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ARTICLE IN PRESS
Journal of Biotechnology xxx (2011) xxx–xxx

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

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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 Arino d , Andrés Moya a , Javier Urchueguía b , Manuel Porcar a,e,∗
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
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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.

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 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 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 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., 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 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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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

light emission was reproducible after a short refraction time (Fig. it is tempting to hypothesize that external electric pulses induce voltage-dependent calcium channel opening and subsequent massive calcium entry. EDTA+) were used. The intensity of light peaks was. coe+). the fact that bioluminescence can be observed without loss of intensity over repeated applications of the voltage (Fig. However.01. Vilanova et al. C. which retrieved no light emission (Fig. and ready for reenactment by an ulterior pulse. This is an indication that the externally induced membrane depolarization does not mimic alkali-based depolarization. similarly to the mechanism supposed to be responsible of electroporation for transforming competent cells with exogenous DNA. Control reactions lacking coelenterazine (Aeq+. coe+) and without (Aeq−. along with a chelating (EDTA)-containing reaction (Aeq+. coelenterazine. Aequorin-expressing yeast emits light under electric control..005 . et al. 1. Thus. coe−) coelenterazine were assayed. and confirm that light emission requires the integrity of the whole calcium-based system. A different mechanism might thus lie behind light production under external electrical stimulation. these results discard the possibility that light production is an artifact. Alkali-triggered stimulation (dashed line) and electro-stimulation (continous line) of yeast strains showing very similar behavior. doi:10. 3).G Model BIOTEC 5663 1–3 2 100 90 80 ARTICLE IN PRESS C. and lacking both coelenterazine and yeast (SD. From our results. pulse-dependent (Fig. it cannot be ruled out that.. Cell suspensions grown as previously described (Viladevall et al. significant light production was also observed (data not shown). free calcium ions must be present in the extracellular medium in order for yeast cells to glow.jbiotec. 2). / Journal of Biotechnology xxx (2011) xxx–xxx 70 60 50 40 30 20 10 0 0 10 20 30 Time (s) 40 50 60 Fig. The quick exponential fall in the bioluminescence signal suggests an active mechanism of calcium sequestration. 2.)/10 6 cells Please cite this article in press as: Vilanova. 2) suggests electrical stimulation is not harmful to the cells. Electro-stimulation was carried out at t = 5 s by supplying 6 V during 5 s to another aequorin-expressing yeast suspension. Further control reactions lacking aequorin. at least under the tested conditions (from 2 to 9 V). Arbitrary units are shown. coe+. One possibility is that remaining calcium channels (and maybe alternative calcium-transporting proteins) might facilitate non-physiological calcium ions entry under artificial depolarization. wild-type yeast strains without aequorin. In any case. electro-stimulation might also trigger transient channelindependent pores to open through the cell membrane. Response of genetically engineered yeasts expressing aequorin to electrical stimulation. Taken together. (2011). Under our experimental conditions. with (Aeq−. However. 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. 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. Luminescence (a. 66 67 68 69 70 71 72 73 74 75 observed shortly after electro-stimulation (Fig. were also electrically stimulated and again no light production was retrieved. 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. which results in a light peak as a consequence of calcium binding to the aequorin–coelenterazine complex. probably by cellular organelles such as vacuoles or endoplasmic reticulum.2011. 1).u. J. coe−). or yeast suspensions. Additionally.. As expected. 2004) were subjected to electrostimulation. Biotechnol. ruling out cell lysis as a reason for increased luminescence of aequorin. 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 Fig.1016/j. 2). indicating that the mechanism underlying calcium entry is triggered by the electric pulse. coe−). deactivated when no electricity is applied. when calcium channel mutant strains mid1 and cch1 (Viladevall et al. yeast (SD..

˜ Viladevall. (2011).. A. Domenech. Pasini. 639. J.. Shimomura. the possibility of electrically controlling the behavior of GMOs. Structure and regulation of voltage-gated Ca2+ channels. Maquieira and M.. 295–303.u.G Model BIOTEC 5663 1–3 ARTICLE IN PRESS C. Arino. 1963. A. O. 2004. Supplementary data 127 128 129 130 131 132 50 Supplementary data associated with this article can be found. Comp. Acad. 524–531. 110 111 112 113 114 115 116 117 118 119 120 The present work is the first step towards electrically controlling bioluminescence.. J. P. Intracellular ionized calcium changes in squid giant axons monitored by Fura-2 and aequorin. Q1 124 Luminescence (a. Biotechnol.. 59. Physiol. 6. F.. Rev. J.005 0 133 134 0 10 20 30 Time (s) 40 50 60 References Catterall. A. A. Cardiovasc. Aequorin-expressing yeast emits light under electric control. Mathematical modeling of calcium homeostasis in yeast cells. Cui. L..A. Y. et al. Luminescence given in arbitrary units.005 . J. Shimomura. Camacho. 1089–1125. including displays with living cells as pixels and second. M. J. Ann.A. for 5 s. R.. purification and properties of aequorin. P. Cell Calcium 39. further studies are needed in order to forecast the potential for controlling gene expression.)/10 6 cells 2V 200 125 Acknowledgements 150 126 100 We are indebted to A. Uncited reference Shimomura et al. Regarding the electrical control of cell physiology.2011. (1963).. Barceló. Whittembury. Characterization of the calcium-mediated response to alkaline stress in Saccharomyces cerevisiae. Serrano.F. J. 279 (42). Biol. Johnson. Brinley Jr. Michelini. F..01. and 9 V. Herman. 112–125. 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. Lechleiter.H.. 337–348. Kaandorp. N. Y.. Res. Measurement of intracellular calcium..J. Mirasoli. Rev.. Takahashi... O. Y. Pulse-response of aequorin-expressing yeast suspensions prepared as described in the main text and subjected to electrical pulses of 2. Microdetermination of calcium by aequorin luminescence. Biol.. 1998. Guardigli. 223–239. G. W. C. J. Given our results.. Cell... Appendix A. 2004. Biotechnological applications of bioluminescence and chemiluminescence. Requena.jbiotec. 37 (2).D. This work is based on the project of the Valencia team that attended the 2009 iGEM competition. Scarpa.H. 22 (6). a bioluminescent protein from the luminous hydromedusan Aequorea.. 79. L. Endoh. E. Please cite this article in press as: Vilanova. opening two novel research directions: first. in the online version.. J... Vilanova et al.A. Science 140. 16. Physiol. Sci.jbiotec. / Journal of Biotechnology xxx (2011) xxx–xxx 3 121 122 123 300 250 9V 6V enzymatic activity or any other desired behavior in GMOs by triggering the massive and re-enactable entry of ions by artificial electrical stimulation. Giraldo. Extraction. J..01. 1339–1340. 3. Roda. 1991. J. at doi:10. B. the implementation of novel bio-electronic lighting devices. Ruiz.. Saiga. Relationship between the increase in Ca2+ transient and contractile force induced by angiotensin II in aequorin-loaded rabbit ventricular myocardium. A. Johnson. UPV) for their technical support.. Saiga. M. 521–555...1016/j. 2006.1016/j. M.. 1999. A. 1962. 43614–43624. We are very grateful to Fabiola Barraclough for correcting the English manuscript. González from IQMA (Instituto de Química Molecular Aplicada.2011. Cell Dev. Mullins. Trends Biotechnol. doi:10. 2000. Chem... see supplementary material Video 1 online)... 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 Fig. Watanabe.. J.