Team:UC Davis/Adding secretion/model 5

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Model 1

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Secretion Model 5:

Click on an individual part for more information.


a.

b.


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.
For more information go to: BBa_K265008 


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: BBa_K103006


RBSRibosome Binding site number 32 (BBa_J61132) from the registry is being used in our secretion system.
For more information go to: 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: BBa_B0015


GFP: We are using Green Fluorescent Protein as a reporter that also serves as a small protein in testing our secretion system.
For more informaiton go to: BBa_K265003 


Luciferase: Luciferase is a firefly protein that also fluoresces, so it serves as a reporter as well as a testable large protein.
For more information go to: BBa_1712019


LacI: One inducible Promoter which was found in the part registry.
For more information go to: 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.
For more infromation go to :BBa_K265002


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.