Team:UC Davis/Adding secretion/model 1

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Revision as of 18:45, 21 October 2009

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INPNC: The ice-nucleation protein (INP) from Pseudomonas syringae is used by its natural host to nucleate ice formation and is implicated in P.syringae-associated pathogenesis.  INP, as well as a truncated derivative lacking the central domain (INPNC), have been used extensively for displaying proteins on the surface of E. coli (7).  For instance, AldO and PhaZ1 have been successfully displayed on the surface of E. coli using INPNC (7, 15).
Park et al. have shown that INPNC, when fused to the phaZ1 gene, including its signal sequence, can serve as a suitable surface delivery and secretion device of the otherwise toxic phaZ1 gene product (15).  This part was synthesized by Mr. Gene (Regensburg, Germany) with codon optimization and subsequently transferred into vector ( pSB1AK3).  As it is expected that this part will be used in the context of the fusion protein, the prefix and suffix for this part are consistent with the BBF RCF-12 standard. 
We have proposed to build and test a general protein secretion system modeled after that developed by Park et al. in which a fusion of INPNC and the signal sequence from the phaZ1 gene are used to secrete any target protein. 
We have modified this protein to be consistent with the BBF RFC-12 Standard. We have submitted this part to the parts registry as part BBa_K265008.

OmpA: OmpA is a protein found on the outer membrane of E. coli (13) and is used as a displaying fusion protein on the cell surface. This part has already been documented on the parts registry; however, it has not been tested as a component of a secretion system (via fusion with a target protein linked with a cleavable signal sequence).
We have modified this protein to be consistent with the BBF RFC-12 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

RBS:  Ribosome 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: This is an inducible promoter which was found in the part registry.
For more information go to: BBa_R0010

SS: The signal sequence (SS) for the phaZ1 gene product of Paucimonas lemoignei, a polyhydroxybutyrate depolymerase (15).  In the native protein the signal sequence is cleaved between residues Ala37 and Leu38.  Park et al. have showed that the fusion of the complete phaZ1 gene (including SS) and a truncated ice nucleation protein from Pseudomonas syringae (BBa_K265008), could lead to stable expression and secretion of the phaZ1 gene product. 
We propose that the signal sequence might be generally useful as a cleavage tag in secretion systems that include a membrane anchor component, such as INPNC (BBa_K265008) or OmpA (BBa_K103006). The proposed constructs would consist of a membrane anchor (INPNC or OmpA) followed by the cleavable signal sequence and finally a target protein marked for secretion. 
Since we expect that this part will be used in the context of a fusion protein, we have modified this protein to be consistent with the BBF RFC-12 Standard. We have submitted this part to the part registry as part 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 the GFP or Luciferase do not work under a plate reader.

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