"
Team:UC Davis/Parts
From 2009.igem.org
Parts related to secretion: Parts related to pH sensor:
| Proteins: |
Promoters: |
Others: |
Proteins: |
Promoters: |
INPNC:
An
exogenous gene,
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, studies 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 be consistent with BBF RFC-12
Standard.
For more information go to: BBa_K265008
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
SS:It
allows protein fused to outer membrane
proteins to become cleaved free. In this experiment 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 be consistent with BBF RFC-12 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.
ChvI
promoter: Gene
fusion studies confirmed that ChvI gene
was induced by acidic conditions (1). Also, it has been known to
implicate in
virulence (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.
