.igem.org/Team:Washington/Team Find us in our wiki] | ||
| - | + | ='''BioBricks'''= | |
| + | === Target Vector === | ||
| - | + | <table><tr><td> | |
| + | [[image:Target_structure.png |300px]] | ||
| + | </td><td> | ||
| - | + | [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" /> | ||
| + | *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 | |
| - | + | </td></tr></table> | |
| + | ---- | ||
| - | BBa_K215105 | + | === Secretion System === |
| + | <table><tr><td> | ||
| + | [[image:Secretion_structure.png |300px]] | ||
| + | </td><td> | ||
| + | [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" /> | ||
| + | *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 | |
| + | </td></tr></table> | ||
| - | + | ---- | |
| - | + | === Display System === | |
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| + | <table><tr><td> | ||
[[image:Display_structure.png |300px]] | [[image:Display_structure.png |300px]] | ||
| + | </td><td> | ||
| - | BBa_K215200 | + | [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" /> | |
| - | + | *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 | |
| - | + | ||
| - | + | ||
| - | BBa_K215250 | + | |
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | |||
| - | - | + | *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 | |
| + | </td></tr></table> | ||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | + | {{Template:Team:Washington/Templates/Footer}} | |
Latest revision as of 20:22, 20 October 2009
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
|
|
[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.
|
Secretion System
|
|
[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].
|
Display System
|
|
[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.
|
