Engineering of a synthetic electron
conduit in living cells
Heather M. Jensena,b, Aaron E. Albersc, Konstantin R. Malleyc,1, Yuri Y. Londerd, Bruce E. Cohenc, Brett A. Helmsc,
Peter Weigeled, Jay T. Grovesa,b,c,e, and Caroline M. Ajo-Franklinb,c,2
aDepartments of Chemistry and
Sciences Divisions, Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720; and
eHoward Hughes Medical Institute, University of California, Berkeley, CA 94720;
bPhysical Biosciences and
dNew England Biolabs, Ipswich, MA 01938
Edited by Charles R. Cantor, Sequenom, Inc., San Diego, CA, and approved September 8, 2010 (received for review July 2, 2010)
Engineering efficient, directional electronic communication be-
tween living and nonliving systems has the potential to combine
the unique characteristics of both materials for advanced biotech-
nological applications. However, the cell membrane is designed by
nature to be an insulator, restricting the flow of charged species;
therefore, introducing a biocompatible pathway for transferring
electrons across the membrane without disrupting the cell is a
significant challenge. Here we describe a genetic strategy to move
intracellularelectrons to an inorganic extracellular acceptoralong a
molecularly defined route. To do so, we reconstitute a portion of
the extracellular electron transfer chain of Shewanella oneidensis
MR-1 into the model microbe Escherichia coli. This engineered
E. coli can reduce metal ions and solid metal oxides ∼8× and
∼4× faster than its parental strain. We also find that metal oxide
reduction is more efficient when the extracellular electron acceptor
has nanoscale dimensions. This work demonstrates that a genetic
cassette can create a conduit for electronic communication from
living cells to inorganic materials, and it highlights the importance
of matching the size scale of the protein donors to inorganic
cytochrome c ∣ nanobioelectronics ∣ synthetic biology ∣ iron reduction ∣
interface that permits electrical communication between living
and nonliving systems would enable previously undescribed op-
portunities in fields such as biosensing, bioenergy, and cellular
engineering. Sophisticated pipette- and electrode array-based
techniques permit transfer of ions from electrogenic and none-
lectrogenic cells to electrodes (1, 2). Although most technological
devices are electronic (i.e., rely on electron flow), a limited set of
techniques are available to permit transfer of electrons from a
variety of cell types to electrodes. Lipid-soluble mediators or
combinations of mediators can be used to transport electrons
from intracellular redox enzymes to extracellular electrodes in
bacterial (3, 4), fungal (5), and mammalian cells (6), but such
mediators rely on diffusion to interact with multiple cellular
substrates, thus obscuring a molecular-level understanding of
the electron path. Alternatively, in the absence of exogenous
mediators, a limited set of bacterial species are able to directly
transfer electrons to electrodes (7–9). However, a general strat-
egy to create cell-electrode connections with a well-defined elec-
tron transfer path that is broadly applicable to many cell types has
To make electrical connections to cells, most approaches rely
on introduction of noncellular redox species (10, 11) or physical
means to abrogate the inherently electrically insulating character
of cellular membranes (12). Here we explore a radically different,
biologically focused approach: to use synthetic biology to intro-
duce an electron transfer pathway that routes electrons along a
well-defined path from the cell interior to an extracellular inor-
ganic material. This approach specifically takes advantage of a
natural electron pathway that has evolved to utilize a variety of
oth organisms and human-made technological devices use
the flow of charge as information and energy. Creating an
solid metals and metal oxides as terminal electron acceptors.
Because such an extracellular electron transfer pathway is absent
in most cell types, we have the ability to create a well-defined
electron path with precise and flexible control over the combina-
tion and localization of the electron-carrying proteins. This ap-
proach is now tractable in part because the advent of genome
sequencing has greatly added to the molecular-level understand-
ing of diverse organisms (13, 14). Also key to this approach, the
growing field of synthetic biology offers more sophisticated tools
available to create and modify genetic systems (15–20). Now
armed with greater control over translation and transcription
of synthetic genes and pathways (21, 22), it is possible to engineer
the living cell as a material for advanced biological systems and
Naturally occurringdissimilatory metal-reducing bacteria, such
as those from the genera Shewanella and Geobacter, have evolved
mechanisms for direct charge transfer to inorganic minerals,
enabling them to use solid metal oxides as terminal electron
acceptors during anaerobic respiration (23–25). The electron
transfer pathway of Shewanella oneidensis MR-1, one of the best
understood pathways, is comprised of c-type cytochromes that
shuttle electrons from cytoplasmic and inner membrane oxidizing
enzymes toward the outside of the cell during anaerobic respira-
tion. Extensive genetic and biochemical data suggest that the
major components of the S. oneidensis MR-1 electron transfer
pathway are an inner membrane tetraheme cytochrome CymA,
a periplasmic decaheme cytochrome MtrA, outer membrane dec-
aheme cytochromes OmcA and MtrC, and an outer membrane
to move electrons from the intracellular quinol pool to extracel-
electron transfer events from quinol to CymA, from CymA to
MtrA, and from MtrA to either MtrC and/or OmcA.
In this work, we set out to determine whether we could convert
a bacterial strain that is incapable of reducing solid metal oxides
to one that can by installing a synthetic electron conduit that
bridges the cytosol to the extracellular space. To do so, we ex-
pressed the MtrC, MtrA, and MtrB proteins from S. oneidensis
MR-1 in Escherichia coli. In this heterologous system, we find that
the mature proteins are functionally expressed, and MtrC and
MtrA are redox active. We present evidence that MtrA interacts
with at least one native E. coli redox protein and that it has a
direct role in accelerating the rate of soluble Fe(III) reduction.
Most importantly, we show that expression of mtrCAB can “wire
Author contributions: H.M.J., Y.Y.L., P.W., J.T.G., and C.M.A.-F. designed research; H.M.J.
and K.R.M. performed research; A.E.A., B.E.C., and B.A.H. contributed new reagents/
analytic tools; H.M.J., Y.Y.L., P.W., and C.M.A.-F. analyzed data; and H.M.J. and C.M.A.-F.
wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
2To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
1Present address: Saint Louis University School of Medicine, St. Louis, MO 63104.
This article contains supporting information online at www.pnas.org/lookup/suppl/
www.pnas.org/cgi/doi/10.1073/pnas.1009645107PNAS Early Edition
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up” E. coli to inorganic solids; i.e., it confers the ability to reduce
solid α-Fe2O3and the rate of electron flow is increased when the
solid has nanometer dimensions.
Design of the Synthetic Electron Conduit Using S. oneidensis MR-1
mtrCAB. Because it is a genetically tractable, gram-negative bac-
terium with readily available tools for heterologous expression of
cytochromes c, E. coli was chosen as a test bed to determine
whether we could genetically introduce a molecularly defined
electron conduit modeled on the extracellular electron transfer
pathway of S. oneidensis MR-1. The physical arrangement of
S. oneidensis MR-1 cytochromes (Fig. 1A) suggests that an inner
membrane, a periplasmic, and an outer membrane cytochrome
are required to achieve extracellular electron transfer in E. coli.
In support of this hypothesis, although CymA (31, 32), MtrA
(32, 33), OmcA (34), and the combination of MtrA and CymA
(32) have been expressed in E. coli, none of these systems has
been shown sufficient to reduce solid Fe(III) oxides to Fe(II).
Additionally, published reports suggest that the outer membrane
28 strand β-barrel protein MtrB is required for correct folding
and localization of MtrC and OmcA (35) and may be involved
in interactions between MtrC and MtrA (36). Yet, because
extensive posttranslational processing is required to correctly in-
corporate the multiple hemes, fold, and localize each of these
cytochromes c, heterologous expression of even a single multi-
heme cytochrome c is a significant technical challenge. To our
knowledge, multiple decaheme cytochromes have not been simul-
taneously expressed in E. coli, so we sought to select a minimal
number of proteins to heterologously express. The work of both
Gescher and Pitts suggest that the native E. coli inner membrane
tetraheme cytochrome NapC, which is 52% sequence similar to
CymA, can reduce heterologously expressed MtrA (31, 33).
Therefore, we selected the mtrCAB genes as a potentially mini-
mal set required to create a synthetic electron conduit that would
allow E. coli to reduce insoluble metal oxides. To allow us to
dissect the electron transfer paths of this heterologous pathway
and to separately investigate the role of MtrA and MtrC in
Fe(III) reduction, we also chose to express MtrA by itself
(Fig. 1 B and C).
Functional Expression of mtrC, mtrA, and mtrB in E. coli. We created
two plasmids containing mtrA and mtrCAB under the control
of a T7 lac promoter (Fig. 1B) (37, 38). Because the E. coli cyto-
chrome c maturation (ccmABCDEFGH) genes are required for
heme insertion but are not transcribed under aerobic conditions
(39), the mtrA and mtrCAB plasmids were cotransformed with
pEC86 (Fig. 1C), a cytochrome c maturation (ccm) plasmid con-
taining ccmA-H under the constitutive tet promoter (Fig. 1B)
(40) into BL21(DE3) cells. BL21(DE3) cells (WT strain) and
cells carrying only the ccm plasmid (ccm strain) were pale yellow
in color; conversely, cells containing both the ccm and mtrA
(mtrA strain) or mtrCAB (mtrCAB strain) plasmids were red
(S4), indicating that the cytochromes were expressed. Addition
of isopropyl β-D-1-thiogalactopyranoside (IPTG), which dere-
presses the T7 lac promoter, would be expected to increase ex-
pression of MtrA and MtrCAB in the mtrA and mtrCAB
strains, respectively. However, even low concentrations of IPTG
(10 μM) resulted in cell pellets with a less intense red color as
compared to the same strain uninduced, suggesting more protein
was expressed under noninducing conditions; therefore, we
performed all subsequent growth in the absence of IPTG.
In S. oneidensis MR-1, the periplasmic and outer membrane
localization of MtrA and MtrC are believed to be crucial for
extracellular electron transfer (26). To probe the localization
of heterologously expressed MtrA and MtrC, aerobically grown
WT, ccm, mtrA, and mtrCAB strains were fractionated into
periplasmic and membrane fractions. These fractions were ana-
lyzed by SDS-PAGE followed by 3,3′,5,5′-tetramethylbenzidine
(TMBZ) staining, which stains proteins with covalently bound
heme (41). The periplasmic and membrane fractions of WT
and ccm strains had no visible bands in the TMBZ stain, suggest-
ing that no or little native c-type cytochrome is present (Fig. 2 A
and B). The periplasmic fractions of both mtrA and mtrCAB
strains had a band at 32 kD, the expected molecular mass of
MtrA, indicating that MtrA is correctly targeted to the periplasm
(Fig. 2A). MtrA is present to a lesser degree in the membrane
fractions of both strains, whereas a band at the expected mole-
cular mass of MtrC, 71 kD, is present only in the membrane frac-
tion of the mtrCAB strain (Fig. 2B). Finally, only the membrane
fraction of the mtrCAB strain produced a band at the expected
molecular mass of MtrB, 77 kD, in an immunoblot with an
MtrB-specific antibody (Fig. 2C). This pattern of localization
for MtrA, MtrB, and MtrC in E. coli is identical to that reported
for S. oneidensis MR-1, indicating that heterologous expression
preserved these proteins’ native localizations (27, 33, 42).
The MtrCAB proteins must also be redox active for functional
electron transport. We probed the redox activity of heterolo-
gously expressed MtrA and MtrC using UV-Vis absorption spec-
troscopy. The visible spectra of the periplasm and membrane
fractions of mtrA and mtrCAB strains were obtained under
oxidizing (air) and reducing conditions (sodium dithionite). The
most prominent features of oxidized c-type cytochromes are the
Soret band at 410 nm and a broad second peak at 530 nm.
After chemical reduction with sodium dithionite, the Soret band
shifts to 420 nm and the β- and α-bands are seen at 525 nm and
lular electron transfer pathway in Shewa-
nella oneidensis MR-1 where ES denotes
the extracellular space, P denotes the
periplasm, and C denotes the cytoplasm.
The silver and black spheres represent ex-
tracellular iron oxide. (B) Schematic of plas-
mids used to create the ccm, mtrA, and
mtrCAB strains in E. coli. (C) Schematic of
the engineered mtrA and mtrCAB strains
for soluble and extracellular metal reduc-
(A) Schematic of proposed extracel-
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552 nm. The periplasmic fractions of mtrA and mtrCAB strains
and the membrane fraction of mtrCAB all exhibited signature
absorption spectra typical of oxidized and reduced c-type cyto-
chrome (Fig. 2 D and E). Additionally, using A552 nmof the peri-
plasmic fractions and the extinction coefficient of purified MtrA
(ε552¼ 28 mM−1cm−1heme−1) (33), we estimate that there are
4,000 and 2,100 redox active MtrA present per cell in the mtrA
and mtrCAB strains, respectively. Assuming the same extinction
coefficient for MtrC, we estimate there are 75 redox active MtrC
per cell. Taken together, these data demonstrate that redox
active, full-length MtrA and MtrC were heterologously expressed
in E. coli with their native localization.
Expression of S. oneidensis MR-1 Cytochromes in E. coli Increases So-
luble Fe(III) Citrate Reduction Rates. We next sought to determine
whether heterologous expression of MtrA and MtrC in E. coli
enabled in vivo reduction of soluble chelated iron species, which
diffuse into the periplasm. To test iron reduction in live cultures,
we added 10 mM Fe(III) citrate separately to sterile media or a
fixed concentration (OD600¼ 0.5) of heat-killed, WT, ccm, mtrA,
and mtrCAB cells under anaerobic conditions and measured the
Fe(II)concentration of the resulting cultures as a function of time
using the ferrozine assay (43). For each time point, the Fe(II)
concentration at that time was subtracted by the corresponding
Fe(II) concentration in media-only sample, representing abiotic
Fe(III) reduction, and normalized by the ratio of the original
OD600to the current OD600to account for the relative number
of cells at each time point.
As shown in Fig. 3, metabolically inactive heat-killed E. coli
showed a small amount of Fe(III) reduction over the 10-d period
that is most likely caused by nonmetabolic processes that have
remained unidentified to date (44). Living strains reduce Fe
(III) citrate at a rate above the metabolically inactive E. coli that
is nearly identically for the first 2 d. After 2 d, the rate of Fe(III)
reduction in the WT strain levels off (10 ? 2 μMd−1). The ccm
strain reduces Fe(III) at a slightly faster rate (33 ? 3 μMd−1).
Because increased expression of native E. coli c-type cytochromes
slightly increases iron reduction (32), we tentatively assign this
rate increase to E. coli c-type cytochromes resulting from the
overexpression of the ccm operon. In striking contrast, the aver-
age rates of reduction in the mtrA (83 ? 3 μMd−1) and mtrCAB
(59 ? 11 μMd−1) strains are ∼8 and ∼6 times greater, respec-
tively, than the rate of WTreduction. We attribute the dramatic
changes in Fe(III) reduction rate in the mtrA and mtrCAB strains
to the presence of the heterologous cytochromes c expressed in
each strain. Surprisingly, the mtrCAB strain reduced Fe(III) at a
lesser rate than the mtrA strain. We suggest that this could be due
to decreased expression of MtrA in the mtrCAB strain and its
preferential ability over MtrC to reduce Fe(III) citrate.
The Redox State of MtrA Is Kinetically Linked to Fe(III) Citrate Reduc-
tion in E. coli. The increase in Fe(III) citrate reduction in the mtrA
strains relative to the ccm strain suggests that MtrA directly
reduces Fe(III) citrate. To confirm this, we simultaneously mea-
sured Fe(II) concentration and monitored the α-band absorption
at 552 nm in high-density anaerobic cell suspensions of the mtrA
strain before and after adding 50 μM Fe(III) citrate. In order to
clearly detect the α-band of MtrA over cell scatter and to observe
Fe(II) formation over a shorter time scale, these experiments
required unusually high cell densities and much lower concentra-
tions of Fe(III) citrate; however, changes in the α-band absorp-
tion could be unambiguously detected even with an OD600
around 3.0 (Fig. 4A). Before the addition of Fe(III) citrate,
the UV-Vis spectrum showed that MtrA is in a reduced state
(black line). Upon Fe(III) addition, the α-band absorption imme-
diately decreased (red line), indicating MtrA is rapidly oxidized.
As time elapsed, the α-band absorption increased (dashed lines),
indicating that MtrA is rereduced, presumably by cellular species.
Closer analysis can be undertaken by plotting ΔA552 nmand Fe
(II) concentration as a function of time relative to Fe(III) citrate
addition (Fig. 4B). Immediately following Fe(III) citrate addition,
the A552 nmdecreases by ∼0.15 OD and dwells in this oxidized
state for 12 min. Over the same time period, ∼30 μM is reduced
to Fe(II). These observations provide a direct link between the
time scales of MtrA oxidation and Fe(III) reduction in a hetero-
logous host and strongly suggest that MtrA directly reduces Fe
(III). They also suggest that movement of Fe(III) and Fe(II)
in and out of the periplasm is extremely fast. Interestingly,
after these fast initial events, the remaining Fe(III) is gradually
converted to Fe(II) while MtrA is slowly rereduced to its initial
redox state. The instantaneous rate of Fe(III) reduction decreas-
ing as a function of time indicates that the reduction rate depends
on the concentration of remaining Fe(III). This would be
expected for any nonzeroth order chemical reaction. The slow
rereduction of MtrA indicates that native E. coli proteins (e.g.,
Heme-stained SDS-PAGE gels of (A) periplasmic fractions and (B) membrane
fractions of the WT, ccm, mtrA, and mtrCAB strains. (C) Anti-MtrB immuno-
blot of membrane fractions of the WT, ccm, mtrA, and mtrCAB strains.
(D) Absorption spectra of the periplasmic fraction of the mtrA strain under
oxidizing and reducing conditions. (E) Absorption spectra of the membrane
fraction of the mtrCAB strain under oxidizing and reducing conditions.
Expression of full-length redox-active MtrA and MtrC in E. coli.
the WT, ccm, mtrA, and mtrCAB E. coli strains. Error bars represent the
standard deviation between triplicates from separate starting cultures.
Reduction of 10 mM Fe(III) citrate to Fe(II) as a function of time for
Jensen et al.PNAS Early Edition
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NapC) are capable of reducing MtrA, but that this process is
quite slow relative to the oxidation of MtrA by Fe(III).
NapC Is not the Only Electron Donor to MtrA in E. coli. Although the
data in Fig. 4B show that MtrA is capable of being rereduced, it
does not indicate which E. coli native protein(s) pass electrons
from the quinol pool to MtrA in the periplasm. Previous work
has suggested that NapC, a native E. coli inner membrane tetra-
heme cytochrome c, could functionally replace CymA, S.oneiden-
sis MR-1’s inner membrane tetraheme cytochrome c, because of
the 52% sequence similarity (31). If NapC is the sole electron
donor to MtrA, then we expect that an E. coli strain expressing
ccmA-H and mtrA but lacking napC would reduce soluble Fe(III)
at the same rate as the ccm strain. To explore this hypothesis, a
napC knockout was made in BL21(DE3) using the λ-red gene
disruption method (45). This strain, ΔnapC, was cotransformed
with ccm and/or mtrA to create the ΔnapC ccm and ΔnapC mtrA
strains, which were analyzed for their ability to reduce soluble Fe
(III) citrate. As shown in Fig. 5, the ΔnapC ccm strain reduces Fe
(III) more slowly than the ccm strain (21 ? 1 vs. 33 ? 3 μMd−1,
respectively), which is in accord with previous reports that sug-
gested increased expression of NapC could enable soluble iron
reduction in E. coli (31). Interestingly, the ΔnapC mtrA strain
reduces Fe(III) more slowly than the mtrA strain (51 ? 6 vs.
83 ? 3 μMd−1, respectively). If NapC were the only protein
transferring electrons from E. coli inner membrane to MtrA, it
would be expected that the ΔnapC mtrA strain reduction rate
would be similar to that of the ΔnapC ccm strain. However,
destroying NapC expression does not completely diminish the
reduction rate to that of the ΔnapC ccm strain, suggesting that
there are other electron donors to MtrA.
MtrCAB in E. coli Reduces Solid α-Fe2O3. Because our primary inter-
est is in exploring a previously undescribed approach to electro-
nically connect living cells and inorganic materials, we sought to
determine whether the mtrCAB cluster is capable of reducing ex-
tracellular metal oxides. To test if heterologous expression of
mtrCAB would reduce solid Fe2O3, we added α-Fe2O3(Fig. 6A,
d ∼ 5 μm) to a final concentration of 2.5 mgmL−1separately to
sterile media or a fixed concentration (OD600¼ 1.0) of WT, ccm,
mtrA, and mtrCAB cells under anaerobic conditions, and mea-
sured the Fe(II) concentration and cfu of the resulting cultures
as a function of time. The Fe(II) concentrations were normalized
by cfu mL−1at each time point. Figure 6B shows a representative
time point (t ¼ 24 d) of bulk α-Fe2O3reduction for all strains.
Very little solid Fe(III) is reduced by the WT, ccm, and mtrA
strains; no solid α-Fe2O3reduction is expected from the E. coli
strains unless there is a complete electron transfer pathway that
crosses both membranes because the E. coli genome does not
encode for any proteins capable of transferring electrons from
tion spectra showing the α-band of MtrA in high-density, anaerobic cell sus-
pensions of the mtrA strain before and after the addition of Fe(III) citrate.
MtrA begins reduced (black line, strong α-band absorption), but is oxidized
upon the addition of 50 μM Fe(III) citrate (red line). Over time, the α-band
absorbance recovers (colored dotted lines). (B) ΔA552 nmand Fe(II) concentra-
tion immediately before and after Fe(III) citrate addition as measured by the
Direct link of MtrA redox state to Fe(III) citrate reduction. (A) Absorp-
(III) citrate to Fe(II) by WT, ccm, ΔnapC ccm, mtrA, and ΔnapC mtrA strains.
NapC is not the sole electron donor to MtrA. Reduction of 10 mM Fe
α-Fe2O3, d ∼ 5 μm. (B) The concentration of bulk α-Fe2O3reduced by WT,
cmm, mtrA, mtrCAB strains normalized by colony forming units after
24 d. (C) Transmission electron microscopy of crystalline α-Fe2O3nanoparti-
cles, d ¼ 13 nm. (B) The concentration of α-Fe2O3nanoparticles reduced by
WT, ccm, mtrA, mtrCAB strains normalized by colony forming units after 24 d.
MtrCAB reduces solid α-Fe2O3. (A) Brightfield optical image of bulk
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the periplasm to the extracellular space. Interestingly, the
mtrCAB strain reduces significant amounts of α-Fe2O3per live
cell (11 ? 5 × 10−6μM∕cfumL−1) in comparison to the WTstrain
(2.6 ? 0.4 × 10−6μM∕cfumL−1). Thus by expressing only three
proteins from S. oneidensis MR-1, we are able to create a pre-
viously undescribed electron transfer pathway in E. coli, which
transfers cytosolic electrons to the surface of extracellular
α-Fe2O3. Additionally, these in vivo data provide further evi-
dence to existing in vitro data that MtrA, MtrB, and MtrC are
necessary and sufficient to reduce extracellular metal oxides.
Electron transfer theory predicts that in order for the mtrCAB
strain to reduce α-Fe2O3, the MtrC-containing outer membrane
must come into physical contact with the solid surface (46).
This suggests that the rate of extracellular iron reduction by
the mtrCAB strain would increase with increased α-Fe2O3surface
area. To test this prediction, we synthesized crystalline α-Fe2O3
nanoparticles (Fig. 6C, d ¼ 13 nm), added these particles to a
final concentration of 0.25 mgmL−1to the WT, ccm, mtrA,
and mtrCAB strains, and measured the formation of Fe(II) as de-
scribed above. As in the bulk Fe2O3experiments, WT, ccm, and
mtrA strains show very little reduction of Fe2O3in comparison to
the mtrCAB strain (27 ? 1 × 10−6μM∕cfumL−1). Moreover, the
amount of Fe(III) reduced was ∼2.5-fold greater for α-Fe2O3na-
noparticles than micron-sized α-Fe2O3over the same time period
despite the fact that Fe2O3concentration was 10-fold lower.
Engineering an efficient means of electronic communication
between living and nonliving systems has the potential to create
hybrid sensors and electronics capable of self-replication and
-repair. Although existing technologies can transfer electrons
from a cell to an electrode, no single approach has achieved what
the next generation applications require: molecularly defined
electron flow across a variety of cell types. Here we have demon-
strated the feasibility of a wholly biological approach that meets
this challenge and provides a previously undescribed blueprint
for cellular-electronic connections. By the addition of previously
undescribed genetic information, we have engineered electronic
communication between living cells and inorganic materials. The
genetic nature of this approach makes it applicable to many cell
types and specifies the route for electron transfer. To transfer the
system to a different prokaryote would simply require the choice
of an appropriate promoter and origin of replication, use of a
host-specific signal sequence to ensure proper localization, and
modification of the ccm genes to achieve their expression under
Another unique advantage is that the cell directs the assembly
of these bioelectronic connections such that they are self-repair-
ing, requiring no experimenter assembly or intervention. Finally,
based on the natural system’s respiratory versatility, we anticipate
that our engineered system should be able to reduce multiple
types of inorganic electrodes.
Although this work achieves a molecularly defined electron
conduit that may be introduced into other cell types, it is useful
to compare the rate at which our engineered strains reduce Fe
(III) to both WT E. coli and S. oneidensis MR-1 as a means of
determining its relative efficiency. The mtrCAB strain reduces
soluble and insoluble Fe(III) ∼6-fold and ∼4-fold faster, respec-
tively, thanWT E.coli;however, compared toS.oneidensis MR-1,
the mtrCAB strain reduces soluble and insoluble Fe(III) ∼30-fold
and ∼10-fold more slowly (47, 48). This rate difference suggests
there is still room to optimize the efficiency and speed of the elec-
tron transfer pathway in our engineered strain.
In the case of soluble Fe(III) reduction, the transfer of
electrons from native proteins of E. coli to MtrA is very likely
the rate limiting step. The observation that no native E. coli
cytochromes are detectable by TMBZ staining whereas MtrA is
readily detectable (Fig. 2) indicates there is a relatively low ratio
of electron donors to electron acceptors. The slow rereduction of
MtrA in the high cell density experiments (Fig. 4) also supports
this hypothesis. The rate of this initial electron transfer step may
potentially be enhanced either by increasing the expression of
native E. coli inner membrane cytochromes that donate electrons
to MtrA (such as NapC) or by additionally expressing the native
electron donor of MtrA, the S. oneidensis MR-1 inner membrane
cytochrome CymA. These approaches could potentially translate
into an increase in soluble Fe(III) reduction rate.
For solid Fe2O3reduction, it is likely that the last step in the
electron transport chain, reduction of Fe(III) by MtrC, is the rate
limiting step. Because our data, as well as other studies (28, 49,
50), indicate that MtrC is the only significant donor of electrons
to Fe2O3(Fig. 6), the relatively low abundance of MtrC relative
to MtrA (75 vs. 2,100 per cell, respectively) is a plausible expla-
nation of why the mtrCAB strain does not reduce solid Fe(III)
at the rates of S. oneidensis MR-1. Given the relative simplicity
of our genetic device, future work will focus on optimizing expres-
sion of MtrC to improve overall electron transfer rates to extra-
cellular metal oxides.
The increase in reduction rate for nanocrystalline α-Fe2O3also
suggests that materials engineering as well as synthetic biology
will play a substantial role in optimizing electron transfer between
engineered cells and inorganic materials. The mtrCAB strain gen-
erated 2.5-fold more Fe(II) from the α-Fe2O3nanoparticles than
bulk α-Fe2O3over the same time period, even though the Fe2O3
concentration was 10-fold less. Although this is a significant rate
enhancement, the surface area of the nanoparticles (d ¼ 13 nm)
is about 4 million times greater thanthe bulk α-Fe2O3(d ∼ 5 μm),
indicating that the reduction rate does not scale linearly with the
surface area. The nanocrystalline Fe2O3is coated with citric acid
to make the nanoparticles water-dispersible, and we speculate
that this organic layer may modulate electron transfer from MtrC
to the solid Fe2O3.
In summary, we have installed a unique electron transfer
pathway in E. coli that allows intracellular electrons to be shuttled
to the outer membrane where extracellular solid metal oxides
can be reduced. These experiments demonstrate that mtrCAB
is a genetic cassette that creates a molecularly defined pathway
for electrons to move between living cells and inorganic materi-
als. We envision that by installing this electronic pathway into
organisms that evolve intracellular electrons in response to light,
we could create extremely cheap, self-replicating photocatalysts
that directly store energy in batteries. Additionally by combining
our platform with organisms that modulate gene expression in
response to small molecules, we could create living biosensors
that provide electrical readouts. More broadly, our approach de-
monstrates that synthetic biology can be used to radically alter
the materials properties of living cells much in the way that
materials engineering can be used to alter physical properties
of materials. Because this is an effective method to functionally
interface cells with inorganic nanomaterials, we anticipate that
this synthetic biology approach will find application in a host
of nanobiotechnologies and bioelectronics.
Materials and Methods
Additional details can be found in SI Text.
Strains and Plasmids. The mtrA gene and mtrCAB cluster were amplified by
PCR using S. oneidensis MR-1 genomic DNA as the template, and the PCR
products were ligated into a modified pET30a+ vector (Novagen). The result-
ing plasmids were simultaneously transformed with a cytochrome c matura-
tion plasmid, pEC86, into E. coli BL21(DE3) (Invitrogen). The ΔnapC deletion
strain was made using the λ-red strategy as described by Datsenko and
Subcellular Fractionation. The periplasmic and membrane fractionation was
performed as described by Londer et al. (51) and Nikaido (52), respectively.
Jensen et al.PNAS Early Edition
5 of 6
Iron Reduction Assay Using Ferrozine. Cells from 50-mL cultures were pelleted,
washed, and resuspended to an OD600of 0.5 in anaerobic supplemented M9
minimal. All subsequent steps were performed in an anaerobic chamber (Coy
Laboratory Products) with an environment of 2% H2balanced in N2. Fe(III)
citrate (Sigma) was added to a final concentration of 10 mM, and at the time
of addition and subsequent time points, aliquots were removed to determine
the optical density at 600 nm and Fe(II) concentration. The Fe(II) concentra-
tion was determined with the ferrozine assay, adapted from Stookey (43).
Cytochrome c Redox Assay in Intact Cells. Dense cell suspensions in anaerobic
M9 minimal media supplemented with 0.4% lactate were transferred into
sealable quartz cuvettes in an anaerobic chamber. The absorption spectrum
of each culture was measured before iron addition, immediately after addi-
tion of 50 μM Fe(III)citrate, and at regular intervals afterward to observe
changes in the redox state of the cytochromes. The concentration of Fe(II)
was simultaneously monitored via the ferrozine assay.
Synthesis of α-Fe2O3ðcitrateÞnNanoparticles. α-Fe2O3ðcitrateÞ nanoparticles
were synthesized using a two-step approach. Oleate passivated nanocrystals
were prepared according to a modified, previously published literature
procedure (53). The oleate shell was subsequently displaced with citric acid
before aqueous transfer into buffer.
Reduction of Bulk and Nano Fe2O3. Cells from 50-mL cultures were pelleted,
washed, and resuspended in anaerobic M9 minimal media to a final
OD600of 1.0. All subsequent steps were performed in an anaerobic chamber.
For the bulk Fe2O3assay, 50 mg of particulate Fe2O3(Sigma) and 20 mL of
anaerobic culture were added to a sterile bottle, yielding a final Fe2O3
concentration of 2.5 mgmL−1. For nanoparticle cultures, the anaerobic
nanoparticle solution (4 mgmL−1) was added to a final concentration
of 0.25 mgmL−1.
ACKNOWLEDGMENTS. We thank Steven W. Singer (Lawrence Berkeley
National Lab, Berkeley, CA) for providing the ccm plasmid pEC86 and
Prof. Daad Saffarini (University of Wisconsin–Milwaukee, Milwaukee, WI)
for a generous gift of the anti-MtrB antibody. This work, carried out at
the Molecular Foundry, and H.M.J. were supported by the Office of Science,
Office of Basic Energy Sciences, of the U.S. Department of Energy under
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www.pnas.org/cgi/doi/10.1073/pnas.1009645107Jensen et al.
Jensen et al. 10.1073/pnas.1009645107
SI Materials and Methods.
Strains and Plasmids. Primers containing EcoRI and XbaI restric-
tion sites (Table S1, primers 1 and 2 for mtrA; primers 3 and 4 for
mtrCAB), and Platinum Pfx Polymerase (Invitrogen) were used to
amplify the respective sequences. After digestion with EcoRI and
XbaI, these DNA fragments were ligated into the modified
pET30a+ vector using T4 DNA Ligase (Roche). The modified
pET30a+ vector had an NdeI site (CATATG) directly upstream
of the EcoRI site used for insertion. The ATG in the NdeI site
was used as the start codon, and the forward primers for PCR
were designed to clone from the second codon of mtrA and mtrC
in the Shewanella genome. This design adds the two codons from
the EcoRI site coding Glu-Phe; however, the extra amino acids
will be cleaved by the Sec system in the process signal sequence
cleavage of MtrA and MtrC in plasmids mtrA and mtrCAB. re-
spectively. The ccm plasmid, pEC86, encodes the genes ccmA-
H under a tet promoter and carries a chloramphenicol resistance
Construction of napC Deletion Strain.The plasmids used in the gene
disruption process, pKD46, pKD3, pKD4, and pCP20, were ob-
tained from the Coli Genetic Stock Center at Yale University.
Primers 5 and 6 (Table S1) contain regions homologous to the
region directly upstream and downstream, respectively. These
primers were used to amplify a cassette containing kanamaycin
resistance flanked by FLP recombinase recognition target sites
off of pKD4 using Platinum Pfx Polymerase (Invitrogen). This
PCR product was electroporated with BL21(DE3) expressing
pKD46, induced with 10 mM L-arabinose, to replace napC in
the E. coli BL21(DE3) genome. Cells were grown with antibiotic
selection on kanamycin LB plates grown at 37°C. Colony PCR
using primers 7 and 8 (Table S1) was performed to verify the re-
placement in the genome. Kanamycin resistance was removed by
electroporating the new strain with pCP20, which removes the
sequence between the FRT sites. After pCP20 inactivation, col-
ony PCR was performed using primers 7 and 8 to verify removal
of the kanamycin resistance gene. The resulting strain is BL21
Cell Growth. All strains, unless otherwise specified, were grown in
2xYT media at 30 °C with 50 μgmL−1kanamycin; strains contain-
ing the ccm plasmid were grown with an additional 30 μgmL−1
chloramphenicol. Glycerol stocks were used to inoculate 5 mL
media, and cultures were grown overnight at 37 °C with 250-rpm
shaking. Then 50 μL of overnight cultures were back-diluted into
5 mL media and grown with 250-rpm shaking for 12 h. For
Fe(III) reduction assays, 50 mL of fresh 2xYTwas inoculated with
250 μL of the previous culture and then grown 16 h with 200-rpm
shaking. For periplasmic and membrane fractionations, 5 mL of
overnight culture were used to inoculate 1 L media and were
grown for 16 h.
Periplasmic Fractionation. The cells from a 1-L culture were
pelleted by centrifugation for 15 min at 4;000 × g and 4 °C.
The resulting cell pellet was slowly resuspended to homogeneity
in 30 mL of ice-cold N-[tris(hydroxymethyl)methyl]-2-aminoetha-
nesulfonic acid, pH 8.0, by pressing the cells with a rubber police-
man on the side of the flask. Chicken egg white lysozyme (Sigma)
was added to the resuspended pellet to a final concentration
0.5 mgmL−1and incubated at room temperature for 15 min.
After addition of 30 mL of ice-cold water, the suspension was
shaken horizontally on ice for 15 min at 100 rpm and then cen-
trifuged at 12;000 × g for 20 min at 4°C. The supernatant was
collected as the periplasmic fraction while the outer membrane
and intact cytoplasm was in the pellet. The periplasmic fraction
was analyzed by SDS-PAGE heme-stained gels and by UV-Vis
for redox spectral properties.
Membrane Fractionation. The cells from a 1-L culture were pel-
leted by centrifugation for 15 min at 4;000 × g and 4 °C and then
washed in 1 L 10 mM Hepes, pH 7.4. The washed pellet was then
resuspended in 120 mL ice-cold 10 mM Hepes, pH 7.4. Chicken
egg white lysozyme was added to the cell suspension to a final
concentration of 20 μgmL−1and incubated at room temperature
for 30 min. The protease inhibitor PMSF (Thermo Scientific) was
added to a final concentration of 1 mM. The cells were disrupted
via ultrasonication (power level 5, 9 min total duty in cycles of
30 s on, 30 s off, Misonix 3000, Misonix Inc.) in an ice bath. Un-
broken cells were removed by centrifugation at 1;000 × g for
15 min, and the resulting supernatant was removed and centri-
fuged at 100;000 × g for 2 h at 4°C to yield a pellet corresponding
to the crude cell envelope, containing both the outer and inner
membrane. This membrane fraction was solubilized in a solution
of 5% ðwt∕volÞ Triton X-100, 50 mM Hepes pH 7.4, 200 mM
NaCl before analysis by SDS-PAGE, Western blotting, and
UV-Vis spectroscopy. The supernatant of this spin was saved
for analysis by SDS-PAGE.
TMBZ Peroxidase Stain of SDS-PAGE. The 3,3′,5,5′-tetramethylben-
zidene (TMBZ) peroxidase stain method was adapted from
Thomas (1) to identify cytochromes c. Protein samples were sus-
pended in lithium dodecyl sulfate without β-mercaptoethanol.
The samples were run in 12.5% Tris HCl polyacrylamide gel
(BioRad) at 16 °C at 200 V for 60 min. TMBZ was dissolved
in methanol to 6.3 mM and mixed 3∶7 TMBZ solution:0.25 M
Sodium acetate, pH 5. The gel was immersed in this mixture
in the dark with occasional mixing for 2 h. Hydrogen peroxide
was added to a final concentration of 30 mM, and bands were
visualized 30 min after peroxide addition.
MtrB Western. Denatured membrane protein samples were elec-
trophoresed in 12.5% polyacrylamide gel and transferred to ni-
trocellulose membranes. The primary antibody for MtrB (Rabbit
Anti-MtrB) was kindly provided by Prof. Daad Saffarini (Univer-
sity of Wisconsin–Madison) and used at 1∶10;000 dilution. The
Immun-Star Goat Anti-Rabbit-HRP Conjugate kit was used as
the secondary antibody at 1∶60;000 dilution. The Western and
visualization of bands was done as per the Immun-Star WesternC
Chemiluminescent Kit (BioRad).
Visible Spectra of Cytochrome Samples. Samples from the periplas-
mic fraction and membrane fractionation were diluted such that
the absorbance was within the linear range. Visible spectra of
air-exposed samples were considered as fully oxidized protein
samples. The protein was chemically reduced by adding sodium
dithionite crystals (Sigma), and the spectrum was taken again.
Membrane fractions were baseline subtracted to consider the
scattering of light caused by Triton X-100 micelles.
Iron Reduction Assay Using Ferrozine. Cells from 50-mL cultures
were pelleted, washed, and resuspended to an OD600of 0.5 in
anaerobic M9 minimal media (12.8 gL−1Na2HPO4-7H2O,
3.0 gL−1KH2PO4, 0.50gL−1NaCl, 1.0 gL−1NH4Cl) (Difco)
supplemented with 0.4% lactate (Alfa Aesar), 1 mM thiamine
Jensen et al. www.pnas.org/cgi/doi/10.1073/pnas.10096451071 of 3
HCl (Sigma), 0.2% casamino acids (Merck), 2.0 mM MgSO4(Al-
drich), and 0.1 mM CaCl2(Aldrich). One aliquot of each culture
was centrifuged at 4;000 × g for 5 min at room temperature in the
anaerobic chamber to pellet the cells, and the supernatant was
acid extracted in 0.5 M HCl for 1 h. The total iron concentration
was determined by a separate acid extraction with 10% hydroxy-
lamine hydrochloride (Aldrich) in 0.5 M HCl for 1 h. An aliquot
of each acid extracted sample was then added to the dye, ferro-
zine (Acros Organics) buffered in 100 mM Hepes, pH 8.0, which
absorbs at 563 nm (ε563 nm¼ 27.9 mM−1cm−1) when bound to Fe
(II). The absorbance of all samples was recorded at 563 nm with a
UV-Vis spectrophotometer (Perkin–Elmer Lambda 35) and was
used to determine the change of Fe(II) concentration over time
as well as monitor total iron concentration available in the media.
The concentration of Fe(II) in each culture was subtracted by any
abiotic iron reduction observed in media controls at each time
point and was normalized to the relative number of cells by multi-
plying by the factor
tion by triplicate cultures.
OD600t. Error bars represent standard devia-
Cytochromec RedoxAssayin Intact Cells.After apparent recovery of
the redox state of the cytochrome, an additional 25 μM of Fe(III)
citrate was added. The spectrum was monitored again with simi-
lar results. All spectra were normalized to the average absorbance
at 561 nm, an approximate isobestic point. ΔA552 nmwas calcu-
lated by subtracting the initial A552 nmfrom all time points.
Synthesis of Fe2O3Nanoparticles. Iron(III) chloride hexahydrate
(544 mg, 2.01 mmol), sodium oleate (1.826 g, 6.00 mmol), oleic
acid (1.0 mL, 3.14 mmol), absolute ethanol (5.0 mL), and H2O
(8.0 mL) were combined in a 20-mL microwave reaction vial and
stirred for 1 h at 25°C, giving an opaque, dark reddish-brown or-
ganic top layer and a clear, pale yellow aqueous bottom layer in
the microwave vial. The reaction vessel was transferred to a mi-
crowave synthesizer (Biotage Initiator 8) and heated at 80 °C for
8 h, then at 180 °C for 15 min. The contents of the vessel were
transferred to a 50-mL conical tube and centrifuged at
4;400 × g for 30 min, affording a dark reddish-brown pellet of
nanocrystals. The liquid portion was decanted off and the pellet
washed sequentially with H2O (5.0 mL) and absolute ethanol
(5.0 mL). The pellet was resuspended in hexanes (50 mL), and
the tube centrifuged at 4;400 × g for 5 min., giving a clear, dark
reddish-brown hexane solution containing smaller, soluble Fe2O3
nanoparticles, and a reddish-brown pellet containing larger, inso-
luble Fe2O3nanoparticles and Fe2O3nanoparticle aggregates.
The hexanes solution was filtered through an Acrodisc Syringe
Filter (0.22 μm, Pall Corporation), giving a clear, dark reddish-
brown solution containing oleic acid-coated Fe2O3nanoparticles.
Dynamic light scattering (DLS) measurements (Malvern Instru-
ments) performed in hexanes at 20 °C showed that the particles
were narrowly distributed, with a mean diameter of 13 nm.
Fe2O3nanoparticles were heated with citric acid (480 mg) in
anhydrous DMSO (2.5 mL) at 100°C for 24 h with stirring. The
resulting solution was added slowly to 100 mL of 100 mM sodium
tetraborate buffer, pH 10.0, with periodic adjustment of pH to 10
with NaOH (1.0 M), giving a clear, reddish-brown aqueous
solution. The nanoparticles were concentrated by spin dialysis
(Amicon Ultra-15 10 K MWCO, Millipore Corporation), and
dialyzed with 5 × 15 mL of 10 mM sodium tetraborate buffer,
pH 8.3. DLS measurements (Malvern Instruments) performed
in buffer at 20 °C showed that the citrate-coated Fe2O3particles
were narrowly distributed, with a mean diameter of 13 nm.
Reduction of Bulk and Nano Fe2O3. At each time point, the colony
forming units and Fe(II) concentration for each culture was mea-
sured. To determine colony forming units, dilutions of each cul-
ture were plated on LB plates supplemented with Kanamycin (to
test for the presence of the mtrA or mtrCAB plasmids). Plates
were grown at 30 °C for 24 h. The concentration of Fe(II) was
determined by the ferrozine assay and was normalized for each
culture by cfu. Error bars represent standard deviation by tripli-
1. Bose S, et al. (2009) Bioreduction of hematite nanoparticles by the dissimilatory iron
reducing bacterium Shewanella oneidensis MR-1. Geochim Cosmochim Acta
and mtrCAB cultures resulted in a less intense red color of the cell pellets and less intense MtrA and MtrC bands in the TMBZ stained whole cell extracts as
compared to cultures containing no IPTG. Thus, basal expression of the mtrA and mtrCAB plasmids yields a higher concentration of correctly folded cytochrome
c than induced expression.
Cell pellets of uninduced WT, mtrA, and mtrCAB strains. The red color is caused by the hemes. Interestingly, addition of as little as 10 μM IPTG to mtrA
Jensen et al. www.pnas.org/cgi/doi/10.1073/pnas.10096451072 of 3
Table S1. Primers Download full-text
Primer no.Sequence (5′–3′)
Table S2. Plasmids
PlasmidPromotor Protein coding regions(s) Antibiotic resistanceSource
Steve Singer (Lawrence Berkeley National Lab, Berkeley, CA)
Coli genetic stock
Coli genetic stock
Coli genetic stock
Coli genetic stock
NA, not applicable.
Table S3. Strains
StrainCell line PlasmidsGene(s)
pSB1ET2 empty vector
pSB1ET2_empty + pEC86
pSB1ET2_MtrA + pEC86
pSB1ET2_MtrCAB + pEC86
pSB1ET2 empty vector
pSB1ET2_empty + pEC86
pSB1ET2_MtrA + pEC86
pSB1ET2_MtrCAB + pEC86
Jensen et al. www.pnas.org/cgi/doi/10.1073/pnas.10096451073 of 3