Journal of Biotechnology 152 (2011) 93–95
Contents lists available at ScienceDirect
Journal of Biotechnology
journal homepage: www.elsevier.com/locate/jbiotec
Short communication
Aequorin-expressing yeast emits light under electric control
Cristina Vilanova a,b , Ángeles Hueso a,b , Carles Palanca a,b , Guillem Marco a,b , Miguel Pitarch a,b ,
Eduardo Otero a,b , Juny Crespo a,b , Jerzy Szablowski a,b , Sara Rivera a,b , Laura Domínguez-Escribà a ,
Emilio Navarro c , Arnau Montagud b , Pedro Fernández de Córdoba b , Asier González d , Joaquín Ariño d ,
Andrés Moya a , Javier Urchueguía b , Manuel Porcar a,e,∗
a
Institut Cavanilles de Biodiversitat i Biologia Evolutiva, Universitat de València, Postal Code 22085, 46071 València, Spain
Interdisciplinary Modeling Group, Intertech, Instituto Universitario de Matemática Pura y Aplicada, IUMPA, Departamento de Matemática Aplicada, Universidad Politécnica de
Valencia, Camino de Vera s/n, 46022 València, Spain
c
Departamento de Lenguajes y Ciencias de la Computación, Campus de Teatrinos, Universidad de Málaga, 29071 Málaga, Spain
d
Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Cerdanyola 08193, Spain
e
Fundació General de la Universitat de València, Spain
b
a r t i c l e
i n f o
Article history:
Received 15 July 2010
Received in revised form
12 December 2010
Accepted 13 January 2011
Available online 1 February 2011
Keywords:
Aequorin
Bioluminescence
Yeast electro-stimulation
Synthetic biology
Bioelectronics
a b s t r a c t
In this study, we show the use of direct external electrical stimulation of a jellyfish luminescent calciumactivated protein, aequorin, expressed in a transgenic yeast strain. Yeast cultures were electrically
stimulated through two electrodes coupled to a standard power generator. Even low (1.5 V) electric
pulses triggered a rapid light peak and serial light pulses were obtained after electric pulses were applied
periodically, suggesting that the system is re-enacted after a short refraction time. These results open
up a new scenario, in the very interphase between synthetic biology and cybernetics, in which complex
cellular behavior might be subjected to electrical control.
© 2011 Elsevier B.V. All rights reserved.
The interface between biology and electronics is a potent yet
unexplored field of synthetic biology. In order to bridge the gap
between these two disciplines, a greater understanding should be
sought of the interaction between electronic devices and biological
components. Electrical impulses are a native language of electronics and are thus well suited for such interactions. Unfortunately,
cells which are naturally equipped with the ability to respond to
electrical signals chemically – neurons and muscle cells (Requena
et al., 1991; Watanabe and Endoh, 1998) – are difficult to culture,
which renders them a poor choice for any real-life applications.
Here we report the use of significantly more robust cells, budding yeast Saccharomyces cerevisiae, reversibly responding to a
programmed electrical stimulus. To date, there are no prior reports
on an electrically induced increase in the intracellular calcium of
yeast. We used a genetically encoded calcium indicator – aequorin
(Takahashi et al., 1999; Roda et al., 2004) – to forecast the potential
∗ Corresponding author at: Institut Cavanilles de Biodiversitat i Biologia Evolutiva,
Universitat de València, Postal Code 22085, 46071 València, Spain.
Tel.: +34 963 544 473; fax: +34 963 543 670.
E-mail address: [email protected] (M. Porcar).
0168-1656/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.jbiotec.2011.01.005
of external electric pulses as a control device of light emission in
genetically engineered S. cerevisiae strains. Aequorin is composed
of two distinct units (Shimomura et al., 1962): apoaequorin, with an
approximate molecular weight of 22 kDa and the prosthetic group,
coelenterazine, a luciferin. The aequorin–coelenterazine complex
is sensitive to Ca2+ because addition of calcium to the complex
triggers fast oxidation of coelenterazine to coelenteramide, which
results in the emission of blue light when coelenteramide relaxes
to the ground state (Shimomura et al., 1963).
A recombinant yeast strain expressing aequorin was grown in
selective Leu-SD (synthetic minimal medium lacking Leucine) and
incubated with coelenterazine as previously described (Viladevall
et al., 2004). Then it was subjected to a controlled depolarization of the cell membrane with KOH, resulting in light emission
as recorded by TD-20e luminometer (Turner BioSystems) (Fig. 1).
Inspired by the adaptation of the Hodgkin–Huxley model, traditionally used to describe the dynamics of electrical pulses in neurons,
we hypothesized that an effective transmembrane potential of a
few volts would produce a similar response in yeast (Catterall,
2000; Cui and Kaandorp, 2006). Indeed, the application of lowvoltage (1.5–16 V) electric pulses for 1–5 s triggered very similar
behavior in terms of light emission, as deduced by the sharp peaks
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C. Vilanova et al. / Journal of Biotechnology 152 (2011) 93–95
300
100
90
9V
250
6V
70
Luminescence (a.u.)/10 6 cells
Luminescence (a.u.)/10 6 cells
80
60
50
40
30
20
10
2V
200
150
100
0
0
10
20
30
40
50
60
Time (s)
50
Fig. 1. Alkali-triggered stimulation (dashed line) and electro-stimulation (continous line) of yeast strains showing very similar behavior. Alkali-based stimulation
was performed at t = 5 s by adding 30 ␮l of KOH 100 mM to 170 ␮l of an aequorinexpressing yeast suspension. Electro-stimulation was carried out at t = 5 s by
supplying 6 V during 5 s to another aequorin-expressing yeast suspension.
0
0
10
20
30
40
50
60
Time (s)
Fig. 3. Pulse-response of aequorin-expressing yeast suspensions prepared as
described in the main text and subjected to electrical pulses of 2, 6, and 9 V, for
5 s. Luminescence given in arbitrary units.
observed shortly after electro-stimulation (Fig. 1). As expected,
electrically induced bioluminescence was calcium-dependent as
confirmed by a series of experiments in which electro-stimulation
was performed with yeast suspensions containing calcium chelating agents such as EDTA, which retrieved no light emission (Fig. 2).
Thus, free calcium ions must be present in the extracellular medium
in order for yeast cells to glow. Further control reactions lacking
aequorin, coelenterazine, or yeast suspensions, were also electrically stimulated and again no light production was retrieved. Taken
together, these results discard the possibility that light production
is an artifact, and confirm that light emission requires the integrity
of the whole calcium-based system.
Under our experimental conditions, light emission was reproducible after a short refraction time (Fig. 2), indicating that the
mechanism underlying calcium entry is triggered by the electric
pulse, deactivated when no electricity is applied, and ready for
reenactment by an ulterior pulse. The intensity of light peaks was, at
least under the tested conditions (from 2 to 9 V), pulse-dependent
(Fig. 3).
From our results, it is tempting to hypothesize that external electric pulses induce voltage-dependent calcium channel opening and
100
Aeq+, coe+
90
Aeq+, coe+, EDTA+
80
Aeq+, coe-
Luminescence (a.u)/106 cells
70
Aeq-, coe+
60
Aeq-, coe-
50
SD, coe+
40
SD, coe-
30
20
10
1
0.5
0
0
1
2
Time (min)
3
Fig. 2. Response of genetically engineered yeasts expressing aequorin to electrical stimulation. Cell suspensions grown as previously described (Viladevall et al., 2004) were
incubated in Leu-SD medium with coelenterazine for 5 h and subjected to a 6 V electric pulse of 5 s of duration every minute. Arbitrary units are shown. Control reactions
lacking coelenterazine (Aeq+, coe−), yeast (SD, coe+), and lacking both coelenterazine and yeast (SD, coe−), along with a chelating (EDTA)-containing reaction (Aeq+, coe+,
EDTA+) were used. Additionally, wild-type yeast strains without aequorin, with (Aeq−, coe+) and without (Aeq−, coe−) coelenterazine were assayed.
C. Vilanova et al. / Journal of Biotechnology 152 (2011) 93–95
subsequent massive calcium entry, which results in a light peak as
a consequence of calcium binding to the aequorin–coelenterazine
complex. However, when calcium channel mutant strains mid1
and cch1 (Viladevall et al., 2004) were subjected to electrostimulation, significant light production was also observed (data
not shown). This is an indication that the externally induced membrane depolarization does not mimic alkali-based depolarization. A
different mechanism might thus lie behind light production under
external electrical stimulation. One possibility is that remaining
calcium channels (and maybe alternative calcium-transporting
proteins) might facilitate non-physiological calcium ions entry
under artificial depolarization. However, it cannot be ruled out
that, similarly to the mechanism supposed to be responsible of
electroporation for transforming competent cells with exogenous
DNA, electro-stimulation might also trigger transient channelindependent pores to open through the cell membrane. In any
case, the fact that bioluminescence can be observed without loss of
intensity over repeated applications of the voltage (Fig. 2) suggests
electrical stimulation is not harmful to the cells. The quick exponential fall in the bioluminescence signal suggests an active mechanism
of calcium sequestration, probably by cellular organelles such as
vacuoles or endoplasmic reticulum, ruling out cell lysis as a reason
for increased luminescence of aequorin.
The present work is the first step towards electrically controlling bioluminescence, opening two novel research directions:
first, the implementation of novel bio-electronic lighting devices,
including displays with living cells as pixels and second, the possibility of electrically controlling the behavior of GMOs. Given our
results, it can be concluded that it is theoretically possible to implement a biological light display with photoprotein-expressing cells
responding to electrical stimulation with light emission (for a simulation of a simple display based on real light emitting yeasts,
see supplementary material Video 1 online). Regarding the electrical control of cell physiology, further studies are needed in
order to forecast the potential for controlling gene expression,
enzymatic activity or any other desired behavior in GMOs by
95
triggering the massive and re-enactable entry of ions by artificial
electrical stimulation.
Acknowledgements
We are indebted to A. Maquieira and M.A. González from IQMA
(Instituto de Química Molecular Aplicada, UPV) for their technical
support. We are very grateful to Fabiola Barraclough for correcting
the English manuscript. This work is based on the project of the
Valencia team that attended the 2009 iGEM competition.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.jbiotec.2011.01.005
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