"
Team:UC Davis/Parts 1
From 2009.igem.org
INPNC LacI SS:signal sequence ChvI ChvI promoter
OmpA 6-His-Tag ChvG KatA promoter
Luciferase RBS aopB promoter
GFP Terminator phoA promoter
impA promoter
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).
Note:
A study has shown that "Ice- nucleation Protein (INP), an outer
membrane protein from Pseudomonas syringae, is able to catalyze the ice
crystal
formation of supercooled water.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
Escherichia coli outer membrane are sorted and incorporated into this
membrane”
(10)
For more information go to: http://partsregistry.org/wiki/index.php?title=Part:BBa_J36836
RBS:
Ribosome Binding site number 32 (BBa_J61132) from the registry is being
used in
our secretion system.
For
more information go to: http://partsregistry.org/wiki/index.php/Part: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: http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015
GFP:
We are using Green Fluorescent Protein as a reporter that also serves
as a
small protein in testing our secretion system.
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 can be found in: http://partsregistry.org/wiki/index.php?title=Part: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.
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.
