Team:Washington/Accomplishments

From 2009.igem.org

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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.
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.
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This part is composed of:
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*This part is composed of:
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**BBa_K215000: Protein Expression Vector
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**BBa_K215001: Fusion protein sequence
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BBa_K215000
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*This part is used in:
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* Protein Expression Vector
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**BBa_K215010: GFP fusion protein
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BBa_K215001
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**BBa_K215011: BBa_K215090 (organic phosphotriesterase) fusion protein
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* Fusion protein sequence
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*Current Status: Complete
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This part is used in:
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*Future directions:
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BBa_K215010
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**Make more universal, to work with all proteins
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* GFP fusion protein
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**Optimize secretion-tag and nano-tag for fusion proteins
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BBa_K215011
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* BBa_K215090 (organic phosphotriesterase) fusion protein
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BBa_K215091
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Current Status: Complete
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Future directions:
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-
 
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* Make more universal, to work with all proteins
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* Optimize secretion-tag and nano-tag for fusion proteins
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[[image:Secretion_structure.png |300px | left]]
[[image:Secretion_structure.png |300px | left]]

Revision as of 16:20, 13 October 2009

Uw title logo.png



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
  • 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 structure.png

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 structure.png

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 structure.png

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)