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Team:Washington/Accomplishments
From 2009.igem.org
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
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
| Biobricks Submitted | Current Status Report | Future Directions |
|---|
Quick overview of what was done and the status of everyting. Maybe 3 paragraphs (display, secretion, conclusions)
