(From left) Timothy Ham, Joanna Chen, Rafael Rosengarten and Nathan Hillson have developed j5, the first DNA construction software that not only makes the process faster and more efficient but also identifies which construction strategy would be the most cost-effective. (Photo by Roy Kaltschmidt, Berkeley Lab) |
DNA
construction, also known as DNA cloning or recombinant DNA
technology—among a host of other terms—is one of the principal tools of
modern biotechnology, used for a wide variety of purposes, including
genetic studies, medical research, and the development of advanced
biofuels.
A
number of software programs make the process faster and more efficient,
but Nathan Hillson, a biochemist at the U.S. Department of Energy
(DOE)’s Joint BioEnergy Institute (JBEI), with an eye on the economics
of scientific discovery, has developed the only DNA construction
software that also identifies which strategy would be the most
cost-effective. This unique software program goes by the unassuming name
of j5.
“Our
j5 is the only software package today that both standardizes and
cost-optimizes the DNA construction process,” says Hillson, who directs
JBEI’s Synthetic Biology program and also holds an appointment with the
Lawrence Berkeley National Laboratory (Berkeley Lab)’s Physical
Biosciences Division. “Through the design of short DNA sequences that
can be used to join longer sequences together in recombinant DNA
assemblies, the j5 software improves the accuracy, scalability, and
cost-effectiveness of DNA construction.”
DNA
construction is the process by which multiple genes or fragments of DNA
sequences are physically assembled together. Such constructs are
valuable for developing new medical treatments and for engineering
microbes to efficiently carry out a specific task, such as converting
cellulosic biomass into clean, green, renewable transportation fuels.
DNA
construction incorporates DNA sequence fragments—often referred to as
“parts”—from a variety of organisms into a self-replicating genetic
element, such as a bacterial plasmid, that will propagate the assembled
parts in a host cell. Traditionally, this has been accomplished through
the use of a panoply of restriction enzymes for splicing desired DNA
sequence fragments, and ligation enzymes for bonding the fragments to
plasmid cloning sites.
“As
the size and number of parts to be incorporated into the plasmid
increases, traditional construction of recombinant DNA assemblies
becomes ever more difficult,” says Hillson. “The process must often be
repeated from scratch for alternate combinations of parts, and every
time you clone a different gene or fragment, you might have to use a
different pair of restriction sites. This has been a labor-intensive and
time-consuming process.”
With
modern DNA construction techniques, Hillson says, a small number of
enzymes can be used over and over again, independent of the DNA sequence
fragments being assembled, and thereby enabling automation with robotic
platforms. However, designing protocols for these modern DNA
construction approaches can be as labor-intensive, time-consuming and
error-prone as the traditional approach. Furthermore, it is now
increasingly important to consider outsourcing portions of DNA
construction?to companies that chemically synthesize long sequences of
DNA?as a cost-effective alternative. To address these considerations
Hillson created the j5 software package.
“The
j5 software package is a Web-based computer application that
automatically designs and optimizes state-of-the-art DNA construction
protocols,” Hillson says. “Within minutes it can determine the optimal
flanking sequences that should be attached to each DNA part to produce
the desired recombinant DNA at the least expense, in a manner that is
executable by hand or robotics.”
As
a result, researchers can direct their resources to investigating their
primary interests, rather than preparing the DNA that is merely a tool
in their experiments.
“At
JBEI, we want researchers spending their time designing their DNA
constructs and assaying their function,” Hillson says. “We don’t want
them to waste their time building these things in the lab, so we’re
trying to go after ways of taking that burden off them.”
The j5 software provides a single design for the SLIC, Gibson, CPEC and Golden Gate DNA assembly strategies and determines which would be most advantageous for a given construction project. |
In
addition to identifying the most cost-effective strategies for DNA
cloning, j5 also makes it possible to construct combinatorial
libraries?collections of hundreds to millions of related DNA assemblies,
each with a different combination of genes or parts that perform
similar functions in different organisms. Combinatorial libraries enable
scientists to select the most effective genetic combination for
achieving a desired result, e.g., the most efficient production of a
biofuel or medication in a given host. No other automated DNA cloning
software does this on the same scale and as fast and effectively as j5.
“Combinatorial
libraries can be screened to identify the gene combination that, when
transferred into a desirable host organism, results in the most
productive enzyme pathway,” says Hillson. “The j5 software is the only
program that enables the combinatorial design of scarless DNA
construction methods.”
Traditional
DNA construction methods result in scars?uncontrolled portions of the
DNA sequence?at DNA fragment junctions that can adversely impact
function. Says Hillson, “The gold standard for combinatorial libraries
is the ability to control the DNA sequence at every single base pair and
this is what j5 allows you to do.”
The
j5 software package features a graphical interface that enables users
to design a DNA construct or combinatorial libraries through the
arrangement of individual part icons that abstractly represent
underlying DNA sequences. Outputs are in the form of user-friendly
spreadsheets that detail the resulting designed experimental protocols,
providing instructions that can either be followed by a person in the
laboratory or fed directly into a robotic platform for a machine to
carry out.
“Our
j5 software is already allowing a growing number of scientists to save
financial resources and months of work that was previously devoted to
constructing recombinant DNA, and has now been redirected to other
fruitful aspects of their work,” Hillson says. Currently, over 110
institutions worldwide are registered users of j5.
Other members of Hillson’s j5 development team were Rafael Rosengarten, Joanna Chen, Douglas Densmore and Timothy Ham.
The
Web-based version of j5 is available to non-commercial users under a
no-cost license agreement. Commercial users can access the Web-based
version of j5 on a 14-day trial basis before entering into a licensing
agreement. To view a demonstration video of the software and the j5
user’s manual, visit the j5 Website at http://j5.jbei.org/