Team:Washington/Accomplishments

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Accomplished

  • Create Protein Expression Vector for IPP system
    • 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
    • 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]
  • 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
    • OPDA [4]
  • Gain real-world synthetic biology experience


To Do

  • Test and optimize computationally designed biotin binding molecule.
  • Demonstrate selective secretion of proteins harboring secretion tag

Target Vector

Target structure.png
Is a BioBrick part that was made to work in conjunction with the Secretion System (). 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:
    • Protein Expression Vector
      Fusion protein sequence
  • This part is used in:
    • GFP fusion protein
      (organic phosphotriesterase) fusion protein
  • Current Status: Complete
  • Future directions:
    • Make more universal, to work with all proteins
    • Optimize secretion-tag and nano-tag for fusion proteins



Secretion System



Secretion structure.png
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 . 


  • This part is composed of
    • Gene encoding prtD
      Gene encoding prtE
      Gene encoding prtF
      + prtD + prtE
      + prtD + prtE + prtF
      + in PSB1AK3 prtDEF + Terminator
      + in PSB3T5 prtDEF + Terminator
      + (Medium Promoter + prtDEF-Terminator)
      + (Weak Promoter + prtDEF-Terminator)
  • Current Status: In progress
  • Future directions:
    • Optimize RBS's
    • Move secretion sequence into new plasmid, not TetR
    • Try variety of cell lines.



Display System



Display structure.png
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:
    • Display Vector with 5 trans-membrane OMPA
      Coding Sequence for fusion protein scaffold, 1 trans-membrane OMPA
      Coding Sequence for fusion protein scaffold, 5 trans-membrane OMPA


  • This Part is used in:
    • Display system (1 trans-membrane) in protein expression vector
      Display system (5 trans-membrane) in protein expression vector
      fused to Streptavidin
      fused to Streptavidin
  • Current Status: In progress
  • Future Directions:
    • Use new CDS display vector to display streptavidin
    • Design monomeric protein to bind peptide sequence
    • Utilize other proteins on CDS to test for activity of other proteins




Biobricks Submitted Current Status Report Future Directions


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