Accomplished

Demonstrated that at least one new BioBrick Part of our own design works as expected

• BBa_K215000 Target Expression Vector - expresses a favorite protein fused to streptavidin-binding Nano-Tag, secretion tag that directs export of the protein, and a His tag - all tags are removable by incubation with TEV protease. Find this in our wiki
• BBa_K215091 Expresses OpdA, a nerve agent-degrading enzyme. Find this in our wiki
• BBa_K215000 Protein Expression Vector - when IPTG-induced, expresses a protein with strong promoter and RBS (a good combination of BioBricks not previously found int he Registry). Find this in our wiki

Characterized existing BioBrick Parts and documented this information on the Registry

• Find these in our wiki
• BBa_J36848 2006 Harvard iGEM Streptavidin Cell Surface Display Construct - monomeric streptavidin, one OmpA transmembrane helix
• BBa_J36849 2006 Harvard iGEM Streptavidin Cell Surface Display Construct - dimeric streptavidin, one OmpA transmembrane helix
• BBa_J36850 2006 Harvard iGEM Streptavidin Cell Surface Display Construct - monomeric streptavidin, five OmpA transmembrane helices
• BBa_J36851 2006 Harvard iGEM Streptavidin Cell Surface Display Construct - dimeric streptavidin, five OmpA transmembrane helices

Synthesized and BioBricked type I secretion system from Erwinia Chrysanthemi

• Find these in our wiki
• BBa_K215107, Type I secretion system from Erwinia Chrysanthemi, constitutive expression, low copy, STRONG PROMOTER
• BBa_K215107, Type I secretion system from Erwinia Chrysanthemi, constitutive expression, low copy, MEDIUM PROMOTER
• BBa_K215107, Type I secretion system from Erwinia Chrysanthemi: constitutive expression, low copy, WEAK PROMOTER

Developed assay to quantify protein secretion

• This assay assess for the presence of secreted His-tagged protein in the growth media by running media over a nickel column (His-tagged proteins will be retained), wash column, and elute protein. Eluted protein is quantified by comparing to a calculated standard curve. Find this in our wiki

Designed new modular surface display system

• This BioBrick is designed to express any protein as a cell surface display protein, using the well-characterized Lpp-OmpA cell surface display system. Generated BioBrick, functionality not confirmed. Find this in our wiki

Computationally design novel Biotin-binding protein using Rosetta

Gain real-world synthetic biology experience

BioBricks

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

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

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

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