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One step forward

Unesp creates the first storage and distribution bank for the genes of sugar cane bacteria

A town in the north west of the state of São Paulo encircled by sugar cane plantations, Jaboticabal stashes valuable secrets in an air conditioned room on the campus of the São Paulo State University (Unesp), in the drawers of large freezers at -86o Celsius. Invisible to the naked eye, these secrets stay in yellow colored cylinders of less than 2 millimeters in diameter, kept in small acrylic boxes.

The material interests scientists of the whole world and attracts the curiosity of the sugar millers in the neighborhood. They are the clones of genes sequenced from sugar cane(Saccharum officinarum) and of four bacteria that cause agricultural pests (phytopathogenics): Leifsonia xyli, which provokes rickets on the sugar cane; Xylella fastidiosa, responsible for the yellowing of orange trees or CVC (citrus variegated chlorosis); Xanthomonas citri, which causes citrus canker; and Xanthomonas campestris, which attacks some vegetables. These are the contents of the Brazilian Clone Collection Center (BCCCenter), a storage center inaugurated here in April last with investment of US$ 600,000.00.

Host bacteria
Created in the mold of the American Type Culture Collection (ATCC) and of the Image Consortium of the United States, the BCCCenter confirms the Brazilian position within the elite of the biotechnology world. “We ran the risk of seeing all the work carried out over the last four years being lost from one moment to another, because before the installation of the data center the information was in the memory of the computers, but the clones containing the genes were dispersed throughout various laboratories”, says professor Jesus Aparecido Ferro, of the School of Agrarian and Veterinary Sciences of Unesp, one of the coordinators of the center.

With capacity for 1,612,800 clones, the BCCCenter already stores in eight freezers a total of 65,000 genes, of which 40,000 are from the sugar cane. It is the largest sugar cane gene bank in the world. To obtain all of these unique genes, it was necessary to produce 300,000 clones of sugar cane and another 200,000 of the phytopathogenic bacteria. “The sequenced genes of the plant or of the bacteria”, relates professor Ferro, “are cloned on a circular segment of DNA (deoxyribonucleic acid, which carries the genetic code) called a vector, that is then introduced into the lineage of the bacterium Escherichia coli .”

“These bacteria”, the researcher goes on, “containing the genes of another organism reproduce themselves in vast quantities, each daughter bacterium also carrying the exogenous gene. In this way, not only do they serve as hosts, but they also increase the number of copies of the genes in a manner that they can be distributed without the loss of the original clone.”

Cloning and storage are done in an area of 300m², within three rooms. In the first, there is the main equipment: a robot that cost US$ 252,000 and carries out the manipulation of the material. It works through a mechanical arm a little less than 1 meter long, installed in a glass case of 2m³. Equipped with a video camera, the robot digitally selects the colonies that contain the genes and collects the material and it is able to arrange 27,000 genes in duplicate on a nylon membrane sheet, 22.5 x 22.5 centimeters, the so-called DNA chip. The advantage of the robot is the ability to select and grasp 30 colonies of bacteria per minute, with the needles situated on the tip of its arm. This way, it wipes out the risk of human error and accelerates the process a hundred times.

Beginning with the work of the robot, the samples are organized on micro plates, each one holding 384 genes, stored in the freezers so that the bacteria conserve their genetic material. “Only in a place like this can we follow the multiplication of these bacteria and of each phase of the cloning “, comments professor Paulo Arruda, of the State University of Campinas (Unicamp), the coordinator of the sequencing project of sugar cane.

Besides storing, the center will commercialize genes for research institutions and companies. “To have access to clones”, explains professor Ferro, “one must sign a Term of Transfer of Biological Material, with the promise that the gene or the genes are to be used only in research and not passed on to another researcher. For the private sector, there will be a different negotiation, with the participation of a committee set up by FAPESP.”

An order can be placed via the Internet and payment can be by credit card. The price will vary. The private companies will have to pay between US$ 30 and US$ 100 per clone. The North American bank charges between US$ 32 and US$ 150. The 60 laboratories of the Brazilian network Onsa (Organization for the Sequencing and Analysis of Nucleotides), created by FAPESP, are having free access to any requested material. “At least 500 clones of sugar cane and another 500 of bacteria have already been distributed to members of the Onsa network who generated these clones”, informs Ferro. “From now on, these groups must cover the cost of maintenance and the distribution of the clones which they ask for, which should amount to US$ 5 per clone. For other groups the cost will likely be higher, but still lower than that the one established for the private sector.”

Self sufficient
Since intellectual ownership lies with FAPESP, the income will remain at the center itself, which should become self sufficient within three years, with an annual budget of around R$ 200,000.00. Besides the clones, they will also sell the DNA chips, the high density membranes with all or almost all of the genes of an organism: in this case the of sugar cane or phytopathogenic bacterium. Ferro reveals that a multinational company has already shown interest in the genes of the sugar cane and is negotiating a purchase through FAPESP. Australian researchers are interested in purchasingthe high density membranes containing the genes of the sugar cane. In the United States, a DNA chip costs close to US$ 2,000 and the expectation is that this price will also be lower in Brazil.

Industrial interest
The center is still waiting for a copy of operon, the piece of the genome that controls the expression of a group of genes in bacteria. It comes from a Xylella bacteria which produces a gum similar to xanthan gum, a thickener for food and medicines as well as lubricant oil for the drill head used in petroleum exploration. At the request of the Xylella genome team, the center sent a clone of this operon to a North American company that is a large producer of the Xanthan gum. The company is testing the operon with the bacterium Xanthomonas campestri, the most commonly used to produce this gum, while the Brazilian team received the lineage of the bacteria and will carry out the same experiments to evaluate whether or not the properties of the operon and of the resulting gum are of commercial interest.

“The more companies that want to make tests, the better for us”, says professor Arruda. The partnership is feasible since the group from São Paulo has already patented the operon in the United States. “If business goes forward, we are guaranteed.” According to him, the tests should finish by the end of the year, and, if positive, could make the cooperation evolve into a commercial agreement. It only remains to kill the curiosity of the next door miller. The BCCCenter is the second part of a project that began with the sequencing of the sugar cane and of the yellowing bacterium, the pest which causes annual damages of R$ 110 million to citrus agriculture in São Paulo. Since 1997, the projects of sequencing of the sugar cane and the bacteria of agricultural interest have consumed close to R$ 40 million, that FAPESP financed in partnership with private institutions such as the Center of Technology of Copersucar (CTC) and the Fund for the Defense of Citrus Agriculture (Fundecitrus).

Genomes and aircraft
The interest of the genome partners is the same: to search for improvements in order to increase agricultural productivity with new varieties of plants, as well as to reduce the cost of combating pests and diseases. “From the identification of the DNA of the plant, we can know exactly which of the genes are involved in the synthesis of the saccharine of the sugar cane, which are responsible for growth, which give resistance to pests and to drought, among so many others”, explains Ferro. “For this reason, we have to be careful to know into whose hands these genes are going.” With the data in hand, research institutions or companies might develop varieties so that in the end the neighboring sugar miller would benefit.

Just as the installation of the Aeronautical Technology Institute (ITA) in São José dos Campos was fundamental for the creation of Embraer, today one of the major aircraft manufacturer in the world, the center is the witness of the competence which should produce the conditions for a greater strengthening of the biotechnological agricultural industry”, says José Fernando Perez, the Scientific Director of FAPESP, at the inauguration of the BCCCenter. The investments go on. In 2001 the Genome Project will receive another R$ 30 million. Among the latest research is the sequencing of the bacteria Schistosoma mansoni, which causes schistosomiasis, and a study of the sequencing of the eucalyptus.

The project
Storage and distribution laboratory for clones of the project of the sugar cane genome (nº 00/02898-4); Modality Subproject of the Sugar Cane Genome; Coordinator Professor Jesus Aparecido Ferro – Unesp of Jaboticabal; Investment R$ 435,411.47 and US$ 467,553.59