Our two-cell system (pictured in the schematic below) involves a Sender Cell and a Receiver Cell. Although many iGEM teams work with bacteria such as E. coli, we chose Human Embryonic Kidney (HEK) 293T cells for several reasons. HEK 293T cell lines are immortal and very simple to work with – one of our team members called them the “E. coli of mammalian cells”. Working with mammalian cells (human cells specifically), allows easier translation of discoveries into therapeutic benefit. For example, a relevant recent paper in Science (Ye, Daoud-El Baba, Peng, & Fussenegger, 2011) chose HEK 293T cells to demonstrate the controlling of a simple synthetic circuit with blue light. They then proceeded to test this system in mice, finding that they could induce insulin production in diabetic mice when blue light was shone through their skin, which resulted in significant therapeutic effects.

The Sender Cell

The goal of the sender cell was to be able to trigger robust production of blue light. We decided on luciferases – enzymes which catalyze the oxidation of a substrate luciferin and release blue wavelength photons in the process:

Lucifereases are luminescent and not fluorescent, so they do not require excitation to emit light. They simply require the addition of the luciferin substrate. We obtained and tested two different luciferases: Gaussia Luciferase and Renilla Luciferase. The substrate of these luciferases is Coelenterazine, a molecule which easily diffuses through the HEK 293T membrane. We tested the relative light outputs of Gaussia and Renilla and found that Renilla emitted significantly more light , so we chose Renilla as the workhorse of our Sender Cell.

When choosing a promoter for Renilla, we selected the CMV promoter. This promoter is known for its extremely high and robust levels in Mammalian cells. Because we took a long time in the summer to set up our lab and get started, and we had an abundance of CMV ready for Gateway cloning, we were unfortunately not able to report experience with the Registry Biobrick for CMV (BBa_I712004).
We characterized the Sender Cell expressing Renilla under the control of the CMV promoter. We did this by quantifying the emitted light from Sender Cells in an iVIS Lumina II imaging system, finding that it produced much stronger luminescence than the positive and negative controls, and much stronger than another luciferase (Aequorin).

The Receiver Cell

The goal of the Receiver Cell was to be able to successfully receive a blue light signal from the Sender Cell and translate this input into a further output of blue light. First, we needed a component that was highly sensitive to 480nm light and could translate this into an intracellular response. This component would also need to instantaneously trigger a second luciferase or fluorescent molecule. We found a suitable receiver protein which is routinely used in the neuroscience field: Channelrhodopsin-2 (ChR2). ChR2 is a membrane-bound ion channel that is gated by 460-480nm light. Upon application of light, the channel opens and allows cations to enter the cell. This concept paired well with the luciferase Aequorin, which not only requires Coelenterazine to produce light, but also requires 3 bound Ca2+ ions. This pairing was the basis of our Receiver Cell – the light-gated opening of ChR2 would trigger Aequorin luminescence in the presence of Coelenterazine. We subsequently learned from a recent Nature Neuroscience paper (Kleinlogel et al., 2011) that a point mutation in ChR2 can dramatically increase calcium permeability (this mutant has been named CatCh). We secured CatCh and co-transfected it with Aequorin, both being controlled by separate CMV promoters (so that we would not sacrifice expression of either during initial experiments).

We first attempted to characterize the final system of Sender Cells activating Receiver Cells using an iVIS Lumina II imaging system. We did not see a significant increase in Aequorin luminescence in the Receiver Cell after pulsing with blue light, so we started trouble-shooting by testing the components of the receiver. Aequorin alone worked – upon the addition of Coelenterazine and Calcium it luminesced. We then attempted to characterize ChR2’s and CatCh’s calcium influx in response to blue light. We conducted these experiments by loading HEK293T cells expressing ChR2-eYFP or CatCh-eYFP with Fura-2 AM dyes, which measure intracellular calcium. We imaged these cells for 340/380nm emission ratio while illuminating them with pulses of 470nm light. To test whether the Sender Cells could activate the Receiver Cells, we co-cultured Sender Cells with ChR2-eYFP or CatCh-eYFP expressing HEK 293T cells and conducted Fura-2 imaging while adding Coelenterazine to activate the Sender Cells.

Pre-Coelenterazine

Since the Receiver Cell relies on the addition of Coelenterazine, it is not truly dependent on light signaling from the Sender Cell. We attempted to solve this problem by developing a genetically encoded Coelenterazine. A patent from the lab of Martin Chalfie showed that Coelenterazine can actually be derived from Green Fluorescent Protein (GFP) (Ward, 1998) – a GFP molecule with two point mutations is naturally cleaved into Coelenterazine. With site-directed mutagenesis, we attempted to express this “Pre-coelenterazine” in our HEK 293T cells, but found that it did not function as normal Coelenterazine – it did not result in luminescence in the presence of Renilla or Aequorin and Calcium.

References

Chow, B. Y., & Boyden, E. S. (2011). Synthetic Physiology. Science, 332(6037), 1508-1509. doi:10.1126/science.1208555

Kleinlogel, S., Feldbauer, K., Dempski, R. E., Fotis, H., Wood, P. G., Bamann, C., & Bamberg, E. (2011). Ultra light-sensitive and fast neuronal activation with the Ca2+-permeable channelrhodopsin CatCh. Nature neuroscience, 14(4), 513-8. Nature Publishing Group. doi:10.1038/nn.2776

Ye, H., Daoud-El Baba, M., Peng, R.-W., & Fussenegger, M. (2011). A synthetic optogenetic transcription device enhances blood-glucose homeostasis in mice. Science (New York, N.Y.), 332(6037), 1565-8. doi:10.1126/science.1203535

Ward, W. W. (1998). U.S. Patent No. 5,741,668. Washington, DC: U.S. Patent and Trademark Office