Team:Valencia/The Jellyfish Factory

From 2009.igem.org

The Jellyfish Factory

Cutting the jellyfish rings with scissors was impossibly slow; we could not produce the amount of aequorin that we needed using this technique. This problem was solved primarily by Dr. Johnson. He constructed the first model of a jellyfish-cutting machine in the summer of 1967; it was essentially a strip of wire screen that worked like a grater. An average jellyfish has about 100 light organs the size of poppy seeds located under the edge of its umbrella. By sliding the jellyfish over the screen, we hoped that the light organs would be scraped off the body and collected in a tray under the screen. We found, however, that the light organs were not scraped off by the wire screen. The next version of the cutting machine had a strip of coarse sandpaper over which seawater flowed slowly; the sandpaper was connected to one end of the first version. When jellyfish were slid down-first over the sandpaper, then the screen-most of the light organs were indeed scraped off. But the material accumulated in the tray contained an excessive amount of slime, and the quality of the material was much poorer than that of the hand-cut rings. Thus, the manufacture of a machine based on the principle of a grater was abandoned.

Dr. Johnson next purchased two circular meat-slicing blades ( 10” diameter) at a local hardware store and began to build a cutting machine; this project took the next two summers to complete. The basic plan was to install a meatslicing blade perpendicular to a black Lucite board, and with the blade slowly rotating, cut the ring off the jellyfish. The motor from a small laboratory shaker was used to rotate the blade. The jellyfish were rotated with a hand tool called a “peg,” a small disk with several short nails on one side and a 2-inch-long, stick-shaped handle attached in the center on the other side. A jellyfish on the Lucite board was grasped by the nails of the disk and rotated by the stick, which was held between the index finger and thumb. The setup worked, at least in principle.

A number of improvements were made over the next two years. A razor blade was installed at the edge of the Lucite board; the razor blade and the rotating circular blade were in contact each other on their flat sides and the jellyfish was cut at the intersection of the two cutting edges. It made cutting so sharp and smooth that the jellyfish might not even feel that their rings were being cut off. To make the rotation ofjellyfish easy, a seawater outlet was installed near the center of the board to lubricate its surface. An ice bath was installed to cool the ring reservoir; this prevented a loss of activity from the rings and also served as a preparation for the extraction process. In the summer of 1969, the quality of the machine-cut rings finally surpassed that of the hand-cut ones. We therefore set up two cutting machines and used them, thereafter, to cut all of the jellyfish.

With machines that could cut rings at 10 times the speed of a hand-cutter, and with a sufficient supply of jellyfish, our mode of operation had to be changed. We needed a large working space, and we also did not want to disturb other researchers with our messy, smelly, and noisy experimental processes. Fortunately, we were assigned to use the Gear Locker, a small, isolated building that had been used for storage in the past. Two large tanks installed outside the building were used for temporary storage of collected jellyfish.

Ring cutting was probably the most important step in determining the quality and yield of purified aequorin. Cutting too thick would increase the amount of impurities. Cutting too thin would decrease the yield because some of the light organs were cut through and destroyed. Therefore, we always assigned the best workers to do this job. Of the many excellent helpers we had in our jellyfish operation, I remember particularly three girls who worked for many summers and cut rings extremely skillfully and fast: Debby Nash, Liz Illg, and Laura Norris; the first was from the town and the other two were daughters of biology professors.

Our jellyfish cutting usually began at 11 AM. A counting person would put 80 jellyfish into each bucket, already half-full of seawater, and would then take the buckets to the cutters. Two cutters cut the jellyfish with the machines that were installed side-by-side: the cutter would place a jellyfish onto the cutting board, quickly rotate it with a peg to spread out the edge of the umbrella where the light organs are located, and then-pushing the jellyfish to the cutting blade while simultaneously rotating the jellyfish quickly-cut off the rings, all in less than 5 seconds. The rings would fall automatically into the ice-cold reservoir, and the ringless jellyfish body was slid down into a waste bucket. These buckets, each filled with about 200 spent jellyfish, were carried to the nearest seashore about 50 yard away, called by us “jellyfish cliff,” and dumped onto the rocks below. The heaps of jellyfish bodies, several thousands of them, were carried away by the next high tide.

The process of extracting aequorin from rings began at 2 PM; it was carried out by a team of two persons. The extraction was done in batches of 480 rings (i.e., six buckets). The first person would drain the rings on a nylon gauze, then mix the drained rings with a cold EDTA solution saturated with ammonium sulfate. The rings shrank quickly and were also desensitized by the salt. They were cut with scissors into pieces l-2 inches long, then stirred with a cake mixer for 10 minutes to dislodge the granular light organs from the tissue. The mixture was squeezed through a nylon gauze to remove the shrunken ring tissue, and then the turbid liquid obtained was filtered on a Buchner funnel using some Celite. The filter cake, containing the light organs, was given to the second person, who was responsible for the rest of the extraction process. The second person put the filter cake into a 2-liter flask containing cold EDTA solution (1 liter), then shook the flask vigorously to extract aequorin from the light organs into the EDTA solution. Finally, the mixture was filtered through a large Buchner funnel, and the filtrate containing aequorin was saturated with ammonium sulfate to precipitate the protein. The first person in the team would start a new batch of rings every 20 minutes, and the second person’s work would also take 20 minutes. Thus, 3360 jellyfish rings could be extracted in about 2 hours and 40 minutes.

The precipitates of crude aequorin were purified at our laboratory in Princeton. The purification was done in several steps of column chromatography, mainly by Sephadex gel filtration and DEAE-cellulose chromatography, all at 0°C. It was indeed a lengthy, time-consuming process, notwithstanding the fact that aequorin should be purified as quickly as possible because it is constantly decomposing through spontaneous weak luminescence, even in the presence of a high concentration of EDTA. To purify an extract of 50,000 jellyfish, which contains a large amount of total protein, chromatography had to be repeated 30 times for only the first gel filtration step, and the total number of chromatography runs required for complete purification was more than 60. An extract of 50,000 jellyfish yielded only 150-200 mg of purified aequorin in the early ’70s but as the techniques improved, the yield gradually increased, exceeding 500 mg by 1980. Since 1975, all of the steps in the purification have been done by my wife, Akemi, who is highly knowledgeable in handling aequorin.

The purified aequorin was used in various studies of luminescence in our laboratory. Thus, the chemical structure of the light-emitter was determined in 1973. Then the structure of the aequorin chromophore “coelenterazine” was elucidated and the regeneration of spent aequorin into active aequorin was accomplished, both in 1975. The molecular characterization of various aequorin isoforms was reported in 1986. The improved forms of aequorin-“semisynthetic aequorins” with widely different calcium sensitivities-were produced in 1988-1989. Purified aequorin has also been supplied to hundreds of cell biologists and physiologists who study intracellular calcium, leading to many important findings about intracellular calcium. Aequorin was cloned in 1985 by two groups simultaneously, one in Georgia and another in Japan. With the recent progress in molecular genetics, studies involving recombinant aequorin are now flourishing.

Acknowledgments

Our work on aequorin was initiated by Dr. Frank H. Johnson, and developed with support and encouragement from many individuals. I thank all the people who helped directly or indirectly with this project. The work was made possible by the excellent facilities of the Friday Harbor Laboratories, University of Washington, and of Princeton University, and was financially supported by research grants from the National Science Foundation and the National Institutes of Health. </div>