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JBEI Seeks Commercial Partner for Automated DNA Cloning Software

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By Uduak Grace Thomas

The Joint BioEnergy Institute said this week that it is looking for companies that are interested in licensing and marketing j5, a software package that automates the process of cloning DNA.

JBEI’s Nathan Hillson told BioInform that in addition to the capabilities of other clone design software packages such as Life Technologies’ Vector NTI, the sequence- and ligase-independent cloning method, and the Gibson assembly method used by researchers at the J Craig Venter Institute to assemble synthetic genomes, j5 can also identify the most cost-effective cloning strategy for a given design: direct DNA synthesis, PCR, or oligo embedding

“There aren’t any existing software packages that do this,” Hillson said.

Hillson said he began designing j5 after spending several frustrating days painstakingly designing a DNA assembly only to discover that the cost of the oligonucleotides he would need to order for PCR-based cloning exceeded the cost of paying a company to synthesize the DNA directly.

“I learned two things coming out of that experience,” he said. “I never wanted to do this by hand again and…I always wanted to take into consideration how much one particular assembly method would cost over just getting the whole thing directly synthesized.”

According to Hillson, the difficulty with traditional cloning approaches is that they aren’t “standardized,” which he argues would make things much easier and far less complicated.

“Every time that you are cloning a different enzyme or gene, you might have to use a different pair of restriction sites,” he said. “With a standardized assembly you would be using that same enzyme over and over again, completely independent of what you are trying to put together.”

When using more traditional protocols, researchers are faced with the time- and labor-intensive task of designing DNA oligos that contain flanking homology sequences, as is the case with the SLIC and Gibson methods; or specified overhang sequences, as is the case with the Golden Gate method. These methods also require a separate validation step to account for mis-primed oligos and mismatched assembly pieces, Hillson said.

One tool that has made an effort to standardize the DNA assembly process is BioBricks, which was developed by the Massachusetts Institute of Technology, Harvard, and the University of California, San Francisco. Hillson noted that although the tool standardizes the process to some degree by letting researchers use the same restriction enzymes for each assembly; it has some “drawbacks.”

“You introduce scar sequences in the plasmids that you are trying to make,” he said. “You’re also really limited in terms of making big combinatorial libraries.”

Hillson said that this is not the case with j5. In addition to making sure researchers end up with the plasmid they want and generating large combinatorial libraries, j5 addresses “all the considerations of traditional multiple cloning site technology.”

He explained that there are multiple methods for cloning DNA. As an example, Vector NTI identifies restriction sites in a plasmid, which works well for the more traditional methods of assembling DNA, such as PCR, but Hillson notes that newer tools like SLIC and the Gibson method don’t use restriction enzymes or multiple cloning sites in their protocols.

Furthermore, with the cost of synthesizing DNA decreasing, Hillson said that simply synthesizing the sequence is becoming a viable option for researchers. For example, gene synthesis services at GenScript start at $0.55 per base pair, with a discounted price of $0.39 per base pair for genes less than 3 kilobases.

But, he pointed out, while a large industrial firm might be able to afford the costs of direct DNA synthesis, for the present it might be too much for an academic scientist in a small lab.

“The way we really want to go at JBEI and more broadly in the field is that we want to have the researchers spending all their time designing the plasmid they want and then assaying it,” he said. “But we don’t want them to waste all their time actually building these things in the lab. So we are trying to go after ways of taking that burden off the researchers.”

Counting Costs and Preventing Promiscuity

Hillson said that his team developed a basic graphical user interface for j5 written in Adobe Flex. The interface generates a set of input files that are uploaded to a server and outputs a zip file containing the results of the assembly design. It includes information such as an annotated GenBank sequence file, the types of oligos needed, as well as the PCR reactions that need to be performed.

“The GUI [is] a symbolic representation of the underlying DNA pieces…you are moving icons around into the order that you want, reflecting the parts that you want and things like that,” he said.

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With the list of DNA sequences to be assembled, j5 determines a “cost-minimizing assembly strategy” for the different protocols, such as direct synthesis, PCR reactions, or oligo embedding. Next the software designs the requisite oligos using the Primer3 program and automatically adds flanking homology sequences or optimized overhang sequences to the oligos and direct synthesis pieces.

As a final step, j5 makes a Blast call to ensure that the sequences aren’t “promiscuous” and that the right pieces end up next to each other. If the wrong sequences are paired together, j5 suggests a “hierarchical subassembly” to correct the situation.

Once the software designs the oligos, it checks the researcher’s existing supply of nucleotides so that “instead of having to reorder the same oligo over and over again, it can tell you to reuse the one that you already have,” Hillson said.

The software also allows users to bundle together multiple independent cloning projects.

“If 20 different people all design their projects with this software [it] can consolidate all those projects onto, for example, a 96-well plate,” said Hillson. “After those constructs are made they go back to the original person who designed them.”

Free for Some

The current version of the software is available for free to academic researchers, not-for-profit organizations, and government entities. Commercial companies who are interested in using the software would be required to acquire a license, according to Hillson.

He speculated that a commercial license could function on several levels. Molecular biology companies that simply want to use the software would pay for a license to do so. Other companies who want to develop the software further could acquire a license to develop j5 for commercial purposes.

“The software that we have right now works but perhaps we are not going to have the personnel to do a lot of customer service or training or tutorials or make it into a really beautiful graphical interface,” he said. “Maybe an existing software company might be interested in partnering and further developing the software to better serve the commercial market.”

He added that if the software were “well developed” and priced low enough, academic researchers or not-for profit organizations might opt for the commercial version over the free one, which will be continually developed but in the long run will likely differ from the commercial version.

“I think for the most part, the underlying functionality might be pretty similar but probably the actual interface and the way that you would use the software might be a little bit different,” he said. “Now going down the road, you could see those software developers actually adding in the features that we don’t have in our existing free version.”

Robin Johnston, who works in JBEI’s Technology Transfer and Intellectual Property Management arm, confirmed that the institute is “talking to companies that are interested in end-user licenses and distribution licenses” but she declined to mention any names.

Johnston added that even if JBEI signs a commercial development agreement for the software, it will retain the rights to distribute it to academic institutions, government labs, and not-for-profit groups.