Team:UC Davis/Parts

 

New parts: 

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 and 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 (part name for AK vector).  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 BBF RFC-12 Standard. We have submitted this part to the parts registry as part BBa_K265008.

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 consists 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 BBF RFC-12 Standard. We have submitted this part to the part registry as part BBa_K265002.

INPNC + SS: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 (15).
We propose that this system might be generally useful for the secretion of other target proteins in E. coli and have therefore created a fusion of parts BBa_K265008 (INPNC) and BBa_K265002 (SS) which is compatible with the BBF RFC-12 Standard.
During the construction of this part, two silent mutations were introduced in the coding region of INPNC (T324A and A348T) that differ from those in part BBa_K265008. 
We have submitted this part to the part registry in the BBF RFC-12 Standard as part BBa_K265009.

OmpA + SS:Since OmpA is believed to function similarly to INPNC and 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 (15), we have decided to test and see if OmpA's ability to secret increases when it is used by a signal sequence.
We have modified this protein to be consistent with BBF RFC-12 Standard and have submitted this part to the part registry, BBa_K265011.

OmpA: OmpA is one of the proteins on the outer membrane of E. coli (13),it 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 compnent of secretion system (via fusion with a target protein linked with a cleavable signal sequence)
We have modified this protein to be consistent with 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 (Green Fluorescent Protein) : Mutant of GFP known to be very stable (superfolder), which will let this protein fold quickly so we can use either a fluorescent reader or UV light to detect it. Therefore it has been used as a reporter in our secretion system. It 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

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.

ChvI promoter: Gene fusion studies confirmed that ChvI gene was induced by acidic conditions (1). This gene is one of the candidates to be use in our biological pH sensor as a promoter.

KatA promoter: This Chromosomal gene is located on the linear chromosome (2) and it seems to be induced under an acidic environment as well as being involved in the Agrobacterium tumorigenesis (2).Research has suggested that ChvG is needed for "responsiveness of  gene expression to low pH "(2). This gene has become a candidate to complete our pH sensor device from this evidence.

AopB promoter: This Chromosomal gene located on the circular chromosome (2) encodes an outer member protein exposed on the bacterial cell surface (2). Also, ChvG was shown to be absolutely required for this gene expression (2)It seems to get induced under an acidic environment as well as being involved in the Agrobacterium tumorigenesis (2). Therefore, we have chosen this gene to be one of our candidates to complete our pH sensor device.

PhoA promoter: There has been a suggestion that ChvI can activate AP activity by activating transcription of this gene, PhoA (3). Therefore, this gene has become one of our candidates to complete our pH sensor device.

ImpA promoter:Gene fusion studies confirmed that impA genes was induced by acidic conditions (1), therefore, this is one of our candidates to complete our pH sensor device.

For more information go to: UCDAVIS_Parts