Team:Washington/Accomplishments

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

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

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

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

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