Living cells make decisions based on information processing genetic programmes. Many of these programmes execute digital functions1,2,3,4,5,6,7,8. The capability to build synthetic digital systems in living cells could allow engineers to build novel decision-making regulatory networks for use in a variety of applications9, ranging from gene therapies that modify cell state based on sensed information10,11 to entirely new developmental programmes for tissue engineering12,13. In electronics a compositional approach has allowed the construction of digital circuits of great complexity to be quickly designed and implemented. Here, we have developed set of low-variability genetic parts that can be routinely composed to create large digital circuits in yeast cells.
Genetic components that implement simple logical operations, which in principle could be interconnected to form complex logic functions, have been demonstrated14,15,16,17,18,19,20,21,22,23. DNA-binding domains (DBDs) such as zinc fingers and TALEs (transcription activator-like effectors) have been used to construct libraries of transcription factors in eukaryotes19,24,25,26,27. However, scaling with DBDs in eukaryotes has been difficult because of challenges in synthesizing libraries of orthogonal parts28,29. Libraries of DBD-based parts have been shown in prokaryotes, but extensive part characterization and computer-aided design (CAD) was necessary to identify part combinations that yielded functional logic circuits22. Recently, programmable and orthogonal CRISPR-dCas9 transcription factors have been employed18,20,30,31,32,33,34 to build up to five component circuits using dCas9-mediated repression in prokaryotes18. Transcriptional repression in these circuits is likely due to steric hindrance of RNA polymerase by dCas9. Although dCas9 allows for programmable interconnections, its response function is leaky leading to signal degradation when layered18. Site-specific recombinases have been employed in genetic circuits as a means to reduce leak35,36,37, but there are a limited number of such enzymes restricting the scalability of this approach. Here, we address these issues, advancing the art of engineering living digital circuits by focusing on two main engineering goals.
In summary, we developed low-variability single-gene NOR gates that can be regularly interconnected into arbitrary topologies that implement large digital circuits in yeast cells. Neither meticulous characterization of individual parts nor sophisticated design tools were necessary to find combinations of NOR gates that conferred functional circuits. Because the technology is essentially generic and easy to rewire, it can in principle be used to implement arbitrary internal logic for a variety of synthetic cellular decision-making systems, such as those being explored for diagnostics11,41, therapeutics41,42 and development43,44.
We observed fluorescence intensity differences in the digital ON and OFF states in various circuits. To distinguish circuit state, value bands for digital ON, OFF and Undefined, fluorescence values were determined with the 16 guide sequences and their cognate pGRR promoters used in circuit construction (Supplementary Fig. 8). For the state of a circuit to be considered ON or OFF we specified that a majority of cell population fall in the expected fluorescence band. Population fraction tables for all circuits can be found in Supplementary Table 3.
(a) Repression cascades of one to seven gRNAs. Cascades were created with sequential genomic integrations of NOT gates. The final output of each cascade is a NOT gate that expresses GFP. Each NOT gate represses the output of a subsequent NOT gate. Cascades with an even number of layers express a high level of GFP, creating a digital ON output, and odd depth cascades express low levels of GFP, creating a digital OFF output. Fluorescence measurements were taken using flow cytometry. The histograms represent population fraction from three different biological replicates measured during a single experiment and were normalized so that area sums to unity. Fluorescence population ratios of the circuits are included in Supplementary Table 3. (b) Temporal dynamics for cascades of one to four gRNAs. Expression of the input gRNA was induced with β-estradiol. A model of the cascade, in which each layer is treated as a Hill function, was used to fit the data. The plot shows the data from one biological replicate. As the number of layers in the cascade increases, signal degradation and increased time to steady state is observed. (c) The steady-state response function for the four inducible cascades. Error bars represent the s.d. of three biological replicates measured over three separate experiments. (d) A representation of the model. The model was used to generate the fits for the steady-state and kinetic inducible cascade experiments.
We introduced a class of dCas9-based modular genetic NOR gates that behave digitally, have low variability and show minimal retroactivity or effects on cell growth. These features made these gates relatively easy to combine into Boolean logic circuits that are among the largest ever built in any organism. In particular, we found that most circuits in Figs 3 and 4 required that only a handful of gate combinations be screened to identify a functional design, and others required only one.
Table 1 compares our technology with selected published circuits. We measured circuit complexity with a combination of two metrics: the number of gates and the number of connections among gates, allowing us to locate circuits in a two-dimensional plot (Supplementary Fig. 14). We can calculate a complexity score using the two metrics, complexity=(gates2+connections2)1/2. For example, the XOR gate had five gates and four connections, producing a complexity of (52+42)1/2=6.4, while the cascade has a complexity of (72+12)1/2=9.2. These complexities compare well with gene circuits developed in Escherichia coli, for example. Our NOR gates enabled extremely simple design and construction of large gene circuits. Before genetic circuits can be made much larger, however, many factors that influence the size and complexity of synthetic genetic circuits must be addressed.
First, the gates in any framework must be well behaved. Gates can suffer from retroactivity, where a downstream gate affects the behaviour of upstream gates to which it is not connected by design55,56,57. In this case it is quite difficult to design large circuits even with CAD because we may not know the source of the retroactivity, how to model it or how to design with it. In addition, gates can be highly variable, where the outputs levels of one gate do not match the input levels of the next. Electrical engineers call this an impedance mismatch. A recent paper22 addressed retroactivity by adding insulators to their gates. By meticulously characterizing the performance each gate, and using CAD, they were able to select compatible subsets of parts out of which they constructed circuits as large as those demonstrated here, despite gate variability. Not all of the circuits predicted to work by the CAD tool functioned correctly, possibly due to residual retroactive effects, requiring the circuits to be screened for function. In contrast, our gates are considerably less variable and do not seem to be confounded by retroactive effects, at least in circuits with complexities
The success or failure of different approaches to building bigger circuits may depend on how well behaved, insulated, simple and scalable the input low-level devices and gates are. In addition, relaxing the requirement that circuits be digital, so that analogue or mixed analogue/digital circuits can be used when appropriate, will likely open up the design space, further increasing the size of the circuits we can build so that one day they can match the size and performance of natural genetic circuits.
Moore's prediction has been used in the semiconductor industry to guide long-term planning and to set targets for research and development, thus functioning to some extent as a self-fulfilling prophecy. Advancements in digital electronics, such as the reduction in quality-adjusted microprocessor prices, the increase in memory capacity (RAM and flash), the improvement of sensors, and even the number and size of pixels in digital cameras, are strongly linked to Moore's law. These ongoing changes in digital electronics have been a driving force of technological and social change, productivity, and economic growth.
One proposed material is indium gallium arsenide, or InGaAs. Compared to their silicon and germanium counterparts, InGaAs transistors are more promising for future high-speed, low-power logic applications. Because of intrinsic characteristics of III-V compound semiconductors, quantum well and tunnel effect transistors based on InGaAs have been proposed as alternatives to more traditional MOSFET designs.
Welcome to Blaauw Lab! Below, we hope to give you a quick overview of the exciting research we are doing, ranging from low power RF, analog mixed-signal and digital circuits, to millimeter-sensors and embedded systems. We are always looking for talented PhD applicants. If you find one of the topics below interesting, feel free to apply to our graduate program and mention our lab in your application. 2b1af7f3a8