Team:Valencia/The Jellyfish Factory
From 2009.igem.org
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- | Cutting the jellyfish rings with scissors was impossibly | + | <span style="color:black; align:justify; font-size:11pt; font-family: Verdana"> Cutting the jellyfish rings with scissors was impossibly |
slow; we could not produce the amount of aequorin that | slow; we could not produce the amount of aequorin that | ||
we needed using this technique. This problem was solved | we needed using this technique. This problem was solved | ||
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- | Dr. Johnson next purchased two circular meat-slicing | + | <span style="color:black; align:justify; font-size:11pt; font-family: Verdana"> Dr. Johnson next purchased two circular meat-slicing |
blades ( 10” diameter) at a local hardware store and began | blades ( 10” diameter) at a local hardware store and began | ||
to build a cutting machine; this project took the next two | to build a cutting machine; this project took the next two | ||
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- | A number of improvements were made over the next | + | <span style="color:black; align:justify; font-size:11pt; font-family: Verdana"> A number of improvements were made over the next |
two years. A razor blade was installed at the edge of the | two years. A razor blade was installed at the edge of the | ||
Lucite board; the razor blade and the rotating circular | Lucite board; the razor blade and the rotating circular | ||
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- | With machines that could cut rings at 10 times the | + | <span style="color:black; align:justify; font-size:11pt; font-family: Verdana"> With machines that could cut rings at 10 times the |
speed of a hand-cutter, and with a sufficient supply of | speed of a hand-cutter, and with a sufficient supply of | ||
jellyfish, our mode of operation had to be changed. We | jellyfish, our mode of operation had to be changed. We | ||
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- | Ring cutting was probably the most important step in | + | <span style="color:black; align:justify; font-size:11pt; font-family: Verdana"> Ring cutting was probably the most important step in |
determining the quality and yield of purified aequorin. | determining the quality and yield of purified aequorin. | ||
Cutting too thick would increase the amount of impurities. | Cutting too thick would increase the amount of impurities. | ||
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- | Our jellyfish cutting usually began at 11 AM. A counting | + | <span style="color:black; align:justify; font-size:11pt; font-family: Verdana"> Our jellyfish cutting usually began at 11 AM. A counting |
person would put 80 jellyfish into each bucket, already | person would put 80 jellyfish into each bucket, already | ||
half-full of seawater, and would then take the buckets to | half-full of seawater, and would then take the buckets to | ||
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- | The process of extracting aequorin from rings began at | + | <span style="color:black; align:justify; font-size:11pt; font-family: Verdana"> The process of extracting aequorin from rings began at |
2 PM; it was carried out by a team of two persons. The | 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). | extraction was done in batches of 480 rings (i.e., six buckets). | ||
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- | The precipitates of crude aequorin were purified at our | + | <span style="color:black; align:justify; font-size:11pt; font-family: Verdana"> The precipitates of crude aequorin were purified at our |
laboratory in Princeton. The purification was done in | laboratory in Princeton. The purification was done in | ||
several steps of column chromatography, mainly by | several steps of column chromatography, mainly by | ||
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- | The purified aequorin was used in various studies of | + | <span style="color:black; align:justify; font-size:11pt; font-family: Verdana"> The purified aequorin was used in various studies of |
luminescence in our laboratory. Thus, the chemical | luminescence in our laboratory. Thus, the chemical | ||
structure of the light-emitter was determined in 1973. | structure of the light-emitter was determined in 1973. | ||
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- | Our work on aequorin was initiated by Dr. Frank H. | + | <span style="color:black; align:justify; font-size:11pt; font-family: Verdana"> Our work on aequorin was initiated by Dr. Frank H. |
Johnson, and developed with support and encouragement | Johnson, and developed with support and encouragement | ||
from many individuals. I thank all the people who helped | from many individuals. I thank all the people who helped |
Revision as of 22:09, 14 October 2009
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.
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