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Journal of Biotechnology
journal homepage: www.elsevier.com/locate/jbiotec

Short communication

Aequorin-expressing yeast emits light under electric control

Cristina Vilanova a,b , ngeles 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 Domnguez-Escrib a , Emilio Navarro c , Arnau Montagud b , Pedro Fernndez de Crdoba b , Asier Gonzlez d , Joaqun Arino d , Andrs Moya a , Javier Urchuegua b , Manuel Porcar a,e,
Institut Cavanilles de Biodiversitat i Biologia Evolutiva, Universitat de Valncia, Postal Code 22085, 46071 Valncia, Spain Interdisciplinary Modeling Group, Intertech, Instituto Universitario de Matemtica Pura y Aplicada, IUMPA, Departamento de Matemtica Aplicada, Universidad Politcnica de Valencia, Camino de Vera s/n, 46022 Valncia, Spain c Departamento de Lenguajes y Ciencias de la Computacin, Campus de Teatrinos, Universidad de Mlaga, 29071 Mlaga, Spain d Departament de Bioqumica i Biologia Molecular, Universitat Autnoma de Barcelona, Cerdanyola 08193, Spain e Fundaci General de la Universitat de Valncia, Spain
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In this study, we show the use of direct external electrical stimulation of a jellysh 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.

Article history: Received 15 July 2010 Received in revised form 12 December 2010 Accepted 13 January 2011 Available online xxx Keywords: Aequorin Bioluminescence Yeast electro-stimulation Synthetic biology Bioelectronics

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The interface between biology and electronics is a potent yet unexplored eld 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 difcult to culture, which renders them a poor choice for any real-life applications. Here we report the use of signicantly 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 Valncia, Postal Code 22085, 46071 Valncia, Spain. Tel.: +34 963 544 473; fax: +34 963 543 670. E-mail addresses: manuel.porcar@uv.es, criviser@gmail.com (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 aequorincoelenterazine 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., 1962). 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 HodgkinHuxley 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 (1.516 V) electric pulses for 15 s triggered very similar behavior in terms of light emission, as deduced by the sharp peaks

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Please cite this article in press as: Vilanova, C., et al., Aequorin-expressing yeast emits light under electric control. J. Biotechnol. (2011), doi:10.1016/j.jbiotec.2011.01.005

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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.

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observed shortly after electro-stimulation (Fig. 1). As expected, electrically induced bioluminescence was calcium-dependent as conrmed 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 conrm 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 subsequent massive calcium entry, which results in a light peak as a consequence of calcium binding to the aequorincoelenterazine complex. However, when calcium channel mutant strains mid1 and cch1 (Viladevall et al., 2004) were subjected to electrostimulation, signicant 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 articial 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.

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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.

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Please cite this article in press as: Vilanova, C., et al., Aequorin-expressing yeast emits light under electric control. J. Biotechnol. (2011), doi:10.1016/j.jbiotec.2011.01.005

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enzymatic activity or any other desired behavior in GMOs by triggering the massive and re-enactable entry of ions by articial electrical stimulation. Uncited reference Shimomura et al. (1963).
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We are indebted to A. Maquieira and M.A. Gonzlez from IQMA (Instituto de Qumica 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

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References
Catterall, W.A., 2000. Structure and regulation of voltage-gated Ca2+ channels. Rev. Cell Dev. Biol. 16, 521555. Cui, J., Kaandorp, J.A., 2006. Mathematical modeling of calcium homeostasis in yeast cells. Cell Calcium 39, 337348. Requena, J., Whittembury, J., Scarpa, A., Brinley Jr., J.F., Mullins, L.J., 1991. Intracellular ionized calcium changes in squid giant axons monitored by Fura-2 and aequorin. Ann. N. Y. Acad. Sci. 639, 112125. Roda, A., Pasini, P., Mirasoli, M., Michelini, E., Guardigli, M., 2004. Biotechnological applications of bioluminescence and chemiluminescence. Trends Biotechnol. 22 (6), 295303. Shimomura, O., Johnson, F.H., Saiga, Y., 1962. Extraction, purication and properties of aequorin, a bioluminescent protein from the luminous hydromedusan Aequorea. J. Cell. Comp. Physiol. 59, 223239. Shimomura, O., Johnson, F.H., Saiga, Y., 1963. Microdetermination of calcium by aequorin luminescence. Science 140, 13391340. Takahashi, A., Camacho, P., Lechleiter, J.D., Herman, B., 1999. Measurement of intracellular calcium. Physiol. Rev. 79, 10891125. Viladevall, L., Serrano, R., Ruiz, A., Domenech, G., Giraldo, J., Barcel, A., Arino, J., 2004. Characterization of the calcium-mediated response to alkaline stress in Saccharomyces cerevisiae. J. Biol. Chem. 279 (42), 4361443624. Watanabe, A., Endoh, M., 1998. Relationship between the increase in Ca2+ transient and contractile force induced by angiotensin II in aequorin-loaded rabbit ventricular myocardium. Cardiovasc. Res. 37 (2), 524531.

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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.

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The present work is the rst step towards electrically controlling bioluminescence, opening two novel research directions: rst, 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,

Please cite this article in press as: Vilanova, C., et al., Aequorin-expressing yeast emits light under electric control. J. Biotechnol. (2011), doi:10.1016/j.jbiotec.2011.01.005