The use of short RNAs in gene circuits is a relatively new development in circuit engineering, the traditional circuit players have been proteins, with directed evolution approaches playing a significant role in adapting these biomolecules for use in specific circuit configurations. Critically, according to Collins and others, any biomolecule to be used as a component or module in a gene circuit should be reliable in behavior, able to adapt to specific parameters, and interface with other biomolecules (as in the case of siRNA).
For some researchers, transcription factors (TFs) present the perfect component to meet these requirements. TFs can be activated at specific times, they can transcribe specific genes, and they interact with protein components of the transcriptional machinery, thus harnessing these molecules has been a goal for many circuit builders.
The first obstacle to overcome with TFs was figuring out how to get synthetic versions of these molecules to specific genomic location to transcribe other circuit components. Enter one of the new workhorses of modern synthetic biology — the zinc finger. Combine a zinc finger with a TF (a so-called synthetic TF or “sTF”) and the result is a TF possessing target site specificity.
In early 2012, Harvard University synthetic biology researcher Pamela Silver described in Nucleic Acids Research how tunable zinc finger TFs could be incorporated into genetic circuits to perform logic computations. Silver and her colleagues generated 15 transcriptional activators and 15 repressors that displayed various levels of repression and induction and then used these to perform a variety of simple OR, AND, NOR, and NAND logic operations.
On the heels of this work, and with the realization that expanding the number of sTFS could expand circuit possibilities, another team of researchers detailed a framework for creating novel sTFs. This work, published in Cell in August 2012by Collins and his colleagues Tim Lu from Massachusetts Institute of Technology (MIT) and Mo Khalil at Boston University, describes the ways in which output strength and transcriptional cooperativity of these TFs can be “tuned.” Surprisingly, the researchers found that even subtle changes in these properties allowed for distinct functional roles.
Although much of the work up to now has focused on creating either RNAi-based or sTF-based circuits, these components are not mutually exclusive. According to Weiss, hybrid approaches where the advantages and specificities of both components are exploited for circuit design could yield novel circuit designs.
While providing a new set of tools to engineer sTFs and, therefore, create new circuits, the Cell paper, along with other recent articles describing increasingly complex circuits, also demonstrates another outcome of today's circuits engineering efforts — these circuits and how they function in cells enhances our basic understanding of how natural cellular networks sense and analyze signals.Insulation from the elements
In August 2012, Chris Voigt, a researcher who works across town from Collins at MIT, and his colleagues described the most complex synthetic genetic circuit to date. Voigt's group engineered the first layered genetic circuits where the circuit components do not interfere with one another. Instead, four sensors for different molecules work together without interfering with one another.
Although layered and complex, at the core, this new circuit is not terribly different in principle from either Collins’ bistable genetic toggle or Elowitz and Leibler's oscillatory network. However, in Voigt's case, the circuit components do not simply not interact, they are engineered to be “insulated” from one another and the rest of the cell — no small feat in the complex environment of a cell.
While many circuit components exist today, one challenge that will always remain when building a genetic circuit is the environment, and how the components of any circuit interact with other molecules, genomic locations, and physical conditions within the cell. “It is impossible to completely isolate circuits, but unwanted interactions can be brought to a minimum,” notes Benenson.
According to Benenson, a crucial component of proper circuit function is a rational design and integration of any gene circuit (see sidebar:“Getting it all together”). And as Collins noted, components should be tested prior to use. But might it be possible to predict how a particular circuit will function in the cell even before integration and experimental testing?