Team:Washington/Accomplishments

From 2009.igem.org

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=== Secretion System ===
=== Secretion System ===
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=== Display System ===
=== Display System ===

Revision as of 07:26, 15 October 2009

Uw title logo.png



Accomplished

  • Create Protein Expression Vector for IPP system
    • [http://partsregistry.org/wiki/index.php/Part:BBa_K215000 BBa_K215000], Strong laq promoter with strong RBS
  • Demonstrate Stable, Functional, His-Tagged Fusion protein from expression vector
    • [1]See Project Page: Target
  • Synthesize and BioBrick type I secretion system from Erwinia Chrysanthemi
    • [http://partsregistry.org/wiki/index.php/Part:BBa_K215107 BBa_K215107], prtDEF from Erwinia Chrysanthemi under constitutive expression in low copy plasmid
  • Develop assay for looking for secreted proteins
    • [2]See Project Page: Secretion
  • Extensively characterize Legacy Surface Display BioBricks in registry[3]
    • [http://partsregistry.org/wiki/index.php/Part:BBa_J36848 BBa_J36848]
    • [http://partsregistry.org/wiki/index.php/Part:BBa_J36849 BBa_J36849]
    • [http://partsregistry.org/wiki/index.php/Part:BBa_J36850 BBa_J36850]
    • [http://partsregistry.org/wiki/index.php/Part:BBa_J36851 BBa_J36851]
  • Develop new modular surface display system
  • Computationally design novel Biotin-binding protein using Rosetta
  • Submit at least one new Biobrick functioning as designed
    • Target Expression Vector (LINK TO REGISTRY)
    • OPDA (LINK TO REGISTRY)
  • Gain real-world synthetic biology experience


Bio-Bricks

Target Vector

Target structure.png

[http://partsregistry.org/wiki/index.php/Part:BBa_K215002 BBa_K215002] Is a BioBrick part that was made to work in conjunction with the Secretion System ([http://partsregistry.org/wiki/index.php/Part:BBa_K215107 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.

  • 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 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 [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 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.

  • 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





Quick overview of what was done and the status of everyting. Maybe 3 paragraphs (display, secretion, conclusions)

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