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]
  • BBa_J36848
  • BBa_J36849
  • BBa_J36850
  • 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
  • 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

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.

• This part is composed of:
  • BBa_K215000: Protein Expression Vector
  • BBa_K215001: Fusion protein sequence

• This part is used in:
  • BBa_K215010: GFP fusion protein
  • BBa_K215011: BBa_K215090 (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

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.

• This part is composed of
  • BBa_K215100: Gene encoding prtD
  • BBa_K215101: Gene encoding prtE
  • BBa_K215102: Gene encoding prtF
  • BBa_K215103: BBa_K21500 + BBa_K21501 prtD + prtE
  • BBa_K215104: BBa_K25103 + BBa_K25102 prtD + prtE + prtF
  • BBa_K215105: BBa_K215104 + BBa_B0014 in PSB1AK3 prtDEF + Terminator
  • BBa_K215106: BBa_K215104 + BBa_B0014 in PSB3T5 prtDEF + Terminator
  • BBa_K215108: BBa_J23114 + BBa_K215106 (Medium Promoter + prtDEF-Terminator)
  • BBa_K215109: BBa_J23113 + BBa_K215106 (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

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:
  • BBa_K215201: Display Vector with 5 trans-membrane OMPA
  • BBa_K215250: Coding Sequence for fusion protein scaffold, 1 trans-membrane OMPA
  • BBa_K215251: Coding Sequence for fusion protein scaffold, 5 trans-membrane OMPA

• This Part is used in:
  • BBa_K215210: Display system (1 trans-membrane) in protein expression vector
  • BBa_K215211: Display system (5 trans-membrane) in protein expression vector
  • BBa_K215260: BBa_K215210 fused to Streptavidin
  • BBa_K215261: BBa_K215211 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)