GENETIC ENGINEERING OF NANOWIRE CYTOCHROMES IN E

weedheightswaistBiotechnology

Dec 11, 2012 (4 years and 7 months ago)

220 views

GENETIC ENGINEERING OF NANOWIRE CYTOCHROMES IN
E. COLI
.
Rachel E.
Lee
2
,
Elaine R. Frawley
1
, Miriam Rosenbaum
3
, Lars T. Angenent
3
,
Robert G. Kranz
1
,
Biology Department, Washington University, St. Louis, MO
1
; Biomedical Engineering
Department, Washington Uni
versity, St. Louis, MO
2
; Energy, Environmental, and Chemical
Engineering Department, Washington University, St. Louis, MO
3
.


The
proteo
bacteria
Shewanella oneidensis
has the ability to grow both aerobically and
anaerobically, using a wide variety of electr
on acceptors
, including
soluble iron in the
periplasm and
metal hydroxides
outside the outer membrane
.
Reduction of
insoluble
metal
hydroxides outside the cell

a
llows
Shewanella

to generate electric current
in bacterial fuel
cells. It has been hypothesized

that this process occurs using nanowires made up of various
proteins

which transport electrons to the outer membrane
.
Most of the proteins that are
included in this model are
c
-
type cytochromes, a research
focus

of
the Kranz lab.

The lab has
engineered a
strain of
E. coli

without its indogenous cytochrome
c

biogenesis
pathway

and
has developed inducible plasmids expressing both System I and System II pathways. In
conjunction with the Angenent lab, the Kranz lab has begun to
create and
study
E. coli
strains

expressing
recombinant
cytochromes from
Shewanella
.

The first two proteins involved in the

putative

Shewanella
nanowire pathway are
CymA

and
MtrA
.
CymA

is a
tetra
heme protein with an N
-
terminal membran
e anchor. It is
predicted that CymA

transports electro
ns from the quinone pool inside of the in
ner
membrane
to

MtrA in

the periplasm
.
MtrA

is a soluble, periplasmic, decaheme
cytochrome

which
transports

electrons from the inner membrane to cytochromes on the outer membrane,
according to the model.

The constr
uction of the
MtrA

expression plasmid
began with purification of
S.
oneidensis
genomic DNA. The applicable DNA sequence was amplified by PCR and cloned
into a chloremphenicol resistant vector, pBad24Cm
R
. The vector was checked for expression
in
E. coli
wit
h either System I or System II maturation proteins, and the
MtrA

protein was
found to be expressed in the periplasm at a size of 34 kDa.
The prot
ein displays the
characteristic

reduced minus oxidized spectrum of a
c
-
type cytochrome.

Similarly, the CymA
gen
e was expressed in
E. coli
with the proper inner membrane location and heme
attatchment.

By reconstructing the nanowire pathway in
E. coli
, the Kranz lab, in conjunction with
the Angenent lab, will determine the minimal protein components of this electron
transport
system.
E. coli

lacks this pathway, which allows for a more careful study of the function of
each component
. As more expression vectors are created, the labs hope to engineer a
strain

of
E. coli
that can generate electricity to power a fuel cell.