Team:UC Davis/Adding secretion/model 3

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Revision as of 04:23, 26 September 2009

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INPNC:Ice-nucleation protein (INP) from Pseudomonas Syringae was suggested to be used for display of foreign proteins on the surface of E. coli(7).Furthermore, researches have shown that an INP derivative constituting the N-and C-terminal domains can and has been used to display foreign proteins on the surface of E. coli(9). In our project we are intending to harness and make use of this feature by fusing a specific protein to it.

We have modified this protein to Biobrick standard, Tom Knights Standard.

OmpA: OmpA is one of the proteins on the outer membrane of E. coli (13). OmpA has been found to be useful as utilizable fusion part that can fuse our protein to and display on the surface of E. coli. This part has already been documented on the parts registry; however, it has not been tested via fusion with a target protein linked with a cleavable signal sequence.

We have modified this protein to Biobrick standard, Tom Knights Standard.

Note: “It has remained essentially unknown how proteins of E. coli outer membrane are sorted and incorporated into this membrane” (10)

For more information go to: http://partsregistry.org/wiki/index.php?title=Part:BBa_J36836

RBS:  Ribosome Binding site number 32 (BBa_J61132) from the registry is being used in our secretion system.

For more information go to: http://partsregistry.org/wiki/index.php/Part:BBa_J61132

Terminator: We are using BBa_B0015, a double terminator, as our terminator in both our secretion and pH system.

For more information go to: http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015

GFP: We are using Green Fluorescent Protein as a reporter that also serves as a small protein in testing our secretion system.

Luciferase: Luciferase is a firefly protein that also fluoresces, so it serves as a reporter as well as a testable large protein.

LacI: One inducible Promoter which was found in the part registry.

More can be found in: http://partsregistry.org/wiki/index.php?title=Part:BBa_R0010

SS:This signal sequence, when placed between INPNC, contains a cleavable site that allows the target fusion protein to ‘secrete’ from INPNC. We will do the same with OmpA.

We have modified this protein to Biobrick standard, Tom Knights Standard.

6-His Tag:The 6-Histidine Tag serves as a tag for Western Blotting if our fluorescent reporters are not expressed as highly as we would like.

Note: We are using this tag, just in case if the GFP or Luciferase does not work under a plate reader.

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