Designing Life's Layered Circuits

strong disease mutations and those devoid a set of 10 cell lines (the test set). The proof- these systems connect is not clear, Gusella of mutations. Hence, they have compared of-concept study showed that differences in says. “The big picture hasn’t yet come to- two groups: patients with HD symptoms and transcript levels can detect the continuous gether.” The work appeared in Human Mo- normal patients (controls). These dichoto- effects of increasing HTT CAG repeat lecular Genetics in April 2013. mous analyses failed to take into account length and provide an approach to discover- differences in CAG repeat length. ing factors contributing to the pathogenic Future: Bigger Datasets Gusella and his colleagues therefore took process, which also increases with HTT In the long run, gaining a more complete a different analytical approach: They corre- CAG repeat length. The expression changes picture of Huntington’s disease progression lated gene expression across the continuum appeared in genes involved in chromatin re- will require a pooling of many different types of HTT CAG repeats from low to high (15 modeling, energy metabolism and axonal of studies—gene expression, fMRI and DWI to 92) in 97 lymphoblastoid cell lines (the transport, suggesting that CAG repeats have and others, Johnson says. “Investigating it training set). They used the results to math- downstream consequences on molecules in- jointly rather than independently is really ematically predict CAG repeat numbers in volved in these pathways. However, how where the future promise is.” n DESIGNING LIFE’S LAYERED CIRCUITS: Tools of the Trade n synthetic biology labs around the world, few inputs to alter genes, Maranas is trying at Virginia Tech’s Bioinformatics Institute, Ibrainstorming has often begun at the same to reconstruct and regulate the entire was working on recreating the genetic net- place: in front of a whiteboard. Marker in repertoire of pathways involved in a mi- works that control cell division in yeast. hand, researchers jot down the parts needed crobe’s metabolism. Like other synthetic biologists, Peccoud to form a new circuit, draw lines and arrows And it’s not just the number of switches viewed the components of the network— to show how they interact, and scrawl notes that adds complexity. Adding new enzyme genes, promoters, ribosome binding sites, about how to assemble the parts into an ap- activity into a bacterium is more compli- and terminators, to name a few—as discrete propriate whole. cated than just adding the enzyme. Take ni- parts, with defined functions, that could be “It’s usually based on intuition, and what trogenase, for example, which Maranas and shuffled around between networks. But he we know has worked in the past,” says Tim- collaborators at Washington University realized that no software existed that could othy Lu, MD, PhD, who heads up the Syn- would like to be able to control within a track which parts worked together, guide thetic Biology Group at the Massachusetts cyanobacterium. Getting the right levels of how the parts could be plugged into genetic Institute of Technology. nitrogenase activity, he adds, doesn’t just circuits, and model how a proposed circuit The whiteboard has been used to design mean having the right levels of gene and would function. many novel genetic programs—whether protein expression, but also accurately re- “It seemed reasonable to assume that aimed at turning bacteria into biosensors or producing the light to dark transitions and synthetic biology would need some com- forming networks of enzymes to churn out a providing sufficient energy in the form of puter-aided-design tools just like any other particular product. But the way of the white- ATP to power the nitrogenase. engineering discipline,” says Peccoud. CAD board might be fading. As circuits become To help manage this complexity, re- programs are heavily relied on by electrical more and more complex, and researchers searchers are developing, refining, and ap- and mechanical engineers, for example, to move toward the design of larger networks plying computerized design programs that design electrical circuits or structures on the and whole-cell programs, it’s becoming track the parts involved in their systems computer before they’re created and tested. harder to manage all the required parts for a and pinpoint the best method to assemble So his lab began developing such a pro- new project in hand-written dry erase. a new circuit. There’s not yet one program gram for biology. The result: GenoCAD, an “When I was looking at a simple circuit that fulfils the dream of “plug and play” bi- open-source, synthetic biology CAD soft- with two inputs, I could by hand iterate ology—where a few simple clicks choose ware. GenoCAD manages lists of genetic through all the possible states of the sys- the parts for the essential biological circuits parts and gives users an interface where tem,” Lu says. “Now, I’m interested in and, voilà, synthetic life! But several pro- they can set design rules, apply them to things with six or eight inputs, and intu- grams are emerging as crucial to the field. their system and then assemble genetic ition starts to fail.” parts into plasmids. It also includes a simu- Costas D. Maranas, PhD, professor of lation engine to test new circuits. chemical engineering at Penn State Uni- Inspired by Engineering In the December 2013 issue of ACS Syn- versity, concurs. While synthetic circuits of In the mid-2000s, Jean Peccoud, PhD, a thetic Biology, Peccoud and two collaborators a decade ago had a single switch and just a computational synthetic biology researcher describe using GenoCAD to create a set of Published by Simbios, the NIH National Center for Physics-Based Simulation of Biological Structures 15 grammatical rules for building novel syn- quire a strong set of software tools and entered into CAD software to make your thetic transcription factors from seven differ- provide an example for the rest of the field computer models accurate. “But in biology ent types of parts. The program was able to to follow. these days, no one has defined what, say, generate eight possible designs that met all the E. coli parameter set is,” he points out. the rules governing what parts were required Even if a circuit is completely worked out and what order they should fall in. The rules, Predictive Power Still to Come in one strain of E. coli, he says, moving it which were derived from experimental infor- Peccoud admits that the weakest part of to a new strain can drastically change how mation, can be revised and updated over GenoCAD is the newest addition to the it functions. time. As new synthetic circuits are tested in program—the simulation engine. “Being Recently, Stanford scientists created a living cells, their success or failure can help able to run simulations of the behavior of a whole-cell computational model of the guide the design of future circuits. synthetic genetic system before making it is circuits within Mycoplasma genitalium, a the holy grail of synthetic biology,” he says. human pathogen. Lu has collaborated “The science is not there yet but it is our with the Stanford team to start putting A Growing Toolbox hope that a tool like GenoCAD can help synthetic circuits into the organism. With In addition to GenoCAD, there are a support the research necessary to under- the whole-cell model at his disposal, he rapidly growing number of synthetic biol- stand gene expression better.” hopes to start predicting how new circuits ogy tools, Peccoud says. He adds that his ul- Lu says that getting more accurate sim- will work in the organism. But even that timate goal isn’t for GenoCAD to beat out ulations of biological circuits will require has been slow going, he says, and it’s just other tools. “I don’t think it should be a more data on how different organisms in- one bacterium. goal to converge to one tool,” he says. “Our teract differently with the various parts that Even without full predictive power field is so new that it is necessary that peo- make up circuits. though, programs like GenoCAD are push- ple explore different avenues.” “In other engineering disciplines, the ing the boundaries of synthetic biology, When designing DNA to characterize manufacturer of a system will give you pa- offering a more modern “whiteboard” to DESIGNING LIFE’S LAYERED CIRCUITS: TOOLS OF THE TRADE THE OF TOOLS CIRCUITS: DESIGNING LIFE’S LAYERED new promoters, George McArthur, PhD, rameters that define that particular sys- sketch out complex circuit designs and or- a chemical engineer at Virginia Common- tem,” Lu says. Those parameters can be ganize growing libraries of biological parts. wealth University, turns to a dif- By giving users a place ferent software program for to organize the gram- nearly every step of the process. matical rules that gov- Aside from GenoCAD, he uses a ern their design process, ribosome binding site (RBS) cal- and the parts that they culator that develops an RBS of want to use, it makes the whatever binding rate he needs; design step of the stan- a tool that produces inert spacer dard engineering “de- sequences; and the automated sign, build, test” cycle DNA assembly program J5 that that much easier. n gives him a list of primers for use in assembling the sequences he Seven molecular building designs in GenoCAD. blocks, each shown here “As a user, I’d love to have in a different color, can everything in one place,” McArthur be arranged in numerous says. “And already it’s great that a ways to form a functional lot of these tools adhere to the transcription factor.

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