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Team:UC Davis/Secretion/parts
From 2009.igem.org
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More Parts
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)
RBS
Ribosome Binding site number 32 (BBa_J61132) from the registry is being used in our secretion system. More
Terminator
We are using BBa_B0015, a double terminator, as our terminator in both our secretion and pH system. More
GFP
We are using Green Fluorescent Protein as a reporter that also serves as a small protein in testing our secretion system. More
Luciferase
Luciferase is a firefly protein that also fluoresces, so it serves as a reporter as well as a testable large protein.
More can be found in:
LacI
One inducible Promoter which was found in the part registry. More
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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