Team:Washington/Accomplishments

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
  • 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

[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

• Future directions:
  • Make more universal, to work with all proteins
  • Optimize secretion-tag and nano-tag for fusion proteins

Secretion System

[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

• Future directions:
  • Optimize RBS's
  • Move secretion sequence into new plasmid, not TetR
  • Try variety of cell lines.

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 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

• 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)