"
Team:UC Davis/Parts
From 2009.igem.org
Secretion Model 1:
Click on an individual part for more information.
a.
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.
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: 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.
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.
