Our Biobrick: Human Optimized Aequorin

This year, our team produced and fully characterized several constructs. However, the majority of our constructs were restricted under “Material Transfer Agreements” (MTAs) that prevented us from submitting these constructs to the registry of standard biological parts. However, we were able to use one of the genes that we synthesized, Aequorin, as the basis for a biobrick that was not limited by any MTAs.

Our goal was to design a completely new cell to cell signaling system that would replace chemical messengers, such as steroid hormones and signal peptides, with light. Therefore, we needed to find a way to get cells to generate their own light, much like miniature fireflies. Furthermore, this light had to be in the blue range of light in order to activate the light-gated ion channel that served as our light sensor. This eliminated several of the most common bioluminescent proteins as viable light emitting molecules, since most of them do not emit light in the correct region of the visible light spectrum.

After much searching we found a suitable light emitting protein, Aequorin. Aequorin was discovered in Aequorea aequorea, a species of bioluminescent jellyfish first purified in 1962 by Osamu Shimomura[1]. This protein emitted light at the correct wavelength (λmax=469 nm)[1], and required calcium as a cofactor[1], which allowed us to easily adapt it for use in our light activated ion channel cell signaling system. In the presence of its substrate, coelenterazine, Aequorin could release blue light that would have, in theory been able to activate the light-gated ion channels our cells would use to receive messages.

Although Aequorin is already in the registry as a biobrick, it was in a form that was optimized for expression in bacteria. Unfortunately, the other components of our cell to cell signaling system could only function in eukaryotic cells. Therefore, we synthesized a new DNA sequence that was optimized for expression in mammalian cells, such as the 293T cells that we used in our experiments.

We characterized our new biobrick with a series of experiments intended to show two things:
1. Our newly synthesized gene could produce a measurable quantity of blue light
2. Our newly synthesized gene could only produce light in the presence of Ca¬2+¬

As our luminometer experiments show, both Ca2+ and coelenterazine are required for substantial production of light. In fact, when compared to Aequorin alone, a mixture of Ca2+ and coelenterazine produced more than 10000 times more light. Furthermore, note that a lack of Ca2+ reduced light output by 1000 times, when compared to the light output of Aequorin in the presence of Ca2+ and coelenterazine. Through this experiment, we have shown that not only can our biobrick produce blue light, but that this production of light is heavily Ca2+ dependent.







For more information, please see our biobrick’s page on the international registry of standard biological parts: BBa_K548000

^[1] Shimomura, Osamu, Frank H Johnson, and Yo Saiga. “Extraction, Purification and Properties of Aequorin, a Bioluminescent Protein from the Luminous Hydromedusan, Aequorea.” Journal of Cellular and Comparative Physiology 59.3 (2005): 223-239. Wiley Online Library. Web. 28 Sept. 2011. <http://onlinelibrary.wiley.com/doi/10.1002/jcp.1030590302/pdf>.