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Hot-wiring cells
Synthetic biologists are designing genetic circuits of increasing complexity. But how did the field get to this point, and where is it going? Nathan Blow examines the challenges, and potential applications, of engineering gene circuits
Nathan Blow, Ph.D.
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Computing circuits

Weiss started his career as a computer scientist. In the mid-1990s, around the time Collins was postulating his bistable toggle switch, Weiss was wondering if it might be possible to program cells like one would program a computer. “Or maybe even use biology as a way to program a computer,” recalls Weiss.

This idea led Weiss into his doctoral studies with Tom Knight, a senior researcher in the MIT Computer Science and Artificial Intelligence Laboratory and a pioneer in the field of artificial intelligence. Around this time, Knight was also making his move into synthetic biology — actually taking classes at MIT to learn key biology concepts and molecular biology lab techniques. It was Weiss who helped Knight establish a “wet-lab” in Knight's computer lab at MIT, and then together, they began exploring genetic circuit design.

Since his time in Knight's lab, Weiss has gone on to design a number of genetic circuits of increasing complexity, but he's still part computer scientist, which explains his interest in trying to inform circuit design through computation analysis.

Computer-assisted design and analysis of gene circuits (think CAD for genetic engineering) is by most accounts in its infancy, something Weiss also acknowledges to some extent. “In principle, I agree. But the prediction capabilities are getting better.”

In fact Weiss says that simple toggles, cascades, and oscillators can now be computationally predicted fairly well. The breakdown in computational predictions comes as the complexity of the circuit increases and more components are added.

Getting it all together

One part of circuit engineering that has become easier for researchers in recent years is getting all those molecular components into cells in the proper location. “Two years ago, cloning was a major challenge, taking much time and effort,” says Yaakov (Kobi) Benenson. But the continuing development of ligation independent cloning (LIC) methodologies, alongside the refinement of nuclease-based approaches for genome engineering (i.e. zinc finger nucleases and TALENs), is greatly enhancing the front-end of circuit engineering.

LIC is a cloning approach wherein the assembly of DNA fragments is accomplished through the use of small overlapping ends and enzymes. In most cases, this approach works with a small number of fragments over a limited size range. But in 2009, Daniel Gibson and colleagues at the J. Craig Venter Institute described an assembly reaction where a larger number of fragments (upward of 10) could be assembled in a single reaction without the need for restriction digests. “Gibson assembly was really transformative for what is possible in a short amount of time,” says Ron Weiss. By using this approach, Weiss and others are now able to assemble and clone a variety of circuit components in a week's time — a rate that was unheard of prior to 2009.

Following cloning and assembly, the decision between transient transfection or stable integration of a given circuit has to be made. Many investigators still use transient transfection since any therapies would require transient transfection. When it comes to stable integration though, genome engineering tools — including ZFNs and TALENs — can be used to integrate a synthetic circuit into specific loci in almost any cell type. Specific integration is important as the genome context can play a role in circuit functionality.

Still, stable integration can cause issues for circuit builders. “Turns out that, as the circuits get longer, there is a greater chance of silencing,” explains Weiss.

Yet another development when it comes to testing your circuits is the iterative plug-and-play method described in 2012 by Jim Collins and his group. Here, a series of vectors were developed to enable the rapid construction and modification of larger gene circuits. The approach is also compatible with LIC and Gibson assembly approaches, thus providing enhancements in both upstream cloning of circuits as well as in their downstream analysis.

All these developments make Benenson hope that one day DNA cloning will become an even more routine task in synthetic biology. “Hopefully, you will be able to type the sequence and get the DNA four weeks later,” he says. “In this way, most of the time will be spent on looking at the circuits rather than cloning.” Which in the end, he adds, is much more time intensive — not to mention interesting. —NB

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