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Proteorhodopsin-cPT-RFP

This is a triple fusion protein containing green light absorbing proteorhodopsin, the cPT region of IcsA, and Fresno RFP. It is intended to function as a unipolarly expressing proton pump. The cPT region directs the localization of the protein to the old pole of E. Coli while the proteorhodopsin pumps protons out of the cell in response to green light (must be grown in 10uM all-trans retinal). RFP has also been tagged for fluorescent visualization. We also have referred to this as the Voltage protein.

Sequence and Features

Introduction

Currently the ISS relies primarily on solar arrays for power, turning to lithium ion batteries when not in direct sunlight. But given the high costs of sending massive objects into space and the hazardous volatility of current batteries, these may not be the most sustainable sources for power. Looking for solutions in nature, we were inspired by the electric eel, whose linearly oriented electrogenic cells unipolarly pump ions in a single direction to generate an electric potential. This inspired our idea of a biobactery. Using E. coli, we mimicked the two key characteristics of eel cells: (1) stimulus-mediated unidirectional ion transport to generate a voltage and (2) linearly oriented cells that propagate this potential, like batteries in sequence. Ultimately, we aimed to use high-density E. coli colonies within a microfluidic device to polarly pump hydrogen ions via optogenetic activation of an ion channel, bacteriorhodopsin, to produce a voltage difference across the device.

Construct Design

The Voltage protein we designed is a triple fusion protein. Its schematic is shown in Figure 1. Each domain has a distinct function. From the amino terminus to the carboxy terminus, these domains are the proteorhodopsin domain, the cPT domain, and the RFP domain. The first domain contains a green-light absorbing proteorhodopsin. We chose to use a proteorhodopsin rather than a channelrhodopsin or a bacteriorhodopsin, because eukaryotic channelrhodopsins are difficult to express in E. Coli due to the requirement of post-translational modifications, and because bacteriorhodopsins form a hexagonal lattice structure which is very important to functionality, less so than the trimeric structure formed by proteorhodopsin.1,2

Figure 1: Schematic of the proteorhodopsin, cPT, RFP fusion protein. The proteorhodopsin is embedded in the inner membrane and linked to the cPT region with a GSx5 linker.

Proteorhodopsin is a transmembrane proton pumping protein endogenous to SAR86 γ-proteobacteria. In response to light, proteorhodopsin pumps protons out of the cell and helps its native organism establish the proton motive force necessary to make ATP.3 It does form a trimeric lattice structure in the inner membrane; however, it has been shown that the individual monomer contains all the necessary components to pump protons, albeit at a potentially lesser efficiency.4 We sourced the sequence for the green-light absorbing proteorhodopsin from the 2012 Caltech iGEM team part BBa_K773002; however, codons towards the carboxy terminus were slightly modified to enhance GC content for IDT synthesis.5 This is the first domain in the protein because the carboxy terminus of proteorhodopsin is intracellular, while the amino terminus is extracellular, and the remaining domains must be intracellular for functionality.6

The second domain of the Voltage protein is the cPT, or central polar targeting BBa_K2485011, region of the larger IcsA protein. In its native organism, Shigella flexneri, the IcsA protein localizes to the outer membrane of the old pole and is responsible of the motility of the organism within mammalian host cells via actin polymerization. The cPT domain within this larger protein, when fused to GFP, has been shown to form a unipolar focus in 64% of E. Coli cells.7 It should be noted that this focus occurs intracellularly since neither the cPT domain, nor the GFP contain export signals.To fuse this domain to the carboxy terminus of the proteorhodopsin, we searched the literature for fusion proteins with proteorhodopsin. After being unable to find any previous attempts at this fusion, we decided to incorporate a flexible 5x Glycine-Serine linker between the proteorhodopsin and the cPT region.

The final domain of the Voltage protein is a fluorescent reporter so that we can visualize the proteins localization. For this reporter, we chose to use FresnoRFP with an ex/em of 553/592.8 We chose this protein because its ex/em is sufficiently far away from the excitation frequency of 525nm of green-light absorbing proteorhodopsin, and we wanted to minimize excitation of the rhodopsin during visualization.3 To link the cPT region to the RFP, we used the same sequence as was used by Dr. Marcia Goldberg, M.D. from Massachusetts General Hospital who graciously gave us her plasmids which contained a cPT-GFP fusion. We showed that this fusion protein exhibited widespread unipolar localization in E. Coli. In order to link cPT to GFP, the last four nucleotides of the cPT region was altered from “TCAT” to “ATCC.” We are unsure of the nature of this modification; however, it has been demonstrated to work efficiently.

By creating this fusion protein and demonstrating both the functionality of the proteorhodopsin and its unipolar localization, we hope to allow E. Coli to generate a voltage differential across its length. Then, by aligning an entire culture of E. Coli such that all the old poles, where the proteorhodopsin localizes, point in the same direction, each voltage will be additive like batteries in series. Although E. Coli are very small, and proton diffusion is very fast, it has been shown that E. Coli expressing proteorhodopsin will generate a voltage drop across the length of a cuvette in response to a unidirectional pulse of light.9 This is hypothesized to be a result of asymmetric excitation of the proteorhodopsin due to E. Coli cells acting as a lens, focusing light on the far end of the cell. By controlling the expression of the proteorhodopsin to be unipolar, we hope to be able to generate this same sort of voltage without the need for the light to be unidirectional.

References

1. Hou, Sing-Yi, et al. “Diversity of Chlamydomonas Channelrhodopsins.” Photochemistry and Photobiology, vol. 88, no. 1, 2011, pp. 119–128., doi:10.1111/j.1751-1097.2011.01027.x.
   
2. Yamashita, Hayato, et al. “Role of trimer–trimer interaction of bacteriorhodopsin studied by optical spectroscopy and high-Speed atomic force microscopy.” Journal of Structural Biology, vol. 184, no. 1, 2013, pp. 2–11., doi:10.1016/j.jsb.2013.02.011.
   
3. Bamann, Christian, et al. “Proteorhodopsin.” Biochimica et Biophysica Acta (BBA) - Bioenergetics, vol. 1837, no. 5, 2014, pp. 614–625., doi:10.1016/j.bbabio.2013.09.010.
   
4. Hussain, Sunyia, et al. “Functional Consequences of the Oligomeric Assembly of Proteorhodopsin.” Journal of Molecular Biology, vol. 427, no. 6, 2015, pp. 1278–1290., doi:10.1016/j.jmb.2015.01.004.
   
5. “Part:BBa_K773002.” Part:BBa K773002, parts.igem.org/Part:BBa_K773002.
   
6. Stone, Katherine M., et al. “Structural Insight into Proteorhodopsin Oligomers.” Biophysical Journal, vol. 104, no. 2, 2013, pp. 472–481., doi:10.1016/j.bpj.2012.11.3831.
   
7. Doyle, Matthew Thomas, et al. “A small conserved motif supports polarity augmentation of Shigella flexneri IcsA.” Microbiology, vol. 161, no. 11, Jan. 2015, pp. 2087–2097., doi:10.1099/mic.0.000165.
   
8. “ProteinPaintbox®.” ATUM, www.atum.bio/products/protein-paintbox#2.
   
9. Sineshchekov, Oleg A., and John L. Spudich. “Light-Induced intramolecular charge movements in microbial rhodopsins in intact E. coli cells.” Photochemical & Photobiological Sciences, vol. 3, no. 6, 2004, p. 548., doi:10.1039/b316207a.
10. Walter, J. M., et al. “Light-Powering Escherichia coli with proteorhodopsin.” Proceedings of the National Academy of Sciences, vol. 104, no. 7, Feb. 2007, pp. 2408–2412., doi:10.1073/pnas.0611035104.