Difference between revisions of "Part:BBa K548000"

Revision as of 23:30, 28 September 2011

Humanized Aequorin

This BioBrick is a human cell line optimized version of Aequorin, a bioluminescent protein found in various jellyfish and other marine organisms. Aequorin was discovered in Aequorea aequorea, a species of bioluminescent jellyfish first purified in 1962 by Osamu Shimomura. The protein requires Ca²⁺ for catalytic activity and oxidizes its substrate, coelenterazine to emit blue light (λmax=469nm). Aequorin is NOT a fluorescent protein, it does not require an excitation light source to emit light. Transfection of the gene into 293T cells using lipofectamine has been confirmed. The part contains EcoRI and PstI restrictions sites, and is ligated to the iGEM pSB1C3 backbone. This backbone provides chloramphenicol resistance.

The Aequorin apoprotein bound to Ca²⁺

The Aequorin apoprotein bound to the coelenterazine prosthetic group

Usage and Biology

This protein emitted light at the correct wavelength, and required calcium as a cofactor, 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 and the Ca2+ cofacter, Aequorin emits a rapid burst of blue light.

Characterization

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 Ca²⁺

To do this, we transfected 293T cells with our gene contained in the pRRL mammalian expression vector using lipofectamine, a common and well characterized transfection reagent. After transfection, cells were lysed using Promega Passive Lysis Buffer (PLB) and the lysate was mixed with either nothing, only coelenterazine, only Ca²⁺, or both coelenterazine and Ca²⁺. Then, samples were placed into a luminometer and the luminescence was recorded. The results are summarized in the graph below.

Results of our luminometer experiments. Note that the y-axis is logarithmic. As shown, Aequorin produces over 1000 times more light with Ca²⁺ than without it.

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.

Properties

Transcript Size: ~22kDa

Gene Length: 618 bp

Optimization: Optimized for use in mammalian cells ONLY. For expression in bacteria see Part:BBa_K222000

Confirmed Working Vectors: pRRL, pSB1C3 (iGEM 2011 biobrick backbone)

Transfected Cells: HEK 293T cells

Transfection Reagents: Lipofectamine

Sequence and Features